Media and methods for the establishment and maintenance of early embryo-like cells

Abstract

This application discloses a medium and method for establishing and maintaining mammalian early embryonic-like cells. The medium can be used to culture mammalian pluripotent stem cells (PSCs) and comprises a chemically defined basal medium for culturing stem cells supplemented with an S-adenosylhomocysteine ​​hydrolase (SAH) / polycomb repressive complex (PRC) / EZH2 inhibitor and a histone deacetylase (HDAC) inhibitor. The medium can be used to convert primate (human and non-human) PSCs into preimplantation ICM-like cells (ICLCs) or 8-cell embryonic-like cells (8CLCs).

Description

[Technical Field]

[0001] The present invention relates to media and methods for establishing and maintaining early mammalian embryonic-like cells. [Background technology]

[0002] Mammalian embryonic development is a complex process of cell division and differentiation that leads to the development of an embryo. Embryogenesis begins with successful fertilization of an oocyte and a sperm. This tightly controlled process results in billions of cells with distinct functions and morphologies arising from a single zygote. The enormous cellular complexity of all sexually reproducing organisms begins with embryogenesis. First, a single zygote divides to form two cells. These two cells then divide to form four, eight, and 16 cells. Further expansion leads to the development of a blastocyst, which consists of two regions: the inner cell mass (ICM) and the trophectoderm (TE). At this stage, embryonic development is referred to as the preimplantation (intrauterine wall) stage. ICM cells form the amnion and all fetal tissues, while TE cells form the placenta during postimplantation development. All of these developmental stages have been well characterized in mice, due to the ease of access to these cells from mouse embryos without ethical concerns. Seminal work by Evans and Kaufman demonstrated the feasibility of extracting cells from the ICM of mouse blastocysts and propagating them indefinitely in vitro under appropriate culture conditions (Non-Patent Document 1). These cells are called embryonic stem cells (ESCs), and they represent the cells within the ICM of mouse blastocysts. Mouse ESCs are pluripotent but not totipotent; they can differentiate into only three germ layers of the embryo (ectoderm, mesoderm, and endoderm) and thus generate cells corresponding to all fetal tissues. In contrast, totipotency refers to the ability of cells to form the entire organism, including embryonic and extraembryonic cells, rather than just fetal tissues, as in the case of pluripotent cells. In early mouse embryos, cells prior to the 4-cell stage are totipotent, whereas in humans, totipotency persists until at least the 8-cell stage (Non-Patent Document 14). Seventeen years after the discovery of Non-Patent Document 1, Thomson et al. successfully generated human ESCs from human ICM (Non-Patent Document 2).

[0003] Due to the great potential of human PSCs for disease modeling and regenerative medicine, much research has been conducted to find alternative sources of these cells that do not require the destruction of human embryos. In 2006, Takahashi and Yamanaka discovered a method to generate induced PSCs from already differentiated cells, eliminating ethical issues (Non-Patent Document 3). ESCs and iPSCs are very similar, and are collectively referred to as PSCs here. Both mouse and human ESCs are derived from the ICM of preimplantation blastocysts, yet they exhibit unique characteristics. Human PSCs cultured under conventional conditions exhibit a primed pluripotent state resembling mouse epiblast stem cells (EpiSCs) derived from the postimplantation epiblast (NPL 4). Primed human PSCs exhibit flat colony morphology, have poor viability when passaged as single cells, require fibroblast growth factor 2 (FGF2) and transforming growth factor β1 (TGFβ1) / activin A / NODAL signaling, and are unable to contribute to human-mouse interspecies chimera formation. In contrast, mouse ESCs exist in a naive state closer to the preimplantation ICM, characterized by domed colonies, increased single-cell clonogenicity, a dependence on Janus kinase / signal transducer and activator of transcription 3 (JAK / STAT3) signaling, a preimplantation ICM-like transcriptome profile, and the ability to form chimerism (NPL 5). Furthermore, mouse ESCs have greater differentiation potential than EpiSCs (Non-Patent Document 6). Furthermore, it has recently been reported that a small population (~0.5%) of 2-cell embryonic-like cells (2CLCs) in mouse ESC cultures exhibits a transcriptional profile similar to that of 2-cell (2C) stage mouse embryos (Non-Patent Document 7). This is important because 2C cells are totipotent.

[0004] Recently, several methods have been published for deriving and maintaining altered states of human and non-human primate PSCs that exhibit (preimplantation-like) human naive characteristics (Non-Patent Document 8). These cells share some morphological and molecular similarities with mouse ESCs. However, whether these reported human naive PSCs truly resemble preimplantation ICM remains under debate. Furthermore, each of the current methods has certain drawbacks, such as lengthy incubation times, variable levels of naive specific genes, transgene dependency for naive induction, genomic instability, imprinting defects, inability to differentiate into multiple lines of lineages, lack of adequate chimera formation, or inefficiency.

[0005] PSCs hold great potential for use in cell therapy in regenerative medicine and for studying disease through patient-specific disease modeling (Non-Patent Document 9). Currently, researchers are using primed PSCs as the source material for these studies. One area where naive cells are beginning to prove useful is the generation of interspecies chimeras. In these studies, PSCs from one species are injected into developing embryos of another species and the proportion of cells contributing to the organism is measured. However, contribution to chimeras is currently exceptionally low (<0.01%). We aim to use PSCs that are transcriptionally and epigenetically closer to the early embryo to improve chimera contribution and, in effect, improve overall PSC function.

[0006] Another area of ​​research using PSCs is blastoid formation. Blastoids are blastocyst-like structures currently formed in vitro by the forced aggregation of ESCs and TE cells (Non-Patent Document 10). These in vitro models of early embryos can be used to focus on developmental processes and model diseases that affect embryonic development. Nevertheless, current state-of-the-art models require the mixing of several cell types, rather than all cells arising from a single cell and self-organizing, resulting in failure to achieve a true blastocyst-like morphology, e.g., the blastocyst cannot gastrulate properly (Non-Patent Document 11). We believe that using cells that are transcriptionally and epigenetically similar to the early embryo could improve this process and allow for proper blastocyst formation.

[0007] Epigenetics is a major regulator of cell fate transitions during development. This means that by manipulating the epigenome, it should be possible to produce cells that correspond to any developmental stage. One of the best examples of this development is the generation of iPSCs from somatic cells. Here, transient expression of transcription factors or chemical compounds is sufficient to convert fully differentiated cells into PSCs (Non-Patent Document 12). Another example is the use of small molecule inhibitors of epigenetic pathways and cytokines to convert the aforementioned primed PSCs into a naive state. One important component of the epigenome is DNA methylation, which plays a central role in gene regulation. The overall DNA methylation levels of cells during early embryogenesis are highly dynamic. It is known that DNA methylation in preimplantation blastocysts is much lower than that in postimplantation embryos, and interestingly, even lower than that in 8C embryos (Non-Patent Document 13). Therefore, to revert primed PSCs to an ICM-like state, global DNA methylation levels must be significantly reduced, but correspondingly, more controlled reduction is required to capture the 8C-like stage. Furthermore, the DNA methylation landscape needs to be correctly rewired during the reversion process, respecting imprinting control regions (ICRs) and maintaining hemimethylation. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Evans, MJ, and Kaufman, MH (1981). ESTABLISHMENT IN CULTURE OF PLURIPOTENTIAL cell FROM MOUSE EMBRYOS. Nature 292, 154-156. [Non-patent document 2] Thomson,JA,Itskovitz-Eldor,J.,Shapiro,SS,Waknitz,MA,Swiergiel,JJ,Marshall,VS,and Jones,JM(1998).Embryonic stem cell lines derived from human blastocysts.Science 282,1145-1147.

Outdoor Tools3

Outdoor Tools 4

Direct Environment 5

Outdoor Configuration6

Direct Environment 7

Outdoor Track 8

Outdoor Tools9

Outdoor Tools 10

[0009] Therefore, the DNA methylation machinery needs to be tightly fine-tuned to produce early embryo-like cells. [Means for solving the problem]

[0010] In one aspect, the present disclosure discloses a chemically defined medium for culturing PSCs, comprising a basal medium for culturing stem cells supplemented with a polycomb repressive complex (PRC) and / or EZH2 inhibitor and a histone deacetylase (HDAC) inhibitor. In one or more embodiments, the PRC and / or EZH2 inhibitor is an S-adenosylhomocysteine ​​hydrolase (SAH) inhibitor. In one or more embodiments, the medium of claim 1 or 2 further comprises one or more components selected from the group consisting of L-ascorbic acid or a derivative thereof, a JAK / STAT3 signaling activator, a mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) signaling inhibitor, and a tankyrase inhibitor, and is optionally further supplemented with one or more components selected from the group consisting of an activin / NODAL signaling activator, a Rho-associated protein kinase (ROCK) inhibitor, and an extracellular matrix.

[0011] In one or more embodiments, the PRC / EZH2 inhibitor is 3-deazaneplanocin A (DZNep) or CPI-1205. In one or more embodiments, the final concentration of the DZNep in the medium is 5 to 80 nM, preferably 5 to 50 nM. In one or more embodiments, the final concentration of the CPI-1205 in the medium is 0.5 to 5 mM, preferably 1 to 3 mM. In one or more embodiments, the HDAC inhibitor is selected from the group consisting of trichostatin A (TSA), valproic acid (VPA), and sodium butyrate (NaB). In one or more embodiments, the final concentration of the TSA in the medium is 3 to 30 nM, preferably 3 to 25 nM. In one or more embodiments, the final concentration of the VPA in the medium is 0.25 to 2 mM, preferably 0.5 to 1.5 mM. In one or more embodiments, the final concentration of NaB in the medium is 0.25 to 2 mM, preferably 0.5 to 1.5 mM. In one or more embodiments, the final concentration of L-ascorbic acid in the medium is 40-70 μg / ml. In one or more embodiments, the final concentration of the activator of JAK / STAT3 signaling in the medium is 10-50 ng / mL. In one or more embodiments, the JAK / STAT3 signaling activator is LIF. In one or more embodiments, the final concentration of PD0325901 in the medium is 0.5-3 μM. In one or more embodiments, the MAPK / ERK signaling inhibitor is PD0325901. In one or more embodiments, the final concentration of the tankyrase inhibitor in the medium is 2-8 μM. In one or more embodiments, the tankyrase inhibitor is IWR1 or XAV939.

[0012] In one or more embodiments, the final concentration of the activator of activin / NODAL signaling is 10-25 ng / ml. In one or more embodiments, the activin / NODAL signaling activator is activin A or NODAL. In one or more embodiments, the final concentration of the ROCK inhibitor in the medium is 0.5-2 μM. In one or more embodiments, the ROCK inhibitor is selected from the group consisting of Y27632, thiazovivin, and hydroxyfasudil. In one or more embodiments, the amount of extracellular matrix in the medium is 0.1 to 0.5% (v / v). In one or more embodiments, the extracellular matrix is ​​selected from the group consisting of Matrigel™, Geltrex™, and ECM™.

[0013] In one or more embodiments, the medium contains the following: DZNep at a final concentration of 5-15 nM or CPI-1205 at a final concentration of 0.5-3 mM; TSA at a final concentration of 3-10 nM or VPA at a final concentration of 0.25-1 mM or NaB at a final concentration of 0.25-1 mM; L-ascorbic acid at a final concentration of 40-70 μg / ml; LIF at a final concentration of 10-30 ng / mL; PD0325901 at a final concentration of 0.5-1.5 μM; and IWR1 or XAV939 at a final concentration of 3-6 μM, and further contains the following: (1) activin A or NODAL at a final concentration of 10 to 25 ng / ml; Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM; and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (2) the activin A or NODAL at a final concentration of 10 to 25 ng / ml; the Y27632, thiazobin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM; or (3) the activin A or NODAL at a final concentration of 10 to 25 ng / ml; and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (4) a final concentration of 0.5 to 2 μM of the Y27632, thiazovivin, or hydroxyfasudil; and an amount of 0.1% to 0.5% (v / v) of the extracellular matrix; or (5) the activin A or NODAL at a final concentration of 10 to 25 ng / ml; the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM; or the extracellular matrix at 0.1% to 0.5% (v / v); has been replenished.

[0014] In one or more embodiments, the medium contains the following: 10 nM of the DZNep or 1 mM of the CPI-1205; 5 nM of the TSA or 0.5 mM of the VPA or 0.5 mM of the NaB; and 50 μg / ml of the L-ascorbic acid; 20 ng / mL of the LIF; 1 μM of the PD0325901; and 5 μM of the IWR1 or 5 μM The XAV939; and further comprising: (1) 20 ng / mL of human activin A or human NODAL, 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, and 0.2% (v / v) of the extracellular matrix; or (2) 20 ng / mL of the activin A or NODAL, 1 μM of the Y27632, thiazovivin, or hydroxyfasudil; or (3) a final concentration of 10 to 25 ng / mL of the activin A or NODAL; and 0.1% to 0.5% (v / v) of the extracellular matrix; or (4) a final concentration of 0.5 to 2 μM of the Y27632, thiazovivin, or hydroxyfasudil; and an amount of 0.1% to 0.5% (v / v) of the extracellular matrix; or (5) The activin A or NODAL is added at a final concentration of 10 to 25 ng / ml; the Y27632, thiazovivin, or hydroxyfasudil is added at a final concentration of 0.5 to 2 μM; or the extracellular matrix is ​​added at 0.1% to 0.5% (v / v).

[0015] In one or more embodiments, the medium contains the following: the DZNep at a final concentration of 40 to 70 nM or the CPI-1205 at a final concentration of 2 to 4 mM; the TSA at a final concentration of 10 to 30 nM or the VPA at a final concentration of 0.5 to 1.5 mM or the NaB at a final concentration of 0.5 to 1.5 mM; the L-ascorbic acid at a final concentration of 40 to 70 μg / ml; the LIF at a final concentration of 10 to 30 ng / mL; the PD0325901 at a final concentration of 0.5 to 1.5 μM; and the IWR1 or XAV939 at a final concentration of 3 to 6 μM, and further contains the following: (1) activin A or NODAL at a final concentration of 10 to 25 ng / ml; Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM; and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (2) the activin A or NODAL at a final concentration of 10 to 25 ng / ml; the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM; or (3) the activin A or NODAL at a final concentration of 10 to 25 ng / ml; and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (4) a final concentration of 0.5 to 2 μM of the Y27632, thiazovivin, or hydroxyfasudil; and an amount of 0.1% to 0.5% (v / v) of the extracellular matrix; or (5) the activin A or NODAL at a final concentration of 10 to 25 ng / ml; or the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM; or the extracellular matrix at 0.1% to 0.5% (v / v) has been replenished.

[0016] In one or more embodiments, the medium comprises the following: 50 nM of the DZNep or 3 mM of the CPI-1205; 20 nM of the TSA or 1 mM of the VPA or 1 mM of the NaB; and; 50 μg / ml of the L-ascorbic acid; 20 ng / mL of the LIF; 1 μM of the PD0325901; and 5 μM of the IWR1 or 5 μM of the XAV939; (1) 20 ng / mL of the activin A or NODAL, 1 μM of the Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v / v) of the extracellular matrix; or (2) 20 ng / mL of the activin A or NODAL, 1 μM of the Y27632, thiazovivin or hydroxyfasudil; or (3) 20 ng / mL of the activin A or NODAL, 0.2% (v / v) of the extracellular matrix; or (4) 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, and 0.2% (v / v) of the extracellular matrix; or (5) 20 ng / mL of the activin A or NODAL, 1 μM of the Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v / v) of the extracellular matrix; has been replenished.

[0017] In one or more embodiments, the basal medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F10, F12, alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (GMEM), Icecove's Modified Dulbecco's Medium, Neurobasal Medium, and DMEM / F12, and combinations thereof, wherein the basal medium is a 1:1 (v / v) mixture of advanced DMEM / F12 and Neurobasal Medium.

[0018] In one or more embodiments, the medium is further supplemented with one or more components selected from the group consisting of serum replacement, an alternative carbon source, non-essential amino acids, L-glutamine or a substitute thereof, and antibiotics. In one or more embodiments, the serum replacement is a mixture selected from the group consisting of Knockout™ Serum Replacement (KOSR), N2, and B27, and combinations thereof, wherein N2 and B27 are mixed in a 1:1 (w / w) ratio; An alternative carbon source is pyruvate, such as sodium pyruvate; L-glutamine or its substitute is Glutamax™ supplement containing L-alanyl-L-glutamine dipeptide in 0.85% NaCl; and / or The antibiotic is selected from the group consisting of penicillin, streptomycin, and a mixture of penicillin and streptomycin.

[0019] In another aspect, the present disclosure discloses methods for converting primate PSCs into preimplantation ICM-like cells (ICLCs) and / or 8-cell embryonic-like cells (8CLCs), including culturing primate PSCs or ICLCs in the presence of a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor. The present disclosure further discloses methods for converting ICLCs into 8CLCs, including culturing ICLCs in the presence of a SAH / PRC / EZH2 inhibitor and a histone deacetylase (HDAC) inhibitor.

[0020] In one or more embodiments, the method comprises culturing primate PSCs or ICLCs in the presence of a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor, and one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a tankyrase inhibitor, and optionally, culturing in the presence of one or more components selected from the group consisting of an activin / NODAL signaling activator, a ROCK inhibitor, and an extracellular matrix.

[0021] In one or more embodiments, the SAH / PRC / EZH2 inhibitor is DZNep or CPI-1205. In one or more embodiments, the HDAC inhibitor is selected from the group consisting of TSA, VPA, and NaB. In one or more embodiments, preferably, primate PSCs or ICLCs are cultured in the presence of the DZNep at a final concentration of 5 to 80 nM, preferably 5 to 50 nM, or in the presence of CPI-1205 at a final concentration of 0.5 to 5 mM, preferably 1 to 3 mM, and in the presence of the TSA at a final concentration of 3 to 30 nM, preferably 3 to 25 nM, or in the presence of VPA at a final concentration of 0.25 to 2 mM, preferably 0.5 to 1.5 mM, or The culture is carried out in the presence of NaB at a final concentration of 0.25 to 2 mM, preferably 0.5 to 1.5 mM.

