Media and methods for the establishment and maintenance of primordial germ cell-like cells

Abstract

This application discloses culture media and methods for establishing and maintaining mammalian early embryonic-like cells. The culture media described herein can be used to culture mammalian pluripotent stem cells (PSCs) and include a chemically defined basal medium for culturing stem cells supplemented with an S-adenosylhomocysteine ​​hydrolase (SAH) / polycomb repressive complex (PRC) / EZH2 inhibitor, a histone deacetylase (HDAC) inhibitor, and a WNT / β-catenin culture signaling / tankyrase inhibitor. The culture media described herein 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 a medium and a method for establishing and maintaining mammalian early embryo-like cells.

Background Art

[0002] Mammalian embryogenesis is a complex process of cell division and differentiation leading to embryo development. When the fertilization of an oocyte and a sperm is successful, embryogenesis is initiated. Through this strictly controlled process, billions of cells with different functions and forms are generated from a single zygote. The vast cellular complexity of all sexually reproducing organisms begins with embryogenesis. First, a single zygote divides to form two cells. Then, these two cells divide to form four cells, eight cells, and sixteen cells. Further expansion results in a blastocyst consisting of two regions, namely the inner cell mass (ICM) and the trophectoderm (TE), and at this stage, embryogenesis is in the pre-implantation (within the uterine wall) stage. ICM cells form the amnion and all fetal tissues, while TE cells form the placenta during the post-implantation developmental stage. All these developmental stages have been well characterized in mice because these cells can be easily accessed from mouse embryos without ethical concerns. Pioneering research by Evans and Kaufman showed that cells can be extracted from the ICM of mouse blastocysts and these cells can be proliferated indefinitely in vitro under appropriate culture conditions (Non-Patent Document 1). These cells are called embryonic stem cells (ESCs) and represent the cells within the ICM of mouse blastocysts. Mouse ESCs are pluripotent but not totipotent and can differentiate only into all three germ layers (ectoderm, mesoderm, endoderm) of the embryo, so they can generate cells corresponding to all fetal tissues. In contrast, totipotency is the ability of cells to form the entire organism, including embryonic and extra-embryonic cells, not just fetal tissues as in the case of pluripotent cells. In early mouse embryos, cells before 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. succeeded in producing human ESCs from human ICM (Non-Patent Document 2).

[0003] Due to the great potential of human PSCs for disease modeling and regenerative medicine, many studies have 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, excluding ethical issues (Non-Patent Document 3). ESCs and iPSCs are very similar and are collectively referred to as PSCs here. Both mouse ESCs and human ESCs are derived from the ICM of pre-implantation blastocysts, but they exhibit unique characteristics. Human PSCs cultured under conventional conditions show a primed pluripotent state similar to mouse epiblast stem cells (EpiSCs) derived from the post-implantation epiblast (Non-Patent Document 4). Primed human PSCs show a flat colony morphology, have low viability when passaged as single cells, require fibroblast growth factor 2 (FGF2) and transforming growth factor β1 (TGFβ1) / activin A / NODAL signaling, and cannot contribute to human-mouse interspecies chimera formation. In contrast, mouse ESCs exist in a naive state similar to that of pre-implantation ICM, which is characterized by dome-shaped colonies, increased clonogenicity of single cells, dependence on Janus kinase / signal transducer and activator of transcription 3 (JAK / STAT3) signaling, a pre-implantation ICM-like transcriptome profile, and chimerism ability (Non-Patent Document 5). Furthermore, mouse ESCs have a greater potential for differentiation than EpiSCs (Non-Patent Document 6). Additionally, in recent years, a small population (~0.5%) of two-cell embryo-like cells (2CLCs) in mouse ESC culture has been reported to show a transcriptional profile similar to that of two-cell (2C) stage mouse embryos (Non-Patent Document 7). This is important because 2C cells are totipotent.