[0022] In one or more embodiments, L-ascorbic acid is present at a final concentration of 40-70 μg / mL. In one or more embodiments, the final concentration of the activator of JAK / STAT3 signaling is 10-50 ng / mL. In one or more embodiments, the JAK / STAT3 signaling activator is LIF. In one or more embodiments, the final concentration of the inhibitor of MAPK / ERK signaling is 0.5-3 μM. In one or more embodiments, the inhibitor of MAPK / ERK signaling is PD0325901. In one or more embodiments, the final concentration of the tankyrase inhibitor is 2-2 μM. In one or more embodiments, the tankyrase inhibitor is IWR1 or XAV939. In one or more embodiments, the final concentration of the activator of activin / NODAL signaling is 10-25 ng / ml. In one or more embodiments, the activin / NODAL signaling activator is activin A or NODAL. In one or more embodiments, the final concentration of the ROCK inhibitor is 0.5-2 μM. In one or more embodiments, the ROCK inhibitor is selected from the group consisting of Y27632, thiazovivin, and hydroxyfasudil. In one or more embodiments, the extracellular matrix is ​​present in an amount of 0.1% to 0.5% (v / v). In one or more embodiments, the extracellular matrix is ​​selected from the group consisting of Matrigel™, Geltrex™, and ECM™.

[0023] In another aspect, the disclosure further provides a method of converting primate PSCs into ICLCs, comprising culturing primate PSCs in a medium disclosed herein, wherein the basal medium of the medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F10, F12, alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (GMEM), Icecove's Modified Dulbecco's Medium, Neurobasal Medium, and DMEM / F12, and combinations thereof, wherein the basal medium is a 1:1 (v / v) mixture of advanced DMEM / F12 and Neurobasal Medium.

[0024] In a further aspect, the disclosure provides a method of converting primate PSCs or ICLCs to 8CLCs, comprising culturing the primate PSCs or ICLCs in a medium disclosed herein, wherein the basal medium of the medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F10, F12, alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (GMEM), Icecove's Modified Dulbecco's Medium, Neurobasal Medium, DMEM / F12, and Advanced DMEM / F12, and wherein the basal medium is a mixture of Advanced DMEM / F12 and Neurobasal Medium in a 1:1 (v / v) ratio. In one or more embodiments, the primate PSCs are: is selected from the group consisting of: (i) cells derived from ESC and / or ECC lines; (ii) cells derived from iPSC lines; (iii) cells derived from the ICM of in vitro cultured preimplantation blastocysts; (iv) cells derived from the ICM of in vitro cultured postimplantation blastocysts; (v) in vitro cultured cells from stage 8C embryos to the morula stage;

[0025] In one or more embodiments, primate PSCs or ICLCs are isolated from a mammalian subject by one of the following methods: Primate PSCs or ICLCs were grown: (i) on feeder cells; (ii) on feeder-free extracellular matrix; (iii) in suspension without feeder cells; (iv) expanded under hypoxic or normoxic conditions at a temperature of approximately 37°C; (v) passaged as single cells every 3-4 days at a split ratio of 1:4 to 1:8; and (vi) with daily changes of medium. The cells are cultured under one or more conditions selected from the group consisting of:

[0026] In a further aspect, the present disclosure provides isolated primate ICLCs with transcriptomes, transposable element profiles, DNA methylomes, chromatin landscapes, and metabolic states that approximate those of corresponding primate preimplantation ICMs.

[0027] In one or more embodiments, the primate ICLC comprises one of the following: 1) they can self-renew and maintain pluripotency in culture; 2) maintaining genome stability in culture according to karyotype; 3) can give rise to cells of the three germ layers; 4) can give rise to primordial germ cell-like cells; 5) They can integrate into mouse embryos and contribute to embryonic and extraembryonic tissues; 6) capable of transitioning to extraembryonic cell behavior in vitro; and 7) capable of forming blastocyst-like structures in vitro; The method is characterized by one or more selected from the group consisting of:

[0028] In one or more embodiments, the 8CLC is obtained by any of the methods described herein for producing 8CLC. The present disclosure also provides a cell culture comprising cells and a medium comprising the primate ICLC and / or 8CLC described in any embodiment herein, preferably comprising a medium specified herein. 1. A kit comprising a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor, optionally comprising: (1) one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a tankyrase inhibitor; and (2) one or more components selected from the group consisting of activin / NODAL signaling activators, ROCK inhibitors, and extracellular matrices; (3) The medium contains one or more components selected from the group consisting of a basal medium, serum replacement, an alternative carbon source, a non-essential amino acid, L-glutamine or a substitute therefor, and an antibiotic. In one or more embodiments, the kit comprises any of the media identified herein. 1. A composition comprising a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor, optionally comprising: (1) one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a tankyrase inhibitor; and (2) one or more components selected from the group consisting of activin / NODAL signaling activators, ROCK inhibitors, and extracellular matrices; Includes. In one or more embodiments, a composition comprising DZNep or CPI-1205, and TSA or VPA or NaB, and optionally L-ascorbic acid, optionally LIFI, optionally PD0325901, and optionally WR1 or XAV939, preferably each of said components being present in a medium comprising said composition such that the medium is one of the following: In one or more embodiments, the 8CLC is obtained by any of the methods for producing 8CLC described herein, including 5-15 nM, preferably 10 nM, DZNep, or 0.5-2 mM, preferably 1 mM, CPI-1205; 4-6 nM, preferably 5 nM, TSA, or 0.25-1 mM, preferably 0.5 mM, VPA; or 0.25-1 mM, preferably 0.5 mM, NaB; and, optionally, 40-90 μg / mL, preferably 50 μg / mL, L-ascorbic acid; optionally, 10-30 ng / mL, preferably 20 ng / mL, LIF; optionally, 0.5-1.5 μM, preferably 1 μM, PD0325901; and optionally, 3-6 μM, preferably 5 μM, IWR1 or XAV939. The present disclosure also provides a cell culture comprising cells and a medium comprising the primate ICLC and / or 8CLC described in any embodiment herein, preferably comprising a medium specified herein. 1. A kit comprising a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor, optionally comprising: (1) one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a tankyrase inhibitor; and (2) one or more components selected from the group consisting of activin / NODAL signaling activators, ROCK inhibitors, and extracellular matrices; (3) The medium contains one or more components selected from the group consisting of a basal medium, serum replacement, an alternative carbon source, a non-essential amino acid, L-glutamine or a substitute therefor, and an antibiotic. In one or more embodiments, the kit comprises any of the media identified herein. 1. A composition comprising a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor, optionally comprising: (1) one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a tankyrase inhibitor; and (2) one or more components selected from the group consisting of activin / NODAL signaling activators, ROCK inhibitors, and extracellular matrices; Includes. In one or more embodiments, a composition comprising DZNep or CPI-1205, and TSA or VPA or NaB, and optionally L-ascorbic acid, optionally LIFI, optionally PD0325901, and optionally WR1 or XAV939, preferably each of said components being present in a medium comprising said composition such that the medium is one of the following: 5-15 nM, preferably 10 nM, DZNep, or 0.5-2 mM, preferably 1 mM, CPI-1205; 4-6 nM, preferably 5 nM, TSA, or 0.25-1 mM, preferably 0.5 mM, VPA; or 0.25-1 mM, preferably 0.5 mM, NaB; and optionally 40-90 μg / mL, preferably 50 μg / mL, L-ascorbic acid; optionally 10-30 ng / mL, preferably 20 ng / mL, LIF; optionally 0.5-1.5 μM, preferably 1 μM, PD0325901; and optionally 3-6 μM, preferably 5 μM, IWR1 or XAV939. The composition is present in an amount that can further include activin A or NODAL, and / or Y27632, thiazovivin or hydroxyfasudil, and / or extracellular matrix, wherein each of the components is present in an amount that can allow the medium containing the composition to contain 10 to 25 ng / mL, preferably 20 ng / mL, of activin A or NODAL, and / or 0.5 to 2 μM, preferably 1 μM, of Y27632, thiazovivin or hydroxyfasudil, and / or 0.1% to 0.5% (v / v) of extracellular matrix.

[0029] In one or more embodiments, a composition comprising DZNep or CPI-1205, and TSA or VPA or NaB, and optionally L-ascorbic acid, optionally LIFI, optionally PD0325901, and optionally IWR1 or XAV939, preferably each of said components being present in a medium comprising said composition such that the medium is one of the following: 40-70 nM, preferably 50 nM DZNep, or 2-4 mM, preferably 3 mM CPI-1205; 10-30 nM, preferably 20 nM TSA, or 0.5-1.5 mM, preferably 1 mM VPA, or 0.5-1.5 mM, preferably 1 mM NaB; 2-8 μM, and optionally 40-90 μg / mL, preferably 50 μg / mL L-ascorbic acid, optionally 10-30 ng / mL, preferably 20 ng / mL LIF, optionally 0.5-1.5 μM, preferably 1 μM PD0325901, optionally preferably 3-6 μM, preferably 5 μM IWR1 or XAV939. The composition is present in an amount that can further include activin A or NODAL, and / or Y27632, thiazovivin or hydroxyfasudil, and / or extracellular matrix, wherein each of the components is present in an amount that can allow the medium containing the composition to contain 10 to 25 ng / mL, preferably 20 ng / mL, of activin A or NODAL, and / or 0.5 to 2 μM, preferably 1 μM, of Y27632, thiazovivin or hydroxyfasudil, and / or 0.1% to 0.5% (v / v) of extracellular matrix.

[0030] The present disclosure also provides the use of an agent capable of promoting the expression of STELLA or improving the activity of STELLA in the manufacture of a reagent, medium, or kit for promoting the conversion of primate PSCs to ICLCs, or promoting the conversion of primate PSCs or ICLCs to 8CLCs, and the use of an agent capable of promoting the expression of STELLA or improving the activity of STELLA for promoting the conversion of primate PSCs to ICLCs, or promoting the conversion of primate PSCs or ICLCs to 8CLCs. In one or more embodiments, the agent capable of promoting STELLA expression or increasing STELLA activity is an inhibitor of SAH / PRC / EZH2, including, but not limited to, DZNep and CPI-1205. Preferably, SAH / PRC / EZH2 inhibitors, such as DZNep and CPI-1205, are used in the above applications in the amounts described in any of the embodiments described herein.

[0031] The present disclosure further provides the use of an agent capable of promoting the expression of genes belonging to the Eutherian Totipotent Cell Homeobox (ETCHbox) family, including KHDC1L, TRIM60, and / or TPRX1 and ARGFX, or wherein KHDC1L, TRIM60, and / or the protein can improve the activity of proteins belonging to the ETCHbox family, including TPRX1 and ARGFX, in the manufacture of a reagent, medium, or kit for promoting the conversion of primate PSCs or ICLCs to 8CLCs, and the use of an agent capable of promoting the expression of genes belonging to the ETCHbox family, including KHDC1L, TRIM60, and / or TPRX1 and ARGFX, or wherein KHDC1L, TRIM60, and / or the protein can improve the activity of proteins belonging to the ETCHbox family, including TPRX1 and ARGFX, for promoting the conversion of primate PSCs and / or ICLCs to 8CLCs.

[0032] In one or more embodiments, the agent capable of promoting expression of KHDC1L, TRIM60, and / or a gene belongs to the ETCHbox family, including TPRX1 and ARGFX, or the agent capable of improving activity of KHDC1L, TRIM60, and / or a protein belongs to the ETCHbox family, including TPRX1 and ARGFX, and is an inhibitor of SAH / PRC / EZH2, including DZNep and CPI-1205. Preferably, SAH / PRC / EZH2 inhibitors, such as DZNep and CPI-1205, are used in the above uses in the amounts described in any of the embodiments described herein. [Brief explanation of the drawings]

[0033] [Figure 1] (A) Schematic diagram of the protocol we used to generate human ICLCs. Briefly, primed human PSCs cultured in mTeSR medium were switched to ICLC conversion medium (4CL) for 12 days, with cells passaged on days 4 and 8. (B) Phase-contrast microscopy images showing the morphology of primed human PSCs (left panel) and ICLCs converted in 4CL medium 1 (right panel). (C) Representative immunofluorescence microscopy images of ICLC colonies. Nuclei were stained with anti-KLF17 (top and middle panels), anti-NANOG (middle and middle panels), or anti-OCT4 (bottom and middle panels), and counterstained with DAPI (left column). Different channels were merged (right column). [Figure 2] (A) 2D scatter plot showing UMAPs of single-cell RNA-seq gene expression in H9 cells at the priming stage (day 0) and days 1, 2, 3, 5, 8, and 12 after culture in 4CL Medium 1. We also included published single-cell RNA-seq data from human embryonic cells at embryonic days 3 (E3), 4 (E4), 5 (E5), 6 (E6), and 7 (E7) (from E-MTAB-3929). (B) Heatmap (from GSE101571) showing the expression levels of known naive markers in primed H9, H9 converted from 4CL Medium 1, and human ICM cells (right panel). [Figure 3] (A) 2D scatter plot showing UMAP of single-cell RNA-seq transposable element (TE) expression in H9 cells at the priming stage (day 0) and days 1, 2, 3, 5, 8, and 12 after culture in 4CL Medium 1. We also included published single-cell RNA-seq TE expression data from human embryonic cells at embryonic days 3, 4, 5, 6, and 7 (from E-MTAB-3929). (B) Heatmap showing expression levels of known native TEs in primed H9, H9 converted from 4CL Medium 1, and human ICM cells (from GSE101571). [Figure 4]Representative images of chromosomes after Giemsa staining show that cells maintain a stable karyotype in long-term culture. Chromosome analysis was performed on primed H9 (upper left panel), H9 transformed in 4CL Medium 1 at passage 15 (upper right panel), primed UH10 (lower left panel), and UH10 transformed in 4CL Medium 1 at passage 15 (lower right panel). [Figure 5] Box plots showing genome-wide CpG methylation levels (left column) and box plots showing the 2 kb positions surrounding the TSS of all genes (right column). [Figure 6] 1 is a heatmap showing CpG methylation levels in selected ICRs of human ICM cells compared with 4CL-converted cells. [Figure 7] (A-D) 2D scatter plots show UMAP visualization of chromatin accessibility at the KLF17, DPPA3 / STELLA, DPPA5, CD70, POU5F1, and THY1 loci in primed and ICLC single cells. [Figure 8] (A) Specific accessible chromatin regions in primed human PSCs and cells during conversion to ICLCs using 4CL Medium 1. Regions close to the primed region are opened during conversion to ICLCs (top panel). Regions open in the primed state are closed during conversion to ICLCs (bottom panel). (B) Motif enrichment analysis showing the selection of motifs enriched in regions close to open regions (top) and regions that transition from open to closed regions (bottom) during the primed state of ICLC conversion. (C) Bar graphs showing the expression levels of TFAP2C, KLF5, SOX3, and ZIC3 in primed human PSCs and ICLCs 12 days after conversion. [Figure 9] (A) Bar graph showing elevated oxidative phosphorylation (OxPhos)-related genes in 4CL Medium 1 converted ICLCs compared to primed human PSCs. (B) Heatmap (from GSE101571) showing expression levels of selected metabolic genes in primed, ICLCs, and human ICMs. [Figure 10]Hematoxylin and eosin staining of ICLC-derived teratoma tissue demonstrates the structure of all three germ layers: mesoderm (left panel), endoderm (middle panel), and ectoderm (right panel). [Figure 11] (A) Bar graph showing the expression levels of priming, ICM, and TSC markers in TSCLC differentiated from H9 ICLC compared to H9 ICLC. (B) Immunofluorescence microscopy images showing the expression of TSC markers: GATA3, TFAP2C, and KRT7. (C) Principal component analysis comparing the transcriptomes of H9 transformed from 4CL (H9-4CL), TSCLC (H9-TSCLC), trophoblast carcinoma cell lines JEG3 and BeWo, and trophoblasts isolated from mid-trimester human placenta (EGFR and HLAG). (D) Methylation plot showing the CpG methylation status of the ELF5 promoter in priming, ICLC, and TSCLC. [Figure 12] (A) Table showing the number of blastocysts injected with primed, 4CL, or e4CL medium-converted cells and the number of embryos that incorporated labeled cells into the ICM and TE. (B) Microscopic images showing phase contrast (left) and red fluorescence (right) of mouse blastocysts injected with DsRed-labeled human PSCs or ICLCs. (C) Immunofluorescence of injected and uninjected embryos counterstained with anti-OCT4, anti-CDX2, or DAPI. [Figure 13] (A) Phase contrast (top) and red fluorescence (bottom) images of an E10.5 mouse embryo (left), placenta (middle), and yolk sac (right). (B) Immunofluorescence images showing the expression of GATA6 (red) and human nuclear antigen (hN) (green) in an E10.5 mouse embryo. Nuclei were counterstained with DAPI (blue). (C) Immunofluorescence images showing the expression of DsRed (red) and GATA3 (green) in an E10.5 mouse placenta. Nuclei were counterstained with DAPI (blue) in placental tissue sections. [Figure 14] (A) Phase-contrast microscopy images of self-forming blastoids from ICLCs. (B) Immunofluorescence images of self-forming blastoids stained with anti-OCT4 (red), anti-GATA3 (green), or the nuclear counterstain DAPI (blue). [Figure 15]1 is a bar graph showing expression levels of ICM and primed markers in H9, H1, HUES1, and WIBR3 human ESC lines converted to ICLCs using 4CL medium 1. [Figure 16] 1 is a bar graph of RT-qPCR data showing that a panel of pre-implantation ICM markers, KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, REX1, are significantly induced in ICLCs transformed on Geltrex™ coated plates using 4CL Medium 1. [Figure 17] Bar graph of RT-qPCR data showing that a panel of preimplantation epiblast markers, KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1, are significantly induced in ICLCs converted to suspension using 4CL Medium 1. For each gene, the left column represents culture in vegetative cells, and the right column represents culture in suspension. [Figure 18] (A-C) Bar graphs of RT-qPCR data show that a panel of preimplantation ICM markers, KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1, are significantly induced in ICLCs transformed with 4CL medium 2, 4CL medium 3, and 4CL medium 4, respectively. [Figure 19] (A) Schematic diagram showing two methods for generating 8CLCs. Briefly, the medium (e.g., mTeSR1) of primed human PSCs is changed to either e4CL or 4CL medium. Cells are then either continuously grown in e4CL or switched to e4CL medium after two passages in 4CL medium. (B) Bar graph showing the expression levels of selected naive pluripotency markers in H9-primed and H9-e4CL cells. (C) Bar graph showing the expression levels of selected naive pluripotency markers in H9-e4CL and H9-4CL cells. (D) Induction of 8CL-specific genes is similar with both methods. (E) Immunofluorescence microscopy images showing the expression of ZSCAN4 (green) or DAPI counterstained nuclei (blue) in primed H9, H9-4CL, and H9-e4CL. [Figure 20] (A) 2D scatter plot showing UMAP converted single-cell RNA-seq data for H9 cells at the priming stage (day 0) and then days 1, 2, 3, and 5 after culturing in e4CL medium. We also included published single-cell RNA-seq gene expression data from human embryonic cells at days E3, 4, 5, 6, and 7 (from E-MTAB-3929). (B) Heatmap showing expression levels of known 8C markers in primed H9, e4CL-converted H9, and human 8C embryonic cells (from E-MTAB-3929). [Figure 21] (A) 2D scatter plot showing UMAP single-cell RNA-seq TE expression in H9 cells at the priming stage (day 0) and then at days 1, 2, 3, and 5 after culturing in e4CL medium. We also included published single-cell RNA-seq TE expression data from human embryonic cells at days 3, 4, 5, 6, and 7 (from E-MTAB-3929). (B) Heatmap showing RNA gene expression of known native TEs in primed H9, e4CL-converted H9, and human 8C embryonic cells (from E-MTAB-3929). [Figure 22] Representative images of chromosomes after Giemsa staining showing normal karyotypes in H9 (top left panel), e4CL-H9 (top right panel), primed UH10 (bottom left panel), and e4CL-UH10 (bottom right panel) cells. [Figure 23] Box plots showing CpG methylation throughout the genome (left panel) and within 2 kb surrounding the TSS (right panel). [Figure 24] 10 is a heatmap showing CpG methylation at selected ICRs in human ICM (from GSE101571) compared to 8CLC. [Figure 25] Specifically accessible chromatin regions in primed human PSCs and cells undergoing conversion to 8CLCs. Regions that are close together in the primed state are released during conversion to 8CLCs (top panel). Regions that are open in the primed state are released during conversion to 8CLCs (bottom panel). [Figure 26] 1 is a heatmap (from E-MTAB-3929) showing expression of selected metabolic genes in primed H9, H9 8CLC, and human 8C embryonic cells. [Figure 27] Hematoxylin and eosin staining of teratoma tissue derived from 8CLC shows structures of all three germ layers: mesoderm (left panel), endoderm (middle panel), and ectoderm (right panel). [Figure 28] 1 is a bar graph showing that multiple TSC markers, such as GATA3, CGA, ELF5, TP63, KRT18, KRT8, PSG6, and CCR7, are significantly induced in TSCLCs compared to undifferentiated 8CLCs. [Figure 29] (A) Microscopy images showing phase contrast (left) or red fluorescence channel (right) of mouse blastocysts injected with DsRed to label primed human PSCs or 8CLCs. (B) Immunofluorescence images of embryos counterstained with anti-OCT4, anti-CDX2, or DAPI. [Figure 30] (A) Microscopic images showing phase contrast (top) or red fluorescence (bottom) of E10.5 mouse embryos (left), placenta (middle), or yolk sacs (right). (B) Immunofluorescence images showing GATA6 (red) and hN (green) expression in mouse embryos or counterstained with DAPI (blue). (C) Immunofluorescence images showing DsRed (red) and KRT7 (green) expression in mouse placenta or counterstained with DAPI (blue). [Figure 31] (A) Phase-contrast microscopy images of self-forming blastoids from 8CLCs. (B) Immunofluorescence images of self-forming blastoids stained with anti-OCT4 (red) and anti-GATA3 (green) antibodies or counterstained with DAPI (blue). [Figure 32] Bar graph of RT-qPCR data showing that 8CLC markers ZSCAN4, ARGFX, TPRX1, ZNF280A, and ZSCAN5B are significantly induced in 8CLCs transformed into suspension using e4CL medium. In the bar graph, the left column for each gene represents vegetative culture, and the right column represents suspension culture. [Figure 33] Bar graphs of RT-qPCR data showing that the 8C markers ZSCAN4, ARGFX, TPRX1, ZNF280A, ZSCAN5B, DUXA, DUXB, and MBD3L2 are significantly induced in 8C CLCs converted from multiple hPSC lines. As shown in Figure 33, the columns for each of these genes in primed HN10 and UH10 are essentially absent, indicating very low expression of these genes in primed HN10 and UH10. [Figure 34] 1 is a bar graph of RT-qPCR data showing that expression levels of preimplantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 in ICLCs transformed in 4CL Medium 1 under normoxia were comparable to those under hypoxia. [Figure 35](A) Heatmap showing the expression levels (log-fold change compared to the primed state) of preimplantation ICM-enhanced genes in cells transformed by 4CL and published naive PSC medium containing NHSM, tt2iLGoe, and 5iLAF. Human preimplantation ICM (ICM embryos) are included as a control. Hierarchical clustering along with the heatmap indicates that 4CL is closer to human preimplantation ICM. Thus, 4CL is significantly better at remodeling the transcriptome to the preimplantation ICM state than published naive PSC medium containing NHSM, tt2iLGoe (umlaut), and 5iLAF. (B) Heatmap showing the expression levels (log-fold change compared to the primed state) of totipotent genes in published naive or expanded PSC medium transformed by e4CL and containing tt2iLGoe, 5iLAF, and EPSCs. Human 8C embryos are included as a control. Hierarchical clustering along with the heatmap indicates that e4CL is much closer to human 8C embryos. Thus, e4CL is significantly superior to published naive or expanded PSC media containing tt2iLGoe, 5iLAF, and EPSCs in remodeling the transcriptome to an 8C embryonic state. (C) Heatmap showing CpG methylation levels in the imprinting control region of human PSCs cultured in primed conditions, 4CL, e4CL, 5iLAF, tt2iLGoe, NHSM, human ICM, and postimplantation embryos. Hierarchical clustering along the heatmap indicates that 4CL and e4CL are closer to human ICM. Thus, 4CL and e4CL are significantly superior to published naive PSC media containing 5iLAF, tt2iLGoe, and NHSM in remodeling CpG methylation in the imprinting control region to an early embryonic state. [Figure 36](A) Phase contrast image showing colonies in 4CL supplemented with WNT signaling activator CHIR99021 (4CL+CHIR) medium are flatter than in 4CL medium. (B) RT-qPCR showing preimplantation ICM genes are less well induced in 4CL+CHIR compared to 4CL. (C) Immunostaining image showing the expression of a shared pluripotency marker (NANOG) and preimplantation ICM marker (KLF17) in 4CL and 4CL+CHIR (NANOG is present in both 4CL and 4CL+CHIR, while KLF17 is only present in 4CL). (D) Heatmap showing the expression of preimplantation ICM genes is less well induced in 4CL+CHIR compared to 4CL. [Figure 37] (A) Phase contrast image showing colonies in 4CL excluding WNT / β-catenin signaling inhibitor (4CL-IWR1) medium. Colonies are flatter than those in 4CL medium. (B) RT-qPCR showing preimplantation ICM genes are less induced in 4CL-IWR1 compared to 4CL. (C) Immunostaining image showing the expression of a shared pluripotency marker (NANOG) and preimplantation ICM marker (KLF17) in 4CL and 4CL-IWR1. NANOG is present in both 4CL and 4CL-IWR1, while KLF17 is only present in 4CL. (D) Heatmap showing the expression of preimplantation ICM genes is less induced in 4CL-IWR1 compared to 4CL. DETAILED DESCRIPTION OF THE INVENTION

[0034] Current methods for the induction and maintenance of naive human PSCs exhibit several characteristics of mouse ESCs, similar to those of mouse preimplantation ICM (Chan, Goke et al. 2013; Takashima, Guo et al. 2014; Theunissen, Powell et al. 2014). However, the induction of naive human PSCs using current methods suffers from limitations, including lengthy cell development, variable levels of specific naive genes, transgene dependency for naive induction, genomic instability and loss of imprinting, inability to differentiate into multiple lines, lack of adequate chimera formation, or inefficiency. None of these studies have reported the generation of cells approaching stage 8C.

[0035] To overcome these problems, we first conducted a screening study using a panel of inhibitors targeting epigenetic regulators and distinct signaling pathways associated with human preimplantation ICM development. We found that three fundamental regulators (JAK / STAT3 activators, MAPK / ERK inhibitors, and tankyrase inhibitors) could activate the molecular network governing the preimplantation ICM-like state of primate PSCs. We also found that SAH / PRC / EZH2 inhibitors and HDAC inhibitors could rewire the epigenetic landscape of cultured PSCs to one more similar to that of human preimplantation ICMs. This resulted in the conversion of conventional primate PSCs into ICLCs, which possess all the key characteristics of human preimplantation ICMs, as described in the Background section.

[0036] Thus, the present application provides multiple methods and chemically defined media that promote the robust induction of primate ICLCs. The methods described herein can be applied to numerous human and non-human primate PSC lines that are either in a primed state, as evidenced by the presence of pluripotency surface markers such as SSEA-3, SSEA-4, TRA-1-81, and TRA-1-60, or in a preimplantation ICM-like state, as evidenced by the expression of genes such as DNMT3L, STELLA, DPPA5, and KLF17. Primate PSC lines used in the present application include, but are not limited to, conventional primate PSCs and ICM-like PSCs. The methods described herein can also be applied to the isolation of ICLCs from primate preimplantation ICMs. The described methods are non-transgenic and straightforward, provided that primate PSCs can be converted to ICLCs in approximately two weeks under one culture condition.

[0037] To our knowledge, there are currently no suitable methods for inducing primate 8CLCs in vitro. To achieve this, the inventors further optimized the formulation for inducing ICLCs and found that primed human PSCs and / or ICLCs can be converted into 8CLCs simply by increasing the dose of a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor in the culture medium. Therefore, the present application provides a chemically defined culture medium that facilitates the induction of primate 8CLCs. The methods described herein can be applied to numerous human and non-human primate PSC lines that are either in a primed state, as verified by the presence of pluripotent surface markers such as SSEA-3, SSEA-4, TRA-1-81, and TRA-1-60, or in a preimplantation ICM-like state, as verified by the expression of genes such as DNMT3L, STELLA, DPPA5, and KLF17. Primate PSC lines used in the present application include, but are not limited to, primed primate PSCs and ICM-like PSCs. The method described herein can also be applied to the isolation of 8CLCs from primate 8C embryos. The provided primate PSCs can be converted into 8CLCs in approximately one week under one culture condition, making the described method transgenic-free and straightforward. Indeed, activation of WNT / β-catenin signaling also inhibits the formation of 8CLCs. Therefore, modulators that activate WNT / β-catenin signaling, such as the GSK inhibitor CHIR99021 (a widely used inhibitor in published naive and expanded PSC culture conditions), should be excluded from the culture conditions, while modulators that suppress WNT / β-catenin signaling, such as IWR1 and XAV939, are necessary. The present invention will now be described in detail. It should be understood that the features described in the various embodiments can be combined with each other to form preferred technical solutions, which are also considered within the scope of the present disclosure.

[0038] I. Terminology Unless otherwise specified, all terms used herein have the meanings commonly understood by those skilled in the art, and to facilitate understanding of the invention, some of the terms used herein are defined as follows: As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. All digital indications, such as pH, temperature, time, concentration, molecular weight, including ranges, are approximate. It is important to understand, although not always explicitly stated, that all digital indications are preceded by the word "about." It should also be understood, although not always explicitly stated, that the reagents described herein are only examples and that equivalents thereof are known in the art. As used herein, the term "basal medium" refers to any medium capable of supporting cell growth. Basal media provide standard inorganic salts, such as zinc, iron, magnesium, calcium, and potassium, as well as vitamins, glucose, a buffer system, and key amino acids. Basal media used herein include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F10, F12, alpha Minimal Essential Medium (αMEM), Glasgow Minimal Essential Medium (GMEM), Eiscove's Modified Dulbecco's Medium, Neurobasal Medium, and DMEM / F12. Those skilled in the art know how to select a basal medium appropriate for culturing cells. In a preferred embodiment, the basal medium used herein is a 1:1 (v / v) mixture of advanced DMEM / F12 and Neurobasal Medium.

[0039] The term "serum-free" means the absence of any serum of any species, including, but not limited to, the absence of fetal bovine serum, calf serum, human serum, etc., or combinations thereof. As used herein, the term "serum replacement" refers to a supplement used in a basal medium to partially or completely replace serum to support cell survival and growth. Serum replacement typically includes factors such as insulin, metalloproteins, trace elements, and vitamins. These factors are generally not included in basal medium but are provided by the serum typically used in cell culture. Serum replacement typically includes at least one of the following components that support cell growth: one or more insulins and insulin substitutes, one or more metalloproteins and metalloprotein substitutes, one or more trace elements, one or more vitamins, one or more amino acids, one or more hormones and hormone-like compounds, serum albumin or serum albumin substitutes, and one or more lipids. Various commercially available serum replacements are known in the art, including KOSR, N2, B27, insulin-transferrin-selenium supplement (ITS), G5, etc., and are readily available to those skilled in the art. Since each replacement has a specific composition, the concentration of each component can be determined according to its respective ratio in the medium. Those skilled in the art can easily determine serum replacement according to the prior art, the type of cells to be cultured, and other aspects. The serum replacement used herein may be a mixed supplement obtained by mixing KOSR, N2, and / or B27 in a certain ratio. More preferably, the serum replacement used herein is a mixture of N2 and B27 in a 1:1 (w / w) ratio.

[0040] As used herein, the terms "primate" or "primate" refer to animals belonging to the class Primates, including humans and non-human primates. Non-human primates include animals from the suborders Prosimians and Apes. Specific non-human primates include, but are not limited to, macaques, lemurs, gibbons, orangutans, and baboons.

[0041] As used herein, the term "pluripotent stem cells" (PSCs) refers to pluripotent cells derived from embryos at any time before gastrulation and iPSCs generated by reprogramming somatic cells. Depending on their source and culture method, PSCs can exist in alternative states, including primed PSCs, naive PSCs, expanded PSCs, and expanded potential stem cells (Gafni et al., 2013; Takashima et al., 2014; Theunissen et al., 2014). PSCs also possess the ability, under appropriate conditions, to generate progeny of different cell types that are derivatives of all three germ layers (endoderm, mesoderm, and ectoderm), according to standard accepted tests, such as the ability to form teratomas in 8-12-week-old SCID mice. They may also generate placenta with different cell types under appropriate conditions. A PSC culture is described as "undifferentiated" when a significant proportion of stem cells and their derivatives within the population display morphological characteristics of undifferentiated cells, distinguishing them from differentiated cells of embryonic or adult origin. It is understood that colonies of undifferentiated cells within a population may be surrounded by neighboring differentiated cells.

[0042] The present application can be implemented using various types of stem cells. Primate PSCs are particularly suitable for use in the present application. Non-limiting examples include primary cultures or established lines of ESCs and iPSCs. PSCs from mammals other than primates can also be used to implement the subject application. In one or more embodiments, the primate PSCs that can be used in this application are: (i) cells derived from ESC and / or ECC lines; (ii) cells derived from iPSC lines; (iii) cells derived from the ICM of in vitro cultured preimplantation blastocysts; (iv) cells derived from the ICM of in vitro cultured postimplantation blastocysts; (v) In vitro cultured cells from embryonic stage 8C to morula stage; is selected from the group consisting of: Non-limiting PSCs include cell lines established in the art, such as human ESC lines, such as H1 (male), H9 (female), HN10 (female), HUES1 (female), and WIBR3 (female), and human iPSC lines, such as CBC14 (female), C11 (female), Phoenix (female), DiPS 1016SevA (male), STiPS O-XX1 (female), and UH10 (male).

[0043] The media (culture media) disclosed herein are chemically defined media capable of efficiently converting primate PSCs from a primed state to a preimplantation ICM-like state and producing ICLCs within two weeks without colony picking. The media described herein can also convert primate PSCs from a primed state and / or a preimplantation ICM-like state to an 8C-like state and produce 8CLCs in approximately one week. Therefore, this type of medium can also be referred to as a "conversion medium" in the present application. In some embodiments, the media described herein can also support the survival, self-renewal, and proliferation of cells in a preimplantation ICM-like state after induction, passaging, and / or regeneration. In other embodiments, the media described herein can also support the survival, self-renewal, and proliferation of cells in a preimplantation ICM-like state after passaging and / or regeneration on an extracellular matrix without the need for feeder cells or conditioned medium. In some embodiments, the media described herein can support the passaging and / or regeneration, self-renewal, and proliferation of cells in a preimplantation ICM-like state in suspension without the need for feeder cells or conditioned medium. In some other embodiments, the media described herein can support the passage and / or renewal, self-renewal, and proliferation of cells in a pre-implantation ICM-like state on feeder cells. The chemically defined media described herein can be serum-free.

[0044] The medium of the present application is a basal medium capable of supporting cell proliferation, particularly the proliferation of human and non-human primate PSCs, supplemented with a PRC inhibitor and / or an EZH2 inhibitor and an HDAC inhibitor, and optionally containing one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a tankyrase inhibitor. The basal medium used in the present application is a 1:1 (v / v) mixture of Advanced DMEM / F12 and Neurobasal Medium. It should be understood that an SAH inhibitor can inhibit PRC and / or EZH2. It should be understood that SAH can be inhibited by inhibiting PRC and / or EZH2. Therefore, in certain embodiments, a PRC inhibitor and / or EZH2 inhibitor (PRC / EZH2 inhibitor) can also be an SAH inhibitor. In the context described herein, "SAH / PRC / EZH2 inhibitor" refers to an inhibitor of SAH, PRC, and / or EZH2.