[0004] Recently, several methods have been reported for deriving and maintaining an altered state of human and non-human primate PSCs that exhibit (pre-implantation-like) human naive properties (Non-Patent Document 8). These cells share some morphological and molecular similarities with mouse ESCs. However, it is still under debate whether these reported human naive PSCs are truly similar to the pre-implantation ICM. Furthermore, current methods each have specific drawbacks, such as being lengthy, producing naive-specific genes at various levels, being transgene-dependent for naive induction, having genomic instability, imprinting defects, being unable to differentiate into multiple lineages, lacking proper chimera-forming ability, or having poor efficiency.

[0005] PSCs are used in cell therapy in regenerative medicine and have great potential for studying diseases through patient-specific disease modeling (Non-Patent Document 9). Currently, researchers are using primed PSCs as raw materials for these studies. One area where naive cells are beginning to prove useful is in the generation of interspecies chimeras. In these studies, certain PSCs are injected into developing embryos of other species, and the proportion of cells that contribute to the organism is measured. However, the contribution to chimeras is currently exceptionally low (<0.01%). We aim to improve the contribution of PSCs that are transcriptionally and epigenetically close to early embryos and actually enhance the overall function of PSCs.

[0006] Another notable research area using PSCs is blastoid formation. Blastoids are currently blastocyst-like structures formed by the forced aggregation of ESCs and TE cells in vitro (Non-Patent Document 10). These in vitro models of early embryos focus on the developmental process and can be used to model diseases that affect embryonic development. Nevertheless, in current state-of-the-art models, instead of all cells arising from a single cell and self-organizing, it is necessary to mix several types of cells, and for example, blastocysts cannot properly undergo gastrulation and do not achieve the actual blastocyst-like state (Non-Patent Document 11). The inventors believe that this process can be improved and proper blastocysts can be formed by using cells that are transcriptionally and epigenetically close to early embryos.

[0007] The major regulators of ongoing cell fate transitions are epigenetic. This means that by manipulating the epigenome, it should be possible to produce cells that correspond to any stage of development. 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 conversion of the aforementioned primed PSCs to a naive state using epigenetic pathways and small molecule inhibitors of cytokines. One of the important components of the epigenome is DNA methylation, which plays a central role in gene regulation. The overall amount of DNA methylation in cells during early embryogenesis is very dynamic. The DNA methylation of pre-implantation blastocysts is much lower than that of post-implantation embryos, and interestingly, it is known to be lower than that of 8C embryos (Non-Patent Document 13). Therefore, to revert primed PSCs to an ICM-like state, it is necessary to significantly reduce the overall DNA methylation level. Correspondingly, to capture an 8C-like stage, more control of the reduction is required. Furthermore, the DNA methylation landscape needs to be correctly rewired during the regression process, respecting imprinting control regions (ICRs) and maintaining hemimethylation.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Summary of the Invention

Problems to be Solved by the Invention

[0009] Therefore, in order to produce initial embryonic-like cells, it is necessary to precisely fine-tune the DNA methylation apparatus.

Means for Solving the Problems

[0010] In one aspect, the present disclosure discloses a chemically defined culture medium for PSCs, which includes a basal medium for culturing stem cells supplemented with an S-adenosylhomocysteine hydrolase (SAH) inhibitor, a histone deacetylase (HDAC) inhibitor, and a WNT / β-catenin signaling inhibitor. In one or more embodiments, the SAH inhibitor is a polycomb repressive complex (PRC) and / or EZH2 inhibitor. In one or more embodiments, the WNT / β-catenin signaling inhibitor is a tankyrase inhibitor.

[0011] In one or more embodiments, further, one or more components selected from the group consisting of L-ascorbic acid or its derivatives, a JAK / STAT3 signaling activator, and a mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) signaling inhibitor are supplemented to the medium according to claim 1 or 2, and optionally, further, 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 are supplemented. In one or more embodiments, the SAH / PRC / EZH2 inhibitor or the SAH inhibitor is 3-deazaneplanocin A (DZNep) or CPI-1205. In one or more embodiments, the final concentration of the DZNep in the culture 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 culture 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 culture medium is 3 to 30 nM, preferably 3 to 25 nM. In one or more embodiments, the final concentration of the VPA in the culture medium is 0.25 to 2 mM, preferably 0.5 to 1.5 mM. In one or more embodiments, the final concentration of the NaB in the culture medium is 0.25 to 2 mM, preferably 0.5 to 1.5 mM. In one or more embodiments, the tankyrase inhibitor is IWR1 or XAV939. In one or more embodiments, the final concentration of the WNT / β-catenin signaling inhibitor in the culture medium is 2 to 8 μM.