[0045] The presence of a SAH / PRC / EZH2 inhibitor in the culture conditions described herein is crucial for inducing multiple regulatory factors, including STELLA, DNMT3L, and MAEL, that govern the human naive pluripotency network. STELLA is a DNA methylation regulator. Its ectopic overexpression in somatic cells can induce global DNA demethylation by inhibiting the function of the DNA methylation regulator UHRF1. Impairment of UHRF1 function caused by STELLA deletion would result in the accumulation of aberrant DNA methylation during oogenesis (Li et al., 2018). STELLA induction was dose-dependent. We further elucidated the functional role of STELLA and found that STELLA knockout prevented the induction of ICLCs and 8CLCs. During the conversion of primed PSCs to ICLCs, preimplantation ICM markers, including KLF17, DPPA5, DNMT3L, TFCP2L1, and MAEL, were not induced upon STELLA deletion. During the conversion of primed PSCs and ICLCs to 8CLCs, 8C markers, including TPRX1, TRIM60, KHDC1L, YPEL2, ALPG, ZNF280F, FAM151A, and CCNA1, are not induced upon STELLA deletion. As shown in this application, during 4CL or e4CL conversion, global DNA methylation levels are significantly higher in STELLA knockout cells compared to wild-type cells. Thus, STELLA is required for regulated DNA demethylation during conversion to ICLCs and 8CLCs. Overall, this application discovers that the addition of SAH / PRC / EZH2 inhibitors promotes the induction of human ICLCs and 8CLCs through rewiring of histone modifications and DNA methylation landscapes.

[0046] Substances that act as SAH / PRC / EZH2 inhibitors can be used in the medium of the present application, including, but not limited to, DZNep (CAS No. 102052-95-9: an inhibitor acting on SAH) and CPI-1205 (CAS No. 1621862-70-1: an inhibitor acting on SAH / PRC / EZH2). SAH / PRC / EZH2 inhibitors may be used alone or in combination with the medium of the present application, and are generally used in amounts that do not result in cell death. For example, the final concentration of DZNep in the medium is 5 to 80 nM, preferably 5 to 50 nM, and the final concentration of CPI-1205 in the medium is 0.5 to 5 mM, preferably 1 to 3 mM. In one or more embodiments, the SAH / PRC / EZH2 inhibitor is a PRC inhibitor. Substances that can act as HDAC inhibitors, including but not limited to TSA, VPA, and NaB, can be used in the culture medium for the test drug. HDAC inhibitors can be used alone or in combination with the culture medium of the present application, and are generally used in amounts that do not cause cell death. For example, TSA can be used in the culture medium at a final concentration of 3 to 30 nM, preferably 3 to 25 nM, VPA can be used at a final concentration of 0.25 to 2 mM, preferably 0.5 to 1.5 mM, and NaB can be used at a final concentration of 0.25 to 2 mM, preferably 0.5 to 1.5 mM. In one embodiment, SAH / PRC / EZH2 inhibitors can be used at relatively high concentrations; for example, DZNep can be used at a final concentration of 5 to 80 nM, preferably 5 to 50 nM, and CPI-1205 can be used in the culture medium at a final concentration of 0.5 to 5 mM, preferably 1 to 3 mM; on the other hand, HDAC inhibitors can be used at relatively low concentrations; for example, TSA can be used at a final concentration of 3 to 10 nM, preferably 4 to 6 nM, more preferably about 5 nM, VPA can be used at a final concentration of 0.25 to 0.5 mM, and NaB can be used at a final concentration of 0.25 to 0.5 mM. In one embodiment, the SAH / PRC / EZH2 inhibitors are used at relatively low concentrations (e.g., DZNep is used in the medium at a final concentration of 5-15 nM, and CPI-1205 is used in the medium at a final concentration of 0.5-2 mM), while the HDAC inhibitors can be used at relatively high concentrations (e.g., TSA is used in the medium at a final concentration of 3-30 nM, preferably 3-25 nM, VPA is used in the medium at a final concentration of 0.25-2 mM, and NaB is used in the medium at a final concentration of 0.25-2 mM).The medium can convert primate PSCs into ICLCs.

[0047] L-ascorbic acid has been found to improve the generation and maintenance of mouse iPSCs (close to mouse ESCs) from somatic cells by enhancing Jumonji domain-containing histone demethylases, as described in Chinese Patent Application No. 200910041331.9, the contents of which are incorporated herein by reference. Therefore, we hypothesize that L-ascorbic acid has a similar effect on the formation of a preimplantation ICM-like state in primates. Through appropriate testing, we found that L-ascorbic acid potently increases the expression levels of ICM-specific genes, such as DNMT3L, STELLA, DPPA5, and KLF17, when used at a final concentration of 40-70 μg / ml. In a preferred embodiment, L-ascorbic acid is used at a final concentration of approximately 50 μg / ml. Derivatives of L-ascorbic acid can also be used in the present application, and refer to analogous compounds that have a similar structure and antioxidant activity to L-ascorbic acid. Such derivatives are more stable or more easily absorbed by cells while maintaining the biological activity of L-ascorbic acid. L-ascorbic acid derivatives include, but are not limited to, organic esters of L-ascorbic acid, such as L-ascorbic acid phosphate and L-ascorbic acid palmitate. The amount of derivative in the medium of the present application is not limited, but should generally be sufficient to produce a sufficient amount of L-ascorbic acid as specified above.

[0048] One or more JAK / STAT3 signaling activators can be added to a medium capable of inducing a subset of the early embryo-specific genes of the present application. Any known JAK / STAT3 signaling activators can be used, with those commonly used in stem cell culture being preferred. The typical final concentration of the JAK / STAT3 signaling activator is in the range of 10 to 50 ng / mL. One such JAK / STAT3 signaling activator is LIF. As used herein, the term LIF refers to leukemia inhibitory factor, a growth factor commonly supplemented to cultured stem cells. LIF may be human LIF. The JAK / STAT3 activator is used in an amount commonly used in stem cell culture. For example, in the case of LIF, particularly human LIF, the final concentration of the test drug in the medium may be in the range of 10 to 50 ng / mL, preferably 10 to 30 ng / mL, and more preferably about 20 ng / mL. One or more inhibitors of MAPK / ERK signaling can be added to the culture medium, which acts in conjunction with other components of the test drug medium to reduce DNA methylation. Any known MAPK / ERK inhibitor can be used, with those commonly used in stem cell culture being preferred. One such MAPK / ERK inhibitor is PD0325901 (CAS No.: 391210-10-9). MAPK / ERK inhibitors are used in amounts commonly used in stem cell culture. The typical final concentration of MAPK / ERK inhibitors is in the range of 0.5 to 3 μM, preferably 0.5 to 1.5 μM. For example, in the case of PD0325901, the final concentration of the test drug in the culture medium is in the range of 0.5 to 3 μM, preferably 0.5 to 1.5 μM, and more preferably about 1 μM.

[0049] One or more tankyrase inhibitors that inhibit canonical WNT signaling can be supplemented into the test drug medium. Any known tankyrase inhibitor can be used, and those commonly used in stem cell culture are preferred, including, but not limited to, IWR1 (CAS No.: 1127442-82-3) and XAV939 (CAS No.: 284028-89-3). The tankyrase inhibitor can be used in an amount commonly used in stem cell culture. Exemplary final concentrations of the tankyrase inhibitor range from 2 to 8 μM, preferably from 3 to 6 μM. Two or more tankyrase inhibitors can be used in combination, allowing the amount of each inhibitor to be reduced.

[0050] In one or more preferred embodiments, the medium described herein comprises DZNep at a final concentration of 5-15 nM or CPI-1205 at a final concentration of 0.5-2 mM; TSA at a final concentration of 3-30 nM, or VPA at a final concentration of 0.25-2 mM or NaB at a final concentration of 0.25-2 mM, preferably TSA at a final concentration of 3-10 nM, or VPA at a final concentration of 0.25-1 mM or NaB at a final concentration of 0.25-1 mM; L-ascorbic acid at a final concentration of 10-70 μg / mL; LIF at a final concentration of 10-30 ng / mL; XAV939 at a final concentration of 0.5-1.5 μM; and IWR1 or XAV939 at a final concentration of 3-6 μM. In one or more embodiments, the medium described herein comprises DZNep at a final concentration of 5-80 nM, 5-50 nM, or CPI-1205 at a final concentration of 0.5-5 mM, preferably 2-4 mM; TSA at a final concentration of 3-10 nM, or VPA at a final concentration of 0.25-0.5 mM, or NaB at a final concentration of 0.25-0.5 mM; L-ascorbic acid at a final concentration of 40-70 μg / mL; LIF at a final concentration of 10-30 ng / mL; PD0325901 at a final concentration of 0.5-1.5 μM; and IWR1 or XAV939 at a final concentration of 3-6 μM, respectively. More preferably, the medium described herein contains 10 nM DZNep or 1 mM CPI-1205, 5 nM TSA or 0.5 mM VPA or 0.5 mM NaB, 50 μg / ml L-ascorbic acid, 20 ng / mL LIF, 1 μM XAV939, and 5 μM IWR1 or 5 μM PD0325901. The medium is preferably used to convert primate PSCs into ICLCs.

[0051] In one or more preferred embodiments, the medium described herein comprises a final concentration of 40-70 nM DZNep or 2-4 mM CPI-1205; a final concentration of 10-30 nM TSA or 0.5-1.5 mM VPA or 0.5-1.5 mM NaB; a final concentration of 40-70 μg / ml L-ascorbic acid; a final concentration of 10-30 ng / mL LIF; a final concentration of 0.5-1.5 μM PD0325901; and a final concentration of 3-6 μM IWR1 or XAV939, each of which may be used as a therapeutic agent. More preferably, the medium described herein contains 50 nM DZNep or 3 mM CPI-1205, 20 nM TSA or 1 mM VPA or 1 mM NaB, 50 μg / ml L-ascorbic acid, 20 ng / mL LIF, 1 μM XAV939, and 5 μM IWR1 or 5 μM PD0325901. The medium is preferably used to convert primate PSCs or ICLCs into 8CLCs. The media described herein may further comprise at least one or more supplements selected from the group consisting of an extracellular matrix, an activin / NODAL signaling activator, and a ROCK inhibitor.

[0052] Compared to primed human PSCs, the expression levels of NODAL (activin / NODAL signaling activator) are increased in ICLCs and 8CLCs induced by the methods described herein. This observation indicates that activin / NODAL signaling is endogenously / automatically activated during the transformation process and self-renewal. Therefore, in some embodiments of the present application, the culture medium further comprises an activin / NODAL signaling activator to accelerate the transformation process. Known activators of activin / NODAL signaling can be added to the culture medium of the present application, including, but not limited to, human activin A and human NODAL, whose amino acid sequences are known in the art. Human activin A or human NODAL can be present in the culture medium of the present application at a final concentration of 10-25 ng / ml, preferably about 20 ng / ml. Human activin A and human NODAL can also be used in combination. Generally, the total concentration of human activin A and human NODAL in the culture medium is in the range of 10-25 ng / ml, approximately 20 ng / ml.

[0053] Once converted to ICLC and / or 8CLC, inhibition of ROCK signaling is no longer required for survival after passage as single cells. Nevertheless, providing a low concentration of a ROCK inhibitor increases the yield of ICLC and 8CLC, which is beneficial for scale-up of the culture. Therefore, in some embodiments of the present invention, the medium further comprises a ROCK inhibitor. Known ROCK inhibitors can be used in the medium described herein, including, but not limited to, Y27632 (CAS No.: 146986-50-7), thiazovivin (CAS No.: 1226056-71-8), and hydroxyfasudil (CAS No.: 105628-72-6). The ROCK inhibitor can be used at a final concentration ranging from 0.5 to 2 μM, preferably about 1 μM. Two or more ROCK inhibitors can be used in combination, with the total concentration in the medium ranging from 0.5 to 2 μM, preferably about 1 μM. The inventors have discovered that when PSCs are cultured in the media of the present application, they can be transformed and maintained in suspension culture without feeder cells, and the transformed cells can self-renew and grow as colonies, such as spheres. Thus, in certain embodiments of the present application, the methods, culture conditions, and media described are feeder-free.

[0054] In another embodiment, the present inventors have found that providing extracellular matrix during conversion and maintenance promotes spherical colony formation. Under these conditions, over 90% of PSCs can be converted into dome-shaped colonies during conversion, and they express ICM markers such as DNMT3L and KLF17. Therefore, in one embodiment, an extracellular matrix is ​​used in the culture medium for culturing ICLCs and 8CLCs. The extracellular matrix can be a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma (Matrigel™, Geltrex™, or ECM™), or a matrix containing human matrix protein collagen IV and at least one member selected from fibronectin, laminin, and vitronectin. The extracellular matrix is ​​typically present in the medium described herein at a concentration of 0.1% to 0.5% (v / v). If necessary, a combination of different types of extracellular matrix can be used, and the total amount in the medium should be in the range of 0.1% to 0.5% (v / v). Preferably, the extracellular matrix is ​​present in the media described herein generally in an amount of about 0.2% (v / v).

[0055] Thus, in one or more preferred embodiments, the media described herein comprise: (A) DZNep at a final concentration of 5 to 15 nM or CPI-1205 at a final concentration of 0.5 to 2 mM, TSA at a final concentration of 3 to 30 nM, or VPA at a final concentration of 0.25 to 1 mM, or NaB at a final concentration of 0.25 to 1 mM; preferably TSA at a final concentration of 3 to 10 nM, or VPA at a final concentration of 0.25 to 1 mM, or NaB at a final concentration of 0.25 to 1 mM; or DZNep at a final concentration of 5 to 80 nM, preferably 5 to 50 nM, or CPI-1205 at a final concentration of 0.5 to 5 mM, preferably 0.5 to 3 mM, TSA at a final concentration of 3 to 10 nM, or VPA at a final concentration of 0.25 to 0.5 mM, or NaB at a final concentration of 0.25 to 0.5 mM; (B) L-ascorbic acid at a final concentration of 40–70 μg / ml; (C) LIF at a final concentration of 10–30 ng / mL; (D) PD0325901 at a final concentration of 0.5–1.5 μM; (E) IWR1 or XAV939 at a final concentration of 3-6 μM; and further: (1) activin A or NODAL at a final concentration of 10 to 25 ng / ml; Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM; and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (2) the activin A or NODAL at a final concentration of 10 to 25 ng / ml; the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM; or More preferably, the media described herein contain 50 nM DZNep or 3 mM CPI-1205; 20 nM TSA or 1 mM VPA or 1 mM NaB; 50 μg / ml L-ascorbic acid; 20 ng / mL LIF; 1 μM PD0325901; and 5 μM IWR1 or 5 μM XAV939; and further supplemented with: (1) 20 ng / mL activin A or NODAL, 1 μM Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v / v) extracellular matrix; or (2) 20 ng / mL activin A or NODAL and 1 μM Y27632, thiazovivin or hydroxyfasudil; or (3) 20 ng / mL activin A or NODAL and 0.2% (v / v) extracellular matrix; or (4) 1 μM Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v / v) extracellular matrix; or (5) 20 ng / mL activin A or NODAL, or 1 μM Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v / v) extracellular matrix. The medium is preferably used to convert primate PSCs or ICLCs into 8CLCs.

[0056] In addition to the above components, the medium of the present application can be supplemented with other supplements commonly used in media for culturing stem cells, including, but not limited to, N2 and / or B27; alternative carbon sources such as pyruvate, e.g., sodium pyruvate; non-essential amino acids; serum replacement, e.g., L-glutamine or a substitute therefor, e.g., GlutaMAX™ supplement containing L-alanyl-L-glutamine dipeptide in 0.85% NaCl; and antibiotics, e.g., penicillin, streptomycin, or a mixture of penicillin and streptomycin. These supplements can be used in amounts commonly used in cell culture, particularly stem cell culture.

[0057] III. Kits and Compositions Also provided are kits containing the media described herein or all or some of the components of the media described herein for preparing the media.

[0058] In some embodiments, the kits described herein include a ready-to-use medium, the components of which are described in any of the medium embodiments above. In some embodiments, the kits described herein include a conversion medium for converting primate PSCs to ICLCs as described in any of the embodiments described herein and / or a conversion medium for converting primate PSCs or ICLCs to 8CLCs as described in any of the embodiments described herein.

[0059] In other embodiments, the kits described herein include at least an SAH / PRC / EZH2 inhibitor and an HDAC inhibitor, which may be individually packaged or provided as a mixture in a single container. The kits may further include one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a tankyrase inhibitor, which, when present, may be individually packaged or provided as a mixture of any combination of components. Preferably, the kits may include an SAH / PRC / EZH2 inhibitor and an HDAC inhibitor, L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a tankyrase inhibitor. The kits may further include one or more components selected from the group consisting of an activin / NODAL signaling activator and a ROCK inhibitor. Matrigel™ 、The kit may also include a conventional extracellular matrix, such as Geltrex™ or ECM™. Preferably, the kit also includes a basal medium, such as one or more of the basal media described herein, such as DMEM / F12 (1:1) or Neurobasal Medium, as well as other components known to be used in stem cell culture, such as serum replacement, such as N2 or B27, an alternative carbon source, such as sodium pyruvate, non-essential amino acids, L-glutamine or its substitute (e.g., Glutamax™ supplement containing L-alanyl-L-glutamine dipeptide in 0.85% NaCl), antibiotics, etc. The amounts of all of these components should be sufficient to formulate the medium of the subject application. The kit may include instructions and may include text regarding the preparation of the media and its use.

[0060] In one embodiment, a composition is provided comprising a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor. The composition may further comprise one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a tankyrase inhibitor. The composition may also comprise one or more components selected from the group consisting of an activin / NODAL signaling activator and a ROCK inhibitor. In a preferred embodiment, the composition comprises a SAH / PRC / EZH2 inhibitor, an HDAC inhibitor, L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a tankyrase inhibitor, and optionally includes an activin / NODAL signaling activator and optionally a ROCK inhibitor. It should be understood that each of the above components, when present in a composition, should be present in an amount acceptable for a medium, including a composition within the range of each medium specified in any embodiment described herein. More preferably, the composition can be used to formulate a medium according to any embodiment described herein.

[0061] In one or more preferred embodiments, the compositions described herein comprise DZNep or CPI-1205, and TSA or VPA or NaB, and optionally L-ascorbic acid, optionally LIFI, optionally PD0325901, and optionally IWR1 or XAV939, preferably each of said components in a medium containing said composition at one of the following concentrations: 5-15 nM, preferably 10 nM DZNep, or 0.5-2 mM, preferably 1 mM CPI-1205; 3-6 nM, preferably 5 nM TSA, or 0.25-1 mM, preferably 0.5 mM VPA (VPA), or 0.25-1 mM, preferably 0.5 mM NaB; optionally, 40-90 μg / mL, preferably 50 μg / mL L-ascorbic acid, preferably 20 ng / mL LIF, optionally, 0.5-1.5 μM, preferably 1 μM PD0325901, and optionally, 3-6 μM, preferably 5 μM IWR1 or XAV939. The composition may further contain activin A or NODAL, and / or Y27632, thiazovivin or hydroxyfasudil, and / or extracellular matrix, each component being present in an amount such that the medium containing the composition can contain 10 to 25 ng / mL, preferably 20 ng / mL, of activin A or NODAL, and / or 0.5 to 2 μM, preferably 1 μM, of Y27632, thiazovivin or hydroxyfasudil, and / or 0.1% to 0.5% (v / v) of extracellular matrix.

[0062] In one or more embodiments, the compositions described herein comprise DZNep or CPI-1205, and TSA or VPA or NaB, and optionally, L-ascorbic acid, optionally, LIF, and optionally, PD0325901 and IWR1 or XAV939; preferably, each of said components is present in a medium containing said composition such that the medium is one of the following: and optionally, 40-90 μg / mL, preferably 50 μM, L-ascorbic acid; optionally, 10-30 ng / mL, preferably 20 ng / mL, LIF; optionally, 0.5-1.5 μM, preferably 1 μM, PD0325901; and optionally, 3-6 μM, preferably 5 μM, IWR1 or XAV939. The composition further comprises activin A or NODAL, and / or Y27632, thiazovivin or hydroxyfasudil, and / or extracellular matrix, wherein each of the components is present in an amount such that the medium containing the composition can comprise 10 to 25 ng / mL, preferably 20 ng / mL, of activin A or NODAL, and / or 0.5 to 2 μM, preferably 1 μM, of Y27632, thiazovivin or hydroxyfasudil, and / or 0.1% to 0.5% (v / v) of extracellular matrix. In some embodiments, the kit can include the composition described above.

[0063] The kits described herein may further include a medium for maintaining PSCs, such as mTeSR1 or E8 medium, and / or a medium for blastoid formation, such as REM medium (REM is modified reconstructed embryo medium (Zhang Shaopeng et al. 2019)) supplemented with or without 8-15 μM Y27632. Reagents commonly used in stem cell culture may also be included in the kit, including, but not limited to, PBS, EDTA solution, and / or TrypLE:0.5 mM EDTA (1:1). Feeder cells and / or extracellular matrix may also be provided in the kit.

[0064] IV. Methods and Uses The media described herein can be used to reprogram primate somatic cells into ICLCs, to convert primate PSCs into ICLCs, and to convert primate PSCs or ICLCs into 8CLCs. Thus, one aspect described herein discloses a method for reprogramming primate somatic cells into ICLCs, comprising culturing the somatic cells in a conversion medium comprising a SAH / PRC / EZH2 inhibitor, an HDAC inhibitor, L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a tankyrase inhibitor, with or without an extracellular matrix, optionally an activin / NODAL signaling activator, and optionally a ROCK inhibitor. In a preferred embodiment, the conversion medium is a medium identified in any of the above embodiments.

[0065] In one or more preferred embodiments, the method is a method for converting primate PSCs into ICLCs, and the conversion medium is a medium identified in any of the above embodiments that has a relatively low concentration of a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor. In some other preferred embodiments, the method is for converting primate PSCs or ICLCs into 8CLCs, and the conversion medium is a medium as defined in any of the above embodiments that contains a relatively high concentration of a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor.

[0066] Conventional conditions for culturing stem cells can be used to convert PSCs into ICLCs or 8CLCs. For example, single primed PSCs can be seeded in conventional media such as mTeSR1 or E8, optionally supplemented with 5-15 μM of a ROCK inhibitor such as Y27632. After a culture period, for example 24 hours, the medium is switched to the medium of the present application, and cell culture is continued until the desired ICLCs or 8CLCs are generated. During culture, the medium is replaced as needed, preferably daily. For passaging, the cells can be dissociated into single cells using conventional methods, and then replated and cultured in the medium of the present application until ICLCs or 8CLCs are formed. The cells are preferably passaged as a single cell every 3 to 4 days at a split ratio of 1:4 to 1:8, preferably 1:6 to 1:8. Generally, primed PSCs are converted to ICLCs in about 2 weeks, primed PSCs are converted to 8CLCs in about 1 week, and ICLCs are converted to 8CLCs in 3 to 5 days after culturing the ICLCs in a medium containing a relatively high concentration of a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor. It should be understood that the ICLCs used to convert to 8CLCs may be ICLCs obtained by culturing primate PSCs using any of the methods described herein, or may be known ICLCs or ICLCs prepared by any method known in the art. Generally, cells can be cultured at 37°C under normoxic conditions (5% CO2) or hypoxic conditions (5% CO2 and 5% O2). The culture time is not particularly limited and can be easily determined by those skilled in the art based on the disclosure of the subject matter and the prior art of those skilled in the art. The seeding concentration can be determined by those skilled in the art according to their common knowledge and actual production conditions.

[0067] In certain embodiments described herein, the cells are grown: (i) on feeder cells; (ii) on feeder-free extracellular matrix; (iii) in suspension without feeder cells; (iv) grown under hypoxic or normoxic conditions at a temperature of about 37°C; (v) passaged as single cells every 3-4 days at a split ratio of 1:4 to 1:8; and (vi) with daily changes of medium. The cells can be cultured under one or more conditions selected from the group consisting of:

[0068] In one embodiment, to convert primate PSCs into ICLCs, single primate PSCs are plated onto feeders in mTeSR1 or E8 medium supplemented with 5 μM to 15 μM of a ROCK inhibitor (e.g., Y27632). After culturing for a period of time, such as 24 hours, the mTeSR1 or E8 medium is switched to the conversion medium of the present application supplemented with relatively low concentrations of a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor. The cells are cultured at approximately 37°C under hypoxic or normoxic conditions with daily medium changes. During culture, the cells are passaged as single cells at a ratio of 1:4 to 1:8 every 3 to 4 days until ICLCs are obtained. In one embodiment, single primed primate PSCs are cultured as described above, except that the cells are plated on DMEM-F12-coated plates in approximately 1% (v / v) extracellular matrix, such as Geltrex™, instead of feeder cells.

[0069] In one embodiment, to convert primate PSCs into ICLCs, single primed primate PSCs are plated in mTeSR1 or E8 medium supplemented with 5 μM to 15 μM of a ROCK inhibitor (e.g., Y27632). After culturing for a period of time, such as 24 hours, the mTeSR1 or E8 medium is switched to the conversion medium of the present application, which contains relatively low concentrations of a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor, and the cells are cultured under hypoxic conditions. After small spheres form, the spheres are transferred to flasks for suspension culture, and the medium is changed daily. Here, the cells are passaged as single cells every 4 to 5 days at a split ratio of 1:4 to 1:8 until ICLCs are obtained.

[0070] In one embodiment, for the conversion of primate PSCs to 8CLCs, single primed PSCs are seeded onto feeders in mTeSR1 or E8 medium supplemented with 5-15 μM of a ROCK inhibitor (e.g., Y27632) for a period of 24 hours or so. The medium is then switched to the conversion medium of the present application, which contains relatively high concentrations of a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor. The cells are cultured under hypoxic or normoxic conditions with daily medium changes. Herein, the cells are passaged every 3-4 days as single cells with a split ratio of 1:4-1:8. In one embodiment, for conversion of ICLCs to 8CLCs, single cells are isolated from the ICLCs and plated onto feeders using the conversion medium of the present application, which contains relatively low concentrations of SAH / PRC / EZH2 inhibitors and HDAC inhibitors, for a period of time such as 24 hours. Thereafter, the medium is switched to the conversion medium of the present application, which contains relatively high concentrations of SAH / PRC / EZH2 inhibitors and HDAC inhibitors, and the cells are cultured for 3 to 5 days without passaging, with daily medium changes. In one embodiment, for conversion of ICLCs to 8CLCs, single cells are isolated from the ICLCs and cultured in suspension for a certain period in a conversion medium of the present application, which contains relatively low concentrations of SAH / PRC / EZH2 inhibitors and HDAC inhibitors for suspension culture. The conversion medium is supplemented with 5-15 μM of a ROCK inhibitor (e.g., Y27632). After the formation of small aggregates, the medium is changed to a conversion medium of the present application, which contains relatively high concentrations of SAH / PRC / EZH2 inhibitors and HDAC inhibitors without supplementing with additional ROCK inhibitors (e.g., Y27632), for several days without passaging, and the medium is changed daily. The application also includes the use of the conversion medium described in any of the embodiments described herein in the reprogramming of primate somatic cells into ICLCs, in the conversion of primate PSCs into ICLCs, or in the conversion of primate PSCs or ICLCs into 8CLCs, or in the manufacture of a medium or kit for reprogramming primate somatic cells into ICLCs, or in the conversion of primate PSCs into ICLCs, or in the conversion of primate PSCs or ICLCs into 8CLCs. In certain embodiments, the subject application also includes the use of a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor in the manufacture of a medium or kit for reprogramming primate somatic cells into iPSCs, or for converting primate PSCs into ICLCs, or for converting primate PSCs or ICLCs into iPSCs. Preferably, the medium or kit can further comprise one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a tankyrase inhibitor, and optionally, an activin / NODAL signaling activator, and optionally, a ROCK inhibitor (such as Y27632), and optionally, an extracellular matrix.

[0071] In some other embodiments, the methods for converting primate PSCs into ICLCs and converting primate PSCs or ICLCs into 8CLCs may include a genetic engineering step to reduce SAH, PRC, and / or EZH2 activity in PSCs and / or reduce HDAC activity in cells by knocking down and / or knocking out one or more relevant genes in the cells before culturing the primate PSCs in the medium of the present application. To reduce SAH, PRC, and / or EZH2 activity in PSCs, the expression of any of the SAH, PRC, and EZH2 regulators may be knocked down, for example, by siRNA technology, or by knocking out any of the genes, for example, by CRISPR / Cas9 technology. Similarly, the expression of HDAC regulators may be knocked down or knocked out by the same means as above. After knockdown or knockout, the resulting cells can be cultured in any medium containing the test drug according to the methods described above. In certain embodiments, if SAH, PRC, and / or EZH2 activity in PSCs is reduced, the medium used to culture recombinant primate PSCs may or may not contain a SAH / PRC / EZH2 inhibitor. Similarly, if HDAC activity in PSCs is reduced, the medium may or may not contain an HDAC inhibitor. If both SAH, PRC, and / or EZH2 activity and HDAC activity are reduced, the medium may contain neither a SAH / PRC / EZH2 inhibitor nor an HDAC inhibitor, or may contain both a SAH / PRC / EZH2 inhibitor and an HDAC inhibitor. Thus, in some embodiments, the present application further provides a medium containing neither a SAH / PRC / EZH2 inhibitor nor an HDAC inhibitor, or containing either a SAH / PRC / EZH2 inhibitor or an HDAC inhibitor, with the other components and amounts being the same as in any of the above embodiments relating to the medium in Part II. In some embodiments, the medium can contain a reagent for liposomal transfection. For example, in the above method, primate PSCs are cultured in a medium containing, in addition to the other components described in the medium in Part II, a vector for expressing shRNA, such as any of SAH, PRC, and EZH2 regulators, and a reagent for liposomal transfection to introduce the vector into PSCs for genetic engineering, and the medium may or may not contain a SAH / PRC / EZH2 inhibitor.

[0072] Biological Functions of V.STELLA STELLA is a regulator of DNA methylation. Its ectopic overexpression in somatic cells can induce global DNA demethylation by inhibiting the function of UHRF1, a DNA methylation regulator. Impairment of UHRF1 function caused by STELLA deletion would result in the accumulation of aberrant DNA methylation during oogenesis (Li et al., 2018). STELLA has also been reported to maintain maternal imprinting by preventing the Tet3-mediated conversion of 5mC to 5hmC at specific loci (Nakamura et al., 2012). In this study, we first discovered that knockout of STELLA prevents the induction of ICLC and 8CLC, indicating that STELLA is required for regulated DNA demethylation during the transformation process. We also found that supplementation with SAH / PRC / EZH2 inhibitors promotes the induction of ICLC and 8CLC through rewiring of histone modifications and DNA methylation landscapes. Thus, in one embodiment, the present application further includes the use of an agent capable of promoting the expression of STELLA or improving the activity of STELLA in the manufacture of a reagent, medium or kit for promoting the conversion of primate PSCs to ICLCs, or the use of an agent capable of promoting the expression of STELLA or improving the activity of STELLA to promote the conversion of primate PSCs or ICLCs to 8CLCs. Also provided is a method for promoting the conversion of primate PSCs to ICLCs or the conversion of primate PSCs or ICLCs to 8CLCs, comprising culturing primate PSCs in the presence of an effective amount of an agent capable of promoting the expression of STELLA or improving the activity of STELLA. The effective amount of the agent can be readily determined by one of skill in the art based on the disclosure of the subject application and the teachings of the prior art.

[0073] In some preferred embodiments, the agent capable of promoting STELLA expression or improving STELLA activity is an SAH / PRC / EZH2 inhibitor, including, but not limited to, DZNep and CPI-1205. SAH / PRC / EZH2 inhibitors may be used alone or in combination and are generally used at a normal dose that does not result in cell death. For example, DZNep can be used in a medium at a final concentration of 5-80 nM, preferably 5-50 nM, and CPI-1205 can be used in a medium at a final concentration of 0.5-5 mM, preferably 1-3 mM. In one or more embodiments, the SAH / PRC / EZH2 inhibitor is commonly known as a PRC inhibitor.

[0074] In some more preferred embodiments, the method for promoting the conversion of primate PSCs to ICLCs comprises culturing primate PSCs in the presence of 5-15 nM, preferably 10 nM, DZNep, or 0.5-2 mM, preferably 1 mM, CPI-1205. In some other preferred embodiments, the method for promoting the conversion of primate PSCs or ICLCs to 8CLCs comprises culturing primate PSCs in the presence of 40-70 nM, preferably 50 nM, DZNep, or 2-4 mM, preferably 3 mM, CPI-1205. In certain other preferred embodiments, the method for promoting the conversion of primate PSCs or ICLCs to 8CLCs comprises culturing primate PSCs in the presence of 40-70 nM, preferably 50 nM, DZNep, or 2-4 mM, preferably 3 mM, CPI-1205. Also included in the present application are SAH / PRC / EZH2 inhibitors for use in methods of promoting the conversion of primate PSCs to ICLCs, or the conversion of primate PSCs or ICLCs to ICLCs.

[0075] VI.Cells The present application also provides isolated primate ICLCs. The ICLCs described herein have a transcriptome similar to that of human preimplantation ICM, a transposable element profile similar to that of human preimplantation ICM, a DNA methylome similar to that of human preimplantation ICM, a chromatin landscape similar to that of human preimplantation ICM, and a metabolic state similar to that of human preimplantation ICM. As used herein, the term "close" is intended to mean "substantially identical" or "without substantial differences." Those skilled in the art, based on their general knowledge in the art, can recognize that the cells of the subject application, including cells from ICLC or 8CLC described herein, are substantially identical to naturally occurring ICM cells or 8C embryonic cells, even if there are some minor differences. Preferably, the ICLCs described herein exhibit significantly higher expression levels of preimplantation ICM markers, including KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, MAEL, and REX1. More preferably, the expression level of at least one of the above preimplantation ICM markers in the ICLCs described herein is 10-fold or more higher than the expression level of the corresponding preimplantation ICM marker in primed human PSCs. More preferably, the expression level of all of the above preimplantation ICM markers in the ICLCs described herein is 10-fold or more higher than the expression level of the corresponding preimplantation ICM marker in primed human PSCs.

[0076] More preferably, the ICLC described herein is one of the following: 1) they can self-renew and maintain pluripotency in culture; 2) maintaining genome stability in culture according to karyotype; 3) can give rise to cells of the three germ layers; 4) can give rise to primordial germ cell-like cells; 5) They can integrate into mouse embryos and contribute to embryonic and extraembryonic tissues; 6) capable of transitioning to extraembryonic cell behavior in vitro; and 7) capable of forming blastocyst-like structures in vitro; The method is characterized by one or more selected from the group consisting of: The ICLCs can be obtained by culturing primate PSCs by any of the methods described in any of the embodiments described herein. Thus, in one embodiment, the present application also includes cells, particularly ICLCs, obtained by any of the methods described herein. The present application also provides isolated 8CLCs that express 8C-state-specific markers, including ZSCAN4, TPRX1, ZIM3, ZSCAN5B, ZNF280A, and ARGFX, at levels substantially higher than cells in a preimplantation ICM-like state or a primed state. Preferably, at least one specific marker exhibits an expression level that is 5-fold or more higher than the expression level of the corresponding 8C-specific marker in primed PSCs or ICLCs. Preferably, all of the above specific markers exhibit an expression level that is 5-fold or more higher than the expression level of the corresponding 8C-specific marker in primed PSCs or ICLCs. The 8CLCs described herein have a transcriptome, transposable element profile, and chromatin landscape similar to that of a human 8C stage embryo. More preferably, the 8CLCs described herein have the following: 1) Maintain genome stability in culture according to karyotype; 2) can give rise to cells of the three germ layers; 3) can give rise to primordial germ cell-like cells; 4) they can integrate into mouse embryos and contribute to embryonic and extraembryonic tissues; 5) capable of transitioning to extraembryonic cell behavior in vitro; and 6) capable of forming blastocyst-like structures in vitro; The method is characterized by one or more selected from the group consisting of: ICLCs obtained by reprogramming somatic cells with the conversion medium described herein are also contemplated in this application. Cell cultures comprising the cells described herein, particularly the ICLC and / or 8CLC described herein, are also contemplated in the present application. Any of the media described herein can also be included in the cell culture. The present invention will be described in the following non-limiting examples. It should be understood that these examples are for illustrative purposes only and do not limit the scope of the invention in any way. Various variations and modifications can be made within the spirit of the present disclosure. Unless otherwise specified, the techniques referred to in this specification are conventional techniques in the fields of molecular biology, cell biology, biochemistry, etc., and are well known to those skilled in the art. [Example]

[0077] Materials and Methods 4CL basal medium Neurobasal medium (Gibco) and advanced DMEM / F12 (Gibco) were mixed in a 1:1 ratio and supplemented with N2 supplement (1X, Gibco), B27 supplement (1X, Gibco) (homemade N2 and B27 can be used), sodium pyruvate (1X, Hydron), non-essential amino acids (NEAA) (Gibco), Glutamax™ (1X, Gibco), and penicillin-streptomycin (1X, Gibco).

[0078] 4CL supplement Add the following to 4CL basal medium: The medium was 4CL medium 1 supplemented with SAH / PRC / EZH2 inhibitor (10 nM DZNep), HDAC inhibitor (5 nM TSA), L-ascorbic acid (50 μg / ml), JAK / STAT3 activator (20 ng / ml human LIF), MAPK / ERK inhibitor (1 μM PD0325901), tankyrase inhibitor (5 μM IWR1), activin A / NODAL activator (20 ng / ml human activin A), extracellular matrix (0.2% (v / v) Geltrex™), and, in some cases, ROCK inhibitor (1 μM Y27632). A catalog of these reagents and their substitutes is provided in Table 1.

[0079] [Table 1] REM medium This medium is a 1:1 mixture of Advanced DMEM F12 (Gibco) and RPMI 1460 (Gibco) supplemented with 17.5% fetal bovine serum (NATOCOR), 1X Glutmax™ (Gibco), 1X NEAA (Gibco), 1X sodium pyruvate (Hyclone), and 1X penicillin-streptomycin (Gibco). REM is modified reconstructed embryo medium (Zhang Shaopeng et al. 2019). cell H9 human ESC line procedure: 1) Maintenance of primed human PSCs All donated human PSCs were routinely maintained in mTeSR1 or E8 medium on Matrigel™ or Geltrex™-coated plates. Generally, cells were passaged every 4–5 days with 0.5 mM EDTA. For passage, cells were washed once with PBS and treated with 0.5 mM EDTA for 5 minutes. The EDTA was then removed, and cells were dissociated into small clumps using a Pasteur pipette in mTeSR1 or E8 medium. Primed human PSCs were cultured under normoxic conditions (37°C, 5% CO). 2) were grown in an incubator at 4°C. 2) Conversion to ICLC on feeders One day before the start of conversion, primed human PSCs were washed once with PBS, dissociated into single cells, and plated on feeders at 1,000–1,500 cells / cm in mTeSR1 or E8 medium supplemented with 10 μM Y27632. 2 After 24 hours, the medium was switched to 4CL Medium 1. The medium was refreshed every 24 hours. Colonies became round and dome-shaped within 24 to 48 hours. Cells were passaged every 3 to 4 days. For passage, cells were dissociated into single cells using TrypLE:0.5 mM EDTA (1:1) and placed on feeders (Geltrex™). / 1,000–1,500 cells / cm on Matrigel™ pre-treated plates 2 The cells were seeded at a density of 100 μg / ml (Figure 1). ICLC induction and maintenance were performed under hypoxic conditions (37°C, 5% CO2、 5% O 2) , or under normoxic conditions (37°C, 5% CO 2、2 1% O 2) (Figure 34), which can preferably be carried out under hypoxic conditions. 3) Formation of a blastocyst-like structure (also called a blastocyst) Primed human PSCs or ICLCs were digested to single cells and filtered through a 40 μm strainer. Cell numbers were counted using a hemocytometer. Each well of a 24-well plate was coated with 200 μl of thawed Geltrex and placed in an incubator at 37°C for 7 minutes to form a semi-solid matrix. For each well, 30,000 cells were evenly resuspended in 500 μl of REM medium for blastoid formation, supplemented with 10 μM Y27632. The cell mixture was then plated on the semi-solid Geltrex and returned to the incubator for incubation at 37°C under 5% CO2. After 24 hours, the medium was replaced with REM medium supplemented with 4% (v / v) Geltrex without Y27632. The medium was changed daily, and the cells were maintained under hypoxic conditions (5% CO2, 5% O2). 2) The cells were cultured at 37°C.

[0080] Experimental results Figure 1(A) is a schematic diagram of ICLC induction from primed human PSCs. Figure 1(B) shows representative images of colony morphology under a phase-contrast microscope for primed human PSCs (left panel) and ICLCs (right panel). Flat primed human PSCs develop into dome-shaped ICLCs after conversion. Figure 1(C) shows RT-qPCR and immunostaining data demonstrating significant induction of a panel of preimplantation ICM markers, including KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1, in ICLCs compared with primed human PSCs. Figure 1(D) shows that ICLCs induced under normoxic or hypoxic conditions have similar preimplantation ICM marker gene expression levels. To characterize the gene expression profile of ICLCs at the single-cell level, we performed single-cell RNA-Seq (scRNA-seq) on cells at the primed stage (Primed-D0) and then at days 1, 2, 3, 5, 8, and 12 after culturing in 4CL medium 1 (4CL-D1 / 2 / 3 / 5 / 8 / 12). Figure 2(A) shows a 2D scatter plot of UMAP analysis of cells at different time points, along with published scRNA-seq data for in vivo human embryos at days 3, 4, 5, 6, and 7 (E3 / 4 / 5 / 6 / 7) (from E-MTAB-3929). This shows that conventional human PSCs in 4CL medium gradually acquire a gene expression profile similar to that of human embryonic day 5 cells, which corresponds to the early stage of the preimplantation blastocyst. Figure 2(B) shows a heatmap of bulk RNA-seq data from primed human PSCs, ICLCs, and human preimplantation ICM cells (GSE101571), demonstrating that the expression levels of known ICM markers in ICLCs are upregulated to the levels of ICM cells. A subgroup of TEs, such as SVA_D, is known in the art to be specifically activated from the 8C to the preimplantation ICM stage of human embryos. To investigate activated TEs in ICLCs, we extracted TE profiles from the scRNA-seq data shown in Figure 2(A).Figure 3(A) shows a 2D scatter plot of UMAP analysis of TE expression profiles in cells at the primed stage (Primed-D0) and in cells at days 1, 2, 3, 5, 8, and 12 (D1 / 2 / 3 / 5 / 8) after culture in 4CL medium 1 in human embryonic cells (from E-MTAB-3929) at days 3, 4, 5, 6, and 7 (E3 / 4 / 5 / 6 / 7). This shows that human PSCs primed in 4CL medium gradually capture a TE profile similar to that of human embryonic cells at embryonic day 4 (morula stage) and day 5 (blastocyst stage). Figure 3(B) further shows that the expression levels of multiple TE subgroups in ICLCs are induced to those of human preimplantation embryos (from GSE101571). Figure 4 shows that ICLCs maintain a normal karyotype even after long-term culture (passage 15, tested for approximately 60 days in 4CL medium 1). One female human ESC line (H9) and one male human iPSC line (UH10) are shown. These results indicate that ICLCs induced by 4CL medium 1 acquire a preimplantation ICM-like gene expression profile and maintain a stable genome even after extended culture. In terms of epigenetic landscape, the genome of preimplantation ICM is hypomethylated, and the chromatin is more open compared to postimplantation ICM. To determine the effect of 4CL Medium 1 on DNA methylation status, we performed reduced-representation bisulfite sequencing (RRBS) for ICLCs and primed human PSCs. Figure 5 shows boxplots showing genome-wide CpG DNA methylation (top left panel). ICLCs were significantly reduced compared to primed human PSCs, while TSS methylation status was slightly different (top right panel). Notably, the global decrease in DNA methylation levels was prevented by knocking out STELLA (bottom left panel). Figure 6 shows that the imprinting status of ICLCs was maintained similarly to that of ICMs. To determine the chromatin accessibility of ICLCs, we performed single-cell ATAC-seq (scATAC-seq) and bulk ATAC-seq. Figure 7(A) shows a clear separation of chromatin accessibility between primed human PSCs and ICLCs at the single-cell level. Figure 7(B) shows that the loci of preimplantation ICM-specific genes, such as KLF17, STELLA, DPPA5, and CD70, are highly open in ICLCs. Figure 7(C) shows that shared pluripotency genes, such as POU5F1, retained similar chromatin openness between primed human PSCs and ICLCs, while postimplantation-specific genes, such as THY1 (Figure 7D), became more closely located. Time-course bulk ATAC-seq in Figure 8 demonstrates the gradual change in chromatin accessibility during the conversion of primed human PSCs to ICLCs. Preimplantation-specific loci (e.g., TFAP2C, KLF5, and TFE3) located close to the primed human PSCs gradually open during conversion, whereas postimplantation-specific loci (e.g., ZIC3 and FOXA2) that remain open in primed human PSCs gradually close (Figure 8A). Motif enrichment analysis revealed that regions close to the open regions may bind to preimplantation ICM-specific transcription factors such as DUX, TFAP2C, and KLF5 (Figure 8B, upper panel), whereas regions closer to the open regions may bind to postimplantation-related transcription factors such as SOX3, NKX6.1, and NEUROD1 (Figure 8, lower panel). Figure 8C shows the correlation between gene expression and chromatin accessibility. These results indicate that 4CL Medium 1 successfully rewires the epigenetic landscape toward the preimplantation ICM.

[0081] We further investigated the metabolic status of ICLCs induced in 4CL Medium 1. Preimplantation ICMs primarily rely on oxidative phosphorylation (OxPhos) as an energy source, whereas postimplantation ICMs primarily rely on glycolysis. Figure 9 shows that the expression levels of genes related to oxidative phosphorylation are significantly upregulated in ICLCs compared to primed human PSCs. These results suggest that oxidative phosphorylation is activated in ICLCs.

[0082] To determine the differentiation potential of ICLCs, we performed teratoma formation assays using nude mice as recipient animals. Figure 10 shows representative images of hematoxylin and eosin-stained teratoma tissues formed two months after subcutaneous injection of one million ICLCs. They demonstrated the presence of cells from all three germ layers: mesoderm (left panel), endoderm (middle panel), and ectoderm (right panel). It is known in the art that human ICLCs can generate trophectoderm. Therefore, we induced trophoblast stem cells (TSCs) from ICLCs using a previously published protocol. As shown in Figure 11(A), multiple TSC markers, including GATA3, CGA, ELF5, TP63, KRT18, KRT8, PSG6, and CCR7, were significantly elevated in TSCs compared with undifferentiated ICLCs. Figure 11(B) shows immunofluorescence images showing the expression of known TSC markers, GATA3, TFAP2C, and KRT7. Figure 11(C) is a scatter plot of principal component analysis (PCA) showing that the transcriptome of ICLC-derived TSCs is closer to that of the human placental choriocarcinoma cell lines JEG3 and BeWo than that of ICLCs and placental cells (EGFR and HLAG). Figure 11(D) shows the DNA methylation status of the ELF5 promoter region in ICLC-derived TSCs and other cell types. These results indicate that ICLCs acquire developmental potential comparable to that of human preimplantation embryos.

[0083] Due to ethical issues, the developmental potential of ICLCs cannot be tested using human embryos. Therefore, we performed interspecies chimera experiments by aggregating ICLCs with mouse 8C stage blastomeres. Human ICLCs were successfully integrated into most mouse embryos after 24 hours of in vitro culture, forming chimeric blastocysts (Figures 12A-C). At this stage, human ICLCs were located in both the ICM and TE regions of chimeric blastocysts. Figure 12(A) shows an overview of the chimera assay using DsRed-labeled human PSCs and DsRed-labeled ICLCs at the blastocyst stage. Figure 12(B) shows representative images showing the phase contrast (left) or red fluorescence channel (right) of blastocysts developed from mouse 8C blastomeres aggregated with DsRed-labeled human PSCs (top) or DsRed-labeled ICLCs (bottom). Figure 12(C) shows immunofluorescence of chimeric blastocysts stained with anti-OCT4 (ICM, green) and anti-CDX2 (TE, gray). The red signal is from integrated DsRed-labeled ICLCs, and DAPI (blue) is used as a nuclear counterstain. When these chimeric blastocysts were implanted into the uterus of pseudopregnant mice and allowed to develop until embryonic day 10.5 (E10.5), human cells developed along with the mouse embryo and could contribute to various tissues, including embryonic tissues, extraembryonic placenta, and yolk sac, as shown in the microscopy images in Figure 13. Figure 13(A) shows representative images showing the phase contrast (top) or red fluorescent channel (bottom) of an E10.5 chimeric embryo (left), placenta (middle), or yolk sac (right). Figure 13(B) shows immunofluorescence images demonstrating that hN (green) differentiated human cells into GATA6 (red)-positive endoderm tissue. Figure 13(C) is an immunofluorescence image showing that DsRed-labeled human cells (red) differentiated into placental tissues indicated by GATA3 (green). Taken together, these results demonstrate that ICLCs can robustly integrate into mouse blastocysts and contribute to mouse E10.5 embryos and extraembryonic tissues in vivo.

[0084] Recently, blastocyst-like structures (called blastoids) were generated from expanded mouse PSCs (Non-Patent Document 11). However, this model using human cells has not yet been thoroughly studied. When ICLCs were applied to an extracellular matrix-rich medium, the inventors observed that blastocyst-like structures developed only from the ICLCs, but not from primed human PSCs (Figures 14A-B). Figure 14(A) shows the morphology of blastocysts developed from ICLCs in REM medium. Figure 14(B) shows immunofluorescence images of self-formed blastocysts stained with anti-OCT4 (ICM, red) antibody, anti-GATA3 (TE, green) antibody, or counterstained with DAPI (blue). GSK inhibitors, such as CHIR99021 (activating the WNT / β-catenin signaling pathway), are widely used in published naive or expanded PSC culture media. However, they inhibit the formation of ICLCs (Figure 36). Figure 36(A) shows that colony morphology is flatter, similar to that of primed human PSCs. Figure 36(B-D) shows that adding a GSK inhibitor to 4CL (4CL+CHIR) blocks the activation of human preimplantation ICM-enhancing genes. Consistently, removing the WNT / β-catenin signaling inhibitor IWR1 (which suppresses the WNT / β-catenin signaling pathway) from 4CL (4CL-IWR1) inhibits ICLC formation (Figure 37). These results indicate that activation of the WNT / β-catenin signaling pathway by GSK inhibitors is detrimental to ICLC formation. [Example]

[0085] Materials and Methods 4CL basal medium Same as Example 1. 4CL supplement Same as Example 1. cell Human ESC lines: H1 (male), HN10 (female), HUES1 (male), WIBR3 (female); human iPSC lines: CBC14 (female generated by the inventors), C11 (female generated by the inventors), Phoenix (female gift from Ulrich Martin's laboratory), DiPS 1016SevA (male purchased from the Harvard Stem Cell Institute), STiPS O-XX1 (female purchased from the Harvard Stem Cell Institute), UH10 (male). procedure The same procedure as in Example 1 was used. Experimental results Figure 15 is a bar graph of RT-qPCR data showing that preimplantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced in ICLCs converted from multiple primed human PSC lines, demonstrating the general applicability of 4CL Medium 1 to human PSCs. [Example]

[0086] Materials and Methods 4CL basal medium Same as Example 1. 4CL supplement Same as Example 1. cell H9 human ESC line. procedure The same procedure as in Example 1 is used, except that cells are plated on 1% (v / v) Geltrex™ in DMEM-F12 (cat#) coated plates instead of feeder cells. Experimental results Figure 16 is a bar graph of RT-qPCR data showing that ICLCs transduced on Geltrex™-coated plates with 4CL Medium 1 significantly induced the preimplantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1, similar to ICLCs transduced on feeders, demonstrating that 4CL Medium 1 is effective even in the absence of feeder cells. [Example]

[0087] Materials and Methods 4CL basal medium Same as Example 1. 4CL supplement Same as Example 1. cell H9 human ESC line. procedure Primed human PSCs were cultured using the same procedure as in Example 1. One day before the start of conversion, primed human PSCs were dissociated into single cells and seeded at 60,000 cells / well in Aggrewell™ 800 plates using mTeSR1 or E8 medium supplemented with 10 μM Y27632. After 24 hours, the medium was changed to 4CL Medium 1, and the culture conditions were switched to hypoxic conditions. The cells formed small spheres within 3 days. The spheres were then lifted and transferred to flasks (Greiner Bio-One, 658190) for suspension culture. The medium was changed daily. The cells were passaged every 4–5 days. For passaging, the cells were dissociated into single cells using TrypLE:0.5 mM EDTA (1:1) and then resuspended in 4CL Medium 1 at a density of 150,000 cells / ml. The resuspended cells were added to flasks (Greiner Bio-One, 658190) for suspension culture. The cells formed small aggregates within 24 hours. Generally, the cells converted to ICLCs approximately 3 weeks after initiation. Experimental results Figure 17 is a bar graph of RT-qPCR data showing that preimplantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced in ICLCs converted to suspension using 4CL Medium 1, demonstrating that 4CL Medium 1 is also effective in suspension cultures. [Example]

[0088] Materials and Methods 4CL basal medium Same as Example 1. 4CL supplement 4CL Medium 2 (excluding extracellular matrix) is 4CL basal medium plus: The medium was supplemented with SAH / PRC / EZH2 inhibitor (10 nM DZNep), HDAC inhibitor (5 nM TSA), L-ascorbic acid (50 μg / ml), JAK / STAT3 activator (20 ng / ml human LIF), MAPK / ERK inhibitor (1 μM PD0325901), tankyrase inhibitor (5 μM IWR1), activin A / NODAL activator (20 ng / ml human activin A), and ROCK inhibitor (1 μM Y27632). 4CL Medium 3 (minus ROCK inhibitor) contains the following in 4CL basal medium: The medium was supplemented with a SAH / PRC / EZH2 inhibitor (10 nM DZNep), an HDAC inhibitor (5 nM TSA), L-ascorbic acid (50 μg / ml), a JAK / STAT3 activator (20 ng / ml human LIF), a MAPK / ERK inhibitor (1 μM PD0325901), a tankyrase inhibitor (5 μM IWR1), an activin A / NODAL activator (20 ng / ml human activin A), and extracellular matrix (0.2% (v / v) Geltrex™). 4CL Medium 4 (without activin / NODAL activator) is 4CL basal medium plus: The medium was supplemented with a SAH / PRC / EZH2 inhibitor (10 nM DZNep), an HDAC inhibitor (5 nM TSA), L-ascorbic acid (50 μg / ml), a JAK / STAT3 activator (20 ng / ml human LIF), a MAPK / ERK inhibitor (1 μM PD0325901), a tankyrase inhibitor (5 μM IWR1), extracellular matrix (0.2% (v / v) Geltrex™), and a ROCK inhibitor (1 μM Y27632). cell H9 human ESC line procedure: The same procedure as in Example 1 was used. Experimental results Figure 18 is a bar graph of RT-qPCR data showing that the preimplantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 were significantly induced in ICLCs transformed with 4CL Medium 2, 4CL Medium 3, and 4CL Medium 4. These results indicate that 4CL Medium lacking Geltrex™, ROCK inhibitors, or activin / NODAL activators is also effective. [Example]

[0089] Materials and Methods 4CL basal medium Same as Example 1. e4CL supplement e4CL medium, 4CL basal medium plus: The media was supplemented with SAH / PRC / EZH2 inhibitors (50 nM DZNep or 3 mM CPI-1205), HDAC inhibitors (20 nM TSA, 1 mM VPA, or 1 mM NaB), JAK / STAT3 activators (20 ng / ml human LIF), MAPK / ERK inhibitors (1 μM XAV939), tankyrase inhibitors (5 μM IWR1), activin A / NODAL activators (20 ng / ml human activin A, 20 ng / ml human NODAL), ROCK inhibitors (1 μM Y27632, 1 μM thiazovivin, or 1 μM hydroxyfasudil), and extracellular matrix (0.2% (v / v) Geltrex™ or Matrigel™). cell H9, H1, and UH10 human ESC lines. procedure: 1) Conversion of primed human PSCs into 8CLCs Primed human PSCs were cultured using the same procedure as in Example 1. One day before the start of conversion, primed human PSCs were dissociated into single cells and plated on feeders at 2,000–3,000 cells / cm using mTeSR1 or E8 medium supplemented with 10 μM Y27632. 2After 24 hours, the medium was changed to e4CL medium, and the cells were cultured in a hypoxic or normoxic incubator at 37°C with 5% CO2. The medium was changed daily. Cells were passaged every 3–4 days. For passage, cells were plated at 2,000–3,000 cells / cm on feeder-coated plates. 2 The cells were dissociated into single cells using TrypLE:0.5 mM EDTA (1:1) seeded at 100°C. Typically, the cells were converted to 8CLCs in approximately 1 week. 2) Conversion of ICLC to 8CLC One day before the start of transformation, dissociate ICLCs into single cells and plate them on feeders at 2,000–3,000 cells / cm using 4CL medium. 2 After 24 hours, the medium was changed to e4CL medium. The medium was changed daily. The cells were converted to 8CLC within 3 to 5 days without subculturing. 3) Blastoid formation The same procedure as in Example 1 was used.

[0090] Experimental results Figure 19(A) shows a scheme of the 8CLC induction procedure: one directly from primed human PSCs and the other from ICLCs. Figure 19(B) and (C) show bar graphs of RT-qPCR data showing that human 8C-specific markers ZSCAN4, TPRX1, ZIM3, ZSCAN5B, ZNF280A, and ARGFX are significantly induced in 8CLCs converted from primed human PSCs (Figure 19B) or ICLCs (Figure 19C). The induction levels of 8C-specific genes are similar in both conversion methods (Figure 19D). Figure 19(E) shows immunofluorescence images showing the expression of ZSCAN4 in 8CLCs. To characterize the gene expression profile of 8CLCs at the single-cell level, we cultured ICLCs in e4CL medium and then performed scRNA-Seq on cells at the primed stage (primed-D0) and days 1, 2, 3, and 5 (e4CL-D1 / 2 / 3 / 5). Figure 20(A) shows 2D scatter plots of UMAP analysis of cells at different time points, along with published scRNA-Seq data for in vivo human embryos at embryonic days 3, 4, 5, 6, and 7 (E3 / 4 / 5 / 6 / 7, left panel) (from E-MTAB-3929). Cells in e4CL medium gradually acquire a gene expression profile similar to that of human embryos at embryonic days 3 (8C) and 4 (morula stage). Figure 20(B) shows that the expression levels of human 8C-specific markers in 8CLCs are upregulated compared to those in human 8C embryos (GSE101571). Taken together, these results demonstrate that 8CLCs derived from e4CL medium acquire a human in vivo morula- and 8C-stage embryo-like gene expression profile.

[0091] To investigate activated TEs in 8CLCs, we extracted TE profiles from the scRNA-seq data shown in Figure 20(A). Figure 21(A) shows a 2D scatter plot of UMAP analysis of TE expression in primed-stage (Primed-D0) cells, followed by TE expression on days 1, 2, 3, and 5 (D1 / 2 / 3 / 5) after culture in e4CL medium and human embryonic cells on embryonic days 3, 4, 5, 6, and 7 (E3 / 4 / 5 / 6 / 7) (from E-MTAB-3929). This shows that cells in e4CL medium gradually acquire a TE expression profile similar to that of human embryonic cells on embryonic days 3 (8C) and 4 (morula stage). Figure 21(B) further demonstrates that the expression levels of multiple TE subgroups in 8CLCs are induced to those of human 8C embryos (from GSE101571). Figure 22 shows that 8CLCs maintain a normal karyotype. One female human ESC line (H9) and one male human iPSC line (UH10) are shown. These results demonstrate that ICLCs derived from 4CL Medium 1 acquire the gene expression and TE profiles of human 8C embryos and maintain a stable genome. To determine the DNA methylation status of 8CLC, we applied RRBS to 8CLC and primed human PSCs. Figure 23 shows boxplots showing genome-wide CpG DNA methylation (top left panel), which is substantially reduced in 8CLC compared to primed human PSCs, while the methylation status of TSSs is slightly different (top right panel). Notably, the reduction in global DNA methylation levels is prevented by knocking out STELLA (bottom left panel). Figure 24 compares the imprinting status of 8CLC with in vivo human embryonic DNA methylation data. In addition to DNA methylation, chromatin accessibility is also altered. Bulk ATAC-seq in Figure 25 shows differences in chromatin accessibility between primed human PSCs and 8CLCs. 8C-specific loci that are close together in primed human PSCs are open in 8CLCs, whereas postimplantation-specific loci that are open in primed human PSCs are close together. These results indicate that 4CL Medium 1 successfully rewired the epigenetic landscape toward an 8C-like state.

[0092] We further investigated the metabolic status of 8CLCs induced in e4CL medium. Human 8C embryos primarily rely on oxidative phosphorylation (OxPhos) as an energy source, but primarily rely on glycolysis after implantation. Figure 26 shows that the expression levels of genes related to oxidative phosphorylation are significantly upregulated in 8CLCs compared to primed human PSCs. These results suggest that oxidative phosphorylation is activated in 8CLCs. To determine the differentiation potential of 8CLCs, we performed a teratoma formation assay using nude mice as recipient animals. Figure 27 shows images of hematoxylin and eosin-stained teratoma tissue formed 8 weeks after injection of 1 million 8CLCs. These images demonstrate the presence of all three germ layer structures: mesoderm (left panel), endoderm (middle panel), and ectoderm (right panel). We also used 8CLCs to induce trophoblast stem cell-like cells (TSCLCs) using a previously published protocol. As shown in Figure 28, compared with undifferentiated 8CLCs, TSCLCs significantly induced multiple TSC markers, including GATA3, CGA, KRT18, KRT8, PSG6, and CCR7. These results demonstrate that 8CLCs possess embryonic and extraembryonic developmental potential.

[0093] Due to ethical issues, we cannot test developmental potential using human embryos. Therefore, we performed cross-species chimera experiments by aggregating 8CLCs with mouse 8C-stage blastomeres. Human 8CLCs were found to successfully integrate into most mouse embryos and form chimeric blastocysts after 24 hours of in vitro culture. At this stage, human 8CLCs were located in both the ICM and TE regions of the chimeric blastocysts. Figure 29(A) shows representative images in the phase contrast (left) or red fluorescence channel (right) of blastocysts developed from mouse 8C blastomeres aggregated with DsRed-labeled primed human PSCs (top) or DsRed-labeled 8CLCs (bottom). Figure 29(B) shows immunofluorescence of chimeric blastocysts stained with anti-OCT4 (ICM, green) and anti-CDX2 (TE, gray). The red signal is from the integrated DsRed-labeled 8CLCs, and DAPI (blue) is used as a nuclear counterstain. When these chimeric blastocysts were implanted into the uterus of pseudopregnant mice and allowed to develop until embryonic day 10.5 (E10.5), the human cells developed along with the mouse embryo and could contribute to various tissues, including embryonic tissues and extraembryonic placenta and yolk sac, as shown in the microscopy images in Figure 30. Figure 30(A) shows representative images of the E10.5 chimeric embryo (left), placenta (middle), or yolk sac (right) in phase contrast (top) or red fluorescent channel (bottom). Figure 30(B) shows immunofluorescence images demonstrating that hN (green) differentiated human cells into GATA6 (red)-positive endodermal tissue. Figure 30(C) shows immunofluorescence images demonstrating that DsRed-labeled human cells (red) differentiated into placental tissues indicated by GATA3 (green). Taken together, these results demonstrate that 8CLCs can robustly integrate into mouse blastocysts and contribute to mouse E10.5 embryonic and extraembryonic tissues in vivo.

[0094] To determine the blastocyst-like structure forming potential of 8CLCs, we applied 8CLCs to matrix-rich medium and observed blastocyst-like structures forming over 5 days, but not primed human PSCs (Figure 31A). Figure 31B shows immunofluorescence images of self-forming blastocysts stained with anti-OCT4 (ICM, red), anti-GATA3 (TE, green) antibodies, or the nuclear counterstain DAPI (blue). 8CLCs serve as a robust model for functional studies of 8C regulators. In pilot studies, we identified three potential novel regulators governing the 8C state: TPRX1, KHDC1L, and TRIM60. Figure 37 shows that the induction of 8C-specific genes during the conversion of ICLCs to 8CLCs is inhibited by knockdown of TPRX1, KHDC1L, or TRIM60. GSK inhibitors, such as CHIR99021 (which activates the WNT / β-catenin signaling pathway), are widely used in published naive or expanded PSC culture media. However, their inclusion inhibits the formation of 8CLCs (Figure 38). Figure 38 shows that adding a GSK inhibitor to e4CL (e4CL+CHIR) inhibits activation of the human 8C embryonic enriched gene. Consistently, removing the WNT / β-catenin signaling inhibitor IWR1 (which suppresses the WNT / β-catenin signaling pathway) from e4CL (e4CL-IWR1) inhibits the formation of 8CLCs. These results indicate that activation of the WNT / β-catenin signaling pathway by GSK inhibitors is detrimental to the formation of 8CLCs. [Example]

[0095] Materials and Methods e4CL basal medium Same as Example 1. e4CL supplements Same as Example 6. cell H9 human ESC line procedure: Conversion of ICLCs in suspension to 8CLCs ICLCs were cultured using the same procedure as in Example 1. One day before the start of conversion, ICLCs were dissociated into single cells and resuspended in 4CL medium at a density of 300,000 cells / ml. The cell suspension was added to a flask and cultured in suspension (Greiner Bio-One, 658190). After 24 hours, the cells formed small aggregates, and the medium was changed to e4CL without the addition of Y27632. The medium was changed daily, and the cells were converted to 8CLC in 3–5 days without passaging. Experimental results Figure 32 is a bar graph of RT-qPCR data showing that 8C markers ZSCAN4, ARGFX, TPRX1, ZNF280A, and ZSCAN5B are significantly induced in 8CLCs converted to suspension culture using e4CL medium, demonstrating that e4CL medium is also effective for suspension cultures. [Example]

[0096] Materials and Methods e4CL basal medium Same as Example 1. e4CL supplement Same as Example 6. cell Human ESC lines: HN10 and UH10 procedure: Same as Example 6. Experimental results Figure 33 is a bar graph of RT-qPCR data showing that the 8C markers ZSCAN4, ARGFX, TPRX1, ZNF280A, ZSCAN5B, DUXA, DUXB, MBD3L2, STELLA, KLF17, and KHDC1L are significantly induced in 8C CLCs converted from multiple hPSC lineages, demonstrating that e4CL medium is generally applicable to human PSCs. [Example]

[0097] Materials and Methods e4CL basal medium Same as Example 1. e4CL supplements Same as Example 6. cell Mouse ESC line: E14 and Mervl-GFP procedure: One day before the start of conversion, mouse ESCs cultured under serum / LIF conditions were dissociated into single cells and plated onto feeders in serum / LIF medium. After 24 hours, the medium was changed to e4CL medium. The medium was changed daily. Cells were converted to a mouse 2C-like state in 3 days without passaging. Experimental results Figure 39 shows that 2C markers such as Zscan4, Zscan4b, Zscan4c, Zscan4d, Dux, Tcstv1, Tcstv3, Gm4340, Zfp352, and Dub1 are significantly induced in 2C-like cells converted from multiple mouse ESC lines, indicating that e4CL medium is also potent in inducing a 2C-like state in mice and is not cell lineage specific. [Example]

[0098] Materials and Methods 4CL basal medium Same as Example 1. 4CL supplement Same as in Example 1, but different doses of either PD0325901, DZNep, or TSA were also used: PD0325901, 0.5 μM; TSA, 20 nM; DZNep, 5 nM, 20 nM, or 50 nM. cell H9 human ESC line procedure: Same as Example 1. Experimental results Figure 40 shows that pre-implantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced in ICLCs transformed with 4CL Medium 1 supplemented with different doses of either PD0325901, DZNep, or TSA compared to primed human PSCs. References

[0099] [Table 2]

[0100] Table 3

[0101] Table 4

Claims

1. The basal medium for culturing stem cells contains the following: (i) one or more of an S-adenosylhomocysteine ​​hydrolase (SAH) inhibitor, a polycomb repressive complex (PRC) inhibitor, and an EZH2 inhibitor; (ii) histone deacetylase (HDAC) inhibitors, and (iii) L-ascorbic acid or a derivative thereof, a JAK / STAT3 signal activator, a MAPK / ERK signal transduction inhibitor, and a tankyrase inhibitor are added; A chemically defined medium for converting primate-primed mammalian pluripotent stem cells (PSCs) into ICM-like cells (ICLCs) and / or 8-cell embryonic-like cells (8CLCs), or for converting ICLCs into 8CLCs.

2. The medium according to claim 1, further comprising one or more components selected from the group consisting of an activin / NODAL signaling activator, a ROCK inhibitor, and an extracellular matrix.

3. below: the PRC inhibitor, EZH2 inhibitor or SAH inhibitor is selected from the group consisting of DZNep and CPI-1205, and / or the HDAC inhibitor is selected from the group consisting of TSA, VPA and NaB; and / or the activator of JAK / STAT3 signaling is LIF, and / or the MAPK / ERK signaling inhibitor is PD0325901, and / or 3. The medium of claim 2, wherein the tankyrase inhibitor is selected from the group consisting of IWR1 and XAV939.

4. below: the final concentration of DZNep in the medium is 5-80 nM, or the final concentration of CPI-1205 in the medium is 0.5-5 mM, and / or the final concentration of TSA in the medium is 3-30 nM, or the final concentration of VPA in the medium is 0.25-2 mM, or the final concentration of NaB in the medium is 0.25-2 mM; and / or the final concentration of L-ascorbic acid in the medium is 40-70 μg / ml; and / or the final concentration of the activator of JAK / STAT3 signaling in the culture medium is 10-50 ng / mL; and / or the final concentration of the MAPK / ERK signaling inhibitor in the culture medium is between 0.5 μM and 3 μM; and / or The medium according to claim 3, wherein the final concentration of the tankyrase inhibitor in the medium is 2 to 8 μM.

5. 4. The medium according to claim 2 or 3, wherein the final concentration of the activator of activin / NODAL signaling is 10 to 25 ng / ml, wherein the activator of activin / NODAL signaling is selected from the group consisting of activin A and NODAL, and / or the final concentration of the ROCK inhibitor is 0.5 to 2 μM, wherein the ROCK inhibitor is selected from the group consisting of Y27632, thiazovivin, and hydroxyfasudil, and / or the amount of the extracellular matrix in the medium is 0.1 to 0.5% (v / v).

6. below: (A) DZNep at a final concentration of 5-15 nM or CPI-1205 at a final concentration of 0.5-2 mM, TSA at a final concentration of 3-10 nM, or VPA at a final concentration of 0.25-1 mM or NaB at a final concentration of 0.25-1 mM; (B) L-ascorbic acid at a final concentration of 40-70 μg / ml; (C) LIF at a final concentration of 10-30 ng / mL; (D) PD0325901 at a final concentration of 0.5-1.5 μM, and (E) IWR1 or XAV939 at a final concentration of 3-6 μM; is added, and furthermore the following: (1) activin A or NODAL at a final concentration of 10 to 25 ng / ml, Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (2) the activin A or NODAL at a final concentration of 10 to 25 ng / ml, and the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, or (3) the activin A or NODAL at a final concentration of 10 to 25 ng / ml and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (4) the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (5) The medium according to claim 3, to which is added activin A or NODAL at a final concentration of 10 to 25 ng / ml, or Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, or the extracellular matrix at 0.1% to 0.5% (v / v).

7. below: (A) (i) DZNep at a final concentration of 5 to 50 nM or CPI-1205 at a final concentration of 0.5 to 3 mM, and (ii) TSA at a final concentration of 3 to 25 nM, or VPA at a final concentration of 0.25 to 1 mM, or NaB at a final concentration of 0.25 to 1.5 mM; (B) L-ascorbic acid at a final concentration of 40-70 μg / ml; (C) LIF at a final concentration of 10-30 ng / mL; (D) PD0325901 at a final concentration of 0.5-1.5 μM, and (E) IWR1 or XAV939 at a final concentration of 3-6 μM; is added, and furthermore the following: (1) activin A or NODAL at a final concentration of 10 to 25 ng / ml, Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (2) the activin A or NODAL at a final concentration of 10 to 25 ng / ml, and the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, or (3) the activin A or NODAL at a final concentration of 10 to 25 ng / ml and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (4) the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (5) The medium according to claim 3, to which is added activin A or NODAL at a final concentration of 10 to 25 ng / ml, or Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, or the extracellular matrix at 0.1% to 0.5% (v / v).

8. below: (i) 10 nM of the DZNep or 1 mM of the CPI-1205; (ii) 5 nM of the TSA or 0.5 mM of the VPA or 0.5 mM of the NaB, and (iii) 50 μg / mL of the L-ascorbic acid, 20 ng / mL of the LIF, 1 μM of the PD0325901, and 5 μM of the IWR1 or 5 μM of the XAV939, and further comprising: (1) 20 ng / mL of activin A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v / v) of the extracellular matrix; or (2) 20 ng / mL of the activin A or NODAL and 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, or (3) 20 ng / mL of the activin A or NODAL and 0.2% (v / v) of the extracellular matrix, or (4) 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, and 0.2% (v / v) of the extracellular matrix, or (5) The medium according to claim 3, supplemented with 20 ng / mL of activin A or NODAL, or 1 μM of Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v / v) of the extracellular matrix.

9. below: (i) the DZNep at a final concentration of 40 to 70 nM or the CPI-1205 at a final concentration of 2 to 4 mM; (ii) the TSA at a final concentration of 10 to 30 nM, the VPA at a final concentration of 0.5 to 1.5 mM, or the NaB at a final concentration of 0.5 to 1.5 mM; and (iii) The L-ascorbic acid having a final concentration of 40 to 70 μg / mL, the LIF having a final concentration of 10 to 30 ng / mL, the PD0325901 having a final concentration of 0.5 to 1.5 μM, and the IWR1 or XAV939 having a final concentration of 3 to 6 μM, and further comprising the following: (1) the activin A or NODAL at a final concentration of 10 to 25 ng / mL, Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix at 0.1% to 0.5% (v / v); or (2) the activin A or NODAL at a final concentration of 10 to 25 ng / mL, the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, or (3) the activin A or NODAL at a final concentration of 10 to 25 ng / mL and the extracellular matrix at 0.1% to 0.5% (v / v); or (4) the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix at a concentration of 0.1 to 0.5% (v / v); or (5) The medium according to claim 3, to which is added activin A or NODAL at a final concentration of 10 to 25 ng / mL, or Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, or the extracellular matrix at 0.1 to 0.5% (v / v).

10. below: (i) 50 nM of the DZNep or 3 mM of the CPI-1205; (ii) 20 nM of the TSA or 1 mM of the VPA or 1 mM of the NaB, and (iii) 50 μg / mL of the L-ascorbic acid, 20 ng / mL of the LIF, 1 μM of the PD0325901, and 5 μM of the IWR1 or 5 μM of the XAV939, and further comprising: (1) 20 ng / mL of the activin A or NODAL, 1 μM of the Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v / v) of the extracellular matrix, or (2) 20 ng / mL of the activin A or NODAL and 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, or (3) 20 ng / mL of the activin A or NODAL and 0.2% (v / v) of the extracellular matrix, or (4) 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, and 0.2% (v / v) of the extracellular matrix, or (5) The medium according to claim 9, supplemented with 20 ng / mL of activin A or NODAL, or 1 μM of Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v / v) of the extracellular matrix.

11. 11. The medium of any one of claims 1 to 10, wherein the basal medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F10, F12, alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium, Neurobasal Medium, DMEM / F12, and Advanced DMEM / F12, and combinations thereof.

12. The medium according to any one of claims 1 to 11, further comprising one or more components selected from the group consisting of serum replacement, an alternative carbon source, a non-essential amino acid, L-glutamine or a substitute therefor, and an antibiotic.

13. 13. A method for converting primate primed PSCs into ICLCs and / or 8CLCs, or for converting ICLCs into 8CLCs, comprising culturing the primate primed PSCs or ICLCs in the presence of a medium according to any one of claims 1 to 12.

14. 9. A method for converting primate primed PSCs into ICLCs, comprising culturing the primate primed PSCs in the medium of claim 7 or 8, wherein the basal medium of the medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F10, F12, alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium, Neurobasal Medium, and DMEM / F12, and combinations thereof.

15. 11. A method for converting primate-primed PSCs or ICLCs to 8CLCs, comprising culturing the primate-primed PSCs or ICLCs in the medium of claim 9 or 10, wherein the basal medium of the medium is selected from the group consisting of Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basal Eagle's Medium (BME), RPMI 1640, F10, F12, alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium, Neurobasal Medium, DMEM / F12 and advanced DMEM / F12, and combinations thereof.

16. A method for converting primate primed PSCs into ICLCs, comprising: (a) modifying primate primed PSCs to reduce SAH, PRC and / or EZH2 activity in the primate primed PSCs by knocking down and / or knocking out one or more relevant genes in the cells; and (b) The recombinant cells obtained in (a) are subjected to the following: (i) TSA at a final concentration of 3-30 nM, or VPA at a final concentration of 0.25-2 mM, or NaB at a final concentration of 0.25-2 mM, and (ii) a final concentration of 5 to 80 nM DZNep or 0.5 to 5 mM CPI-1205, and (iii) culturing in the medium of claim 3 comprising L-ascorbic acid at a final concentration of 40-70 μg / mL, LIF at a final concentration of 10-30 ng / mL, PD0325901 at a final concentration of 0.5-1.5 μM, and IWR1 or XAV939 at a final concentration of 3-6 μM, wherein the medium further comprises: (1) activin A or NODAL at a final concentration of 10 to 25 ng / ml, Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (2) the activin A or NODAL at a final concentration of 10 to 25 ng / ml, and the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, or (3) the activin A or NODAL at a final concentration of 10 to 25 ng / ml and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (4) the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (5) The method, wherein the activin A or NODAL is added at a final concentration of 10 to 25 ng / ml, or the Y27632, thiazovivin, or hydroxyfasudil is added at a final concentration of 0.5 to 2 μM, or the extracellular matrix is ​​added at 0.1% to 0.5% (v / v).

17. The medium may be one of the following: (i) TSA at a final concentration of 3-10 nM, or VPA at a final concentration of 0.25-1 mM, or NaB at a final concentration of 0.25-1 mM, and (ii) DZNep at a final concentration of 5-15 nM or CPI-1205 at a final concentration of 0.5-2 mM, and (iii) the method according to claim 16, to which L-ascorbic acid at a final concentration of 40 to 70 μg / mL, LIF at a final concentration of 10 to 30 ng / mL, PD0325901 at a final concentration of 0.5 to 1.5 μM, and IWR1 or XAV939 at a final concentration of 3 to 6 μM have been added.

18. The medium may be one of the following: (i) 5 nM TSA, or 0.5 mM VPA, or 0.5 mM NaB, and (ii) 50 μg / ml L-ascorbic acid, 20 ng / mL LIF, 1 μM PD0325901, and 5 μM IWR1 or 5 μM XAV939, and (iii) 10 nM DZNep or 1 mM CPI-1205, and wherein the medium further contains one of the following: (1) 20 ng / mL activin A or NODAL, 1 μM Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v / v) extracellular matrix, or (2) 20 ng / mL of the activin A or NODAL and 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, or (3) 20 ng / mL of the activin A or NODAL and 0.2% (v / v) of the extracellular matrix; or (4) 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, and 0.2% (v / v) of the extracellular matrix, or (5) The method of claim 16, wherein 20 ng / mL of activin A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v / v) of the extracellular matrix is ​​added.

19. 1. A method for converting primate primed PSCs or ICLCs into 8CLCs, comprising: (a) modifying primate-primed PSCs or ICLCs to reduce SAH, PRC and / or EZH2 activity in said primate-primed PSCs or ICLCs by knocking down and / or knocking out one or more relevant genes in said cells; and (b) The recombinant cells obtained in (a) are subjected to the following: (i) TSA at a final concentration of 10-30 nM, or VPA at a final concentration of 0.5-1.5 mM, or NaB at a final concentration of 0.5-1.5 mM, and (ii) L-ascorbic acid at a final concentration of 40 to 70 μg / ml, LIF at a final concentration of 10 to 30 ng / mL, PD0325901 at a final concentration of 0.5 to 1.5 μM, and IWR1 or XAV939 at a final concentration of 3 to 6 μM each; and (iii) culturing in the medium of claim 3 containing DZNep at a final concentration of 40-70 nM or CPI-1205 at a final concentration of 2-4 mM, wherein the medium further contains one of the following: (1) activin A or NODAL at a final concentration of 10 to 25 ng / ml, Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (2) the activin A or NODAL at a final concentration of 10 to 25 ng / ml, and the Y27632, thiazobin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, or (3) the activin A or NODAL at a final concentration of 10 to 25 ng / ml and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (4) the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (5) The method, wherein the activin A or NODAL is added at a final concentration of 10 to 25 ng / ml, or the Y27632, thiazovivin, or hydroxyfasudil is added at a final concentration of 0.5 to 2 μM, or the extracellular matrix is ​​added at 0.1% to 0.5% (v / v).

20. The medium may further contain: (i) 20 nM TSA, or 1 mM VPA, or 1 mM NaB, and (ii) 50 μg / ml L-ascorbic acid, 20 ng / mL LIF, and 1 μM PD0325901, 5 μM IWR1, or 5 μM XAV939, and (iii) containing 50 nM DZNep or 3 mM CPI-1205; and Here, the medium contains the following: (1) 20 ng / mL activin A or NODAL, 1 μM Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v / v) extracellular matrix, or (2) 20 ng / mL of the activin A or NODAL and 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, or (3) 20 ng / mL of the activin A or NODAL and 0.2% (v / v) of the extracellular matrix; or (4) 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, and 0.2% (v / v) of the extracellular matrix, or (5) The method of claim 19, wherein 20 ng / mL of activin A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v / v) of the extracellular matrix is ​​added.

21. A method for converting primate primed PSCs into ICLCs, comprising: (a) modifying primate-primed PSCs to reduce the activity of HDAC in the primate-primed PSCs by knocking down and / or knocking out one or more relevant genes in the cells; and (b) The recombinant cells obtained in (a) are subjected to the following: (i) DZNep at a final concentration of 5 to 80 nM or CPI-1205 at a final concentration of 0.5 to 5 mM, and (ii) TSA at a final concentration of 3-30 nM, or VPA at a final concentration of 0.25-2 mM, or NaB at a final concentration of 0.25-2 mM, and (iii) culturing in the medium of claim 3, comprising L-ascorbic acid at a final concentration of 40-70 μg / ml, LIF at a final concentration of 10-30 ng / mL, PD0325901 at a final concentration of 0.5-1.5 μM, and IWR1 or XAV939, each at a final concentration of 3-6 nM, wherein the medium further contains: (1) activin A or NODAL at a final concentration of 10 to 25 ng / ml, Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (2) the activin A or NODAL at a final concentration of 10 to 25 ng / ml, and the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, or (3) the activin A or NODAL at a final concentration of 10 to 25 ng / ml and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (4) the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (5) The method, wherein the activin A or NODAL is added at a final concentration of 10 to 25 ng / ml, or the Y27632, thiazovivin, or hydroxyfasudil is added at a final concentration of 0.5 to 2 μM, or the extracellular matrix is ​​added at 0.1% to 0.5% (v / v).

22. The medium may be one of the following: (i) DZNep at a final concentration of 5-15 nM or CPI-1205 at a final concentration of 0.5-2 mM, and (ii) TSA at a final concentration of 3 to 10 nM, or VPA at a final concentration of 0.25 to 0.5 mM, or NaB at a final concentration of 0.25 to 0.5 mM, and (iii) the method of claim 21, wherein L-ascorbic acid is added at a final concentration of 40 to 70 μg / mL, LIF at a final concentration of 10 to 30 ng / mL, PD0325901 at a final concentration of 0.5 to 1.5 μM, and IWR1 or XAV939 at a final concentration of 3 to 6 μM.

23. The medium may be one of the following: (i) 10 nM DZNep or 1 mM CPI-1205, and (ii) 50 μg / mL L-ascorbic acid, 20 ng / mL LIF, 1 μM PD0325901, and 5 μM IWR1 or 5 μM XAV939, and (iii) 5 nM TSA, or 0.5 mM VPA, or 0.5 mM NaB, and wherein the medium further contains one of the following: (1) 20 ng / mL of activin A or NODAL, 1 μM of Y27632, thiazovivin, or hydroxyfasudil, and 0.2% (v / v) extracellular matrix, or (2) 20 ng / mL of the activin A or NODAL, and 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, or (3) 20 ng / mL of the activin A or NODAL, and 0.2% (v / v) extracellular matrix. 20 ng / mL of activin A or NODAL, or 1 μM of Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v / v) of the extracellular matrix, or (4) 1 μM of Y27632, thiazovivin or hydroxyfasudil and 0.2% (v / v) of the extracellular matrix, or (5) 20 ng / mL of activin A or NODAL, or 1 μM of Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v / v) of the extracellular matrix.

24. 1. A method for converting primate primed PSCs or ICLCs into 8CLCs, comprising: (a) modifying primate-primed PSCs or ICLCs to reduce HDAC activity in the primate-primed PSCs or ICLCs by knocking down and / or knocking out one or more relevant genes in the cells; and (b) subjecting the recombinant cell to one of: (i) DZNep at a final concentration of 40-70 nM or CPI-1205 at a final concentration of 2-4 mM, and (ii) L-ascorbic acid at a final concentration of 40 to 70 μg / ml, LIF at a final concentration of 10 to 30 ng / mL, PD0325901 at a final concentration of 0.5 to 1.5 μM, and IWR1 or XAV939 at a final concentration of 3 to 6 μM each; and (iii) culturing in the medium of claim 3 containing TSA at a final concentration of 10-30 nM, or VPA at a final concentration of 0.5-1.5 mM, or NaB at a final concentration of 0.5-1.5 mM, wherein the medium further contains: (1) activin A or NODAL at a final concentration of 10 to 25 ng / ml, Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (2) the activin A or NODAL at a final concentration of 10 to 25 ng / ml, the Y27632, thiazobin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, or (3) the activin A or NODAL at a final concentration of 10 to 25 ng / ml and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (4) the Y27632, thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM, and the extracellular matrix in an amount of 0.1% to 0.5% (v / v); or (5) The method, wherein the activin A or NODAL is added at a final concentration of 10 to 25 ng / ml, or the Y27632, thiazovivin, or hydroxyfasudil is added at a final concentration of 0.5 to 2 μM, or the extracellular matrix is ​​added at 0.1% to 0.5% (v / v).

25. The medium may be one of the following: (i) 50 nM DZNep or 3 mM CPI-1205, and (ii) 50 μg / ml L-ascorbic acid, 20 ng / mL LIF, 1 μM PD0325901, and 5 μM IWR1 or 5 μM XAV939, and (iii) 20 nM TSA, or 1 mM VPA, or 1 mM NaB, and wherein the medium further contains one of the following: (1) 20 ng / mL activin A or NODAL, 1 μM Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v / v) extracellular matrix, or (2) 20 ng / mL of the activin A or NODAL and 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, or (3) 20 ng / mL of the activin A or NODAL and 0.2% (v / v) of the extracellular matrix; or (4) 1 μM of the Y27632, thiazovivin, or hydroxyfasudil, and 0.2% (v / v) of the extracellular matrix, or (5) The method of claim 21, wherein 20 ng / mL of activin A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v / v) of the extracellular matrix is ​​added.

26. The primate primed PSCs are: (i) cells derived from ESC and / or ECC lines; (ii) cells derived from iPSC lines; (iii) cells derived from the ICM of in vitro cultured preimplantation blastocysts; (iv) cells derived from the ICM of in vitro cultured post-implantation blastocysts; (v) in vitro cultured cells from post-embryonic stage 8C to morula stage, the method according to any one of claims 13 to 22.

27. A kit comprising the medium according to any one of claims 1 to 12.

28. A composition comprising the medium according to any one of claims 1 to 12.

29. Use of the medium according to any one of claims 1 to 11, capable of promoting the expression of STELLA, in the manufacture of a reagent, medium or kit for promoting the conversion of primate primed PSCs to ICLCs, or for promoting the conversion of primate primed PSCs or ICLCs to 8CLCs; or Use of a medium according to any one of claims 1 to 12, which is capable of promoting the expression of STELLA for promoting the conversion of primate primed PSCs to ICLCs, or for promoting the conversion of primate primed PSCs and / or ICLCs to 8CLCs.

30. Use of the culture medium according to any one of claims 1 to 12, which promotes expression of genes belonging to the ETCHbox family including KHDC1L, TRIM60, and / or TPRX1 and ARGFX, in the manufacture of a reagent, culture medium, or kit for promoting the conversion of primate primed PSCs or ICLCs to 8CLCs; or Use of the medium according to any one of claims 1 to 12, which promotes the expression of KHDC1L, TRIM60, and / or genes belonging to the ETCHbox family, including TPRX1 and ARGFX, for promoting the conversion of primate PSCs and / or ICLCs to ocCLCs.

Images