[0012] In one or more embodiments, the final concentration of L-ascorbic acid in the culture medium is 40 to 70 μg / ml. In one or more embodiments, the final concentration of the JAK / STAT3 signaling activator in the culture medium is 10 to 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 culture medium is 0.5 to 3 μM. In one or more embodiments, the MAPK / ERK signaling inhibitor is PD0325901. In one or more embodiments, the final concentration of the activin / NODAL signaling activator is 10 to 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 culture medium is 0.5 to 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 culture 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 culture medium comprises the following: the DZNep at a final concentration of 5 to 15 nM or CPI-1205 at a final concentration of 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 1 mM or NaB at a final concentration of 0.25 to 1 mM; 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 2 to 8 μM, preferably 3 to 6 μM, and further comprises 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) 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; or (3) 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) 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) 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; or the extracellular matrix in an amount of 0.1% to 0.5% (v / v); is supplemented.

[0014] In one or more embodiments, the culture medium comprises 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 further comprises 5 μM of the IWR1 or 5 μM of the XAV939; and further comprises the following: (1) 20 ng / mL of human activin A or human NODAL, 1 μM of the Y27632, thiazovivin or hydroxyfasudil, 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) the activin A or NODAL at a final concentration of 10-25 ng / ml; and 0.1%-0.5% (v / v) of the extracellular matrix; or (4) the Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2 μM; and 0.1%-0.5% (v / v) of the extracellular matrix; or (5) the activin A or NODAL at a final concentration of 10-25 ng / ml; the Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2 μM; or 0.1%-0.5% (v / v) of the extracellular matrix; is supplemented.

[0015] In one or more embodiments, the culture medium comprises the following: the DZNep at a final concentration of 40-70 nM or the CPI-1205 at a final concentration of 2-4 mM; the TSA at a final concentration of 10-30 nM or the VPA at a final concentration of 0.5-1.5 mM or the NaB at a final concentration of 0.5-1.5 mM; the L-ascorbic acid at a final concentration of 40-70 μg / ml; the LIF at a final concentration of 10-30 ng / mL; the PD0325901 at a final concentration of 0.5-1.5 μM; and further comprises, and preferably, 2-8 μM, preferably 3-6 μM, of the IWR1 or the XAV939; and further comprises the following: (1) Activin A or NODAL at a final concentration of 10 - 25 ng / ml; Y27632, Thiazovivin or Hydroxyfasudil at a final concentration of 0.5 - 2 μM; and the extracellular matrix in an amount of 0.1% - 0.5% (v / v); or (2) The Activin A or NODAL at a final concentration of 10 - 25 ng / ml; the Y27632, Thiazovivin or Hydroxyfasudil at a final concentration of 0.5 - 2 μM; or (3) The Activin A or NODAL at a final concentration of 10 - 25 ng / ml; and the extracellular matrix in an amount of 0.1% - 0.5% (v / v); or (4) The Y27632, Thiazovivin or Hydroxyfasudil at a final concentration of 0.5 - 2 μM; and the extracellular matrix in an amount of 0.1% - 0.5% (v / v); or (5) The Activin A or NODAL at a final concentration of 10 - 25 ng / ml; the Y27632, Thiazovivin or Hydroxyfasudil at a final concentration of 0.5 - 2 μM; or the extracellular matrix in an amount of 0.1% - 0.5% (v / v); is supplemented.

[0016] In one or more embodiments, the culture medium is 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 contains, and 5 μM of the IWR1 or 5 μM of the XAV939; and further, the following: Or, contains the IWR1 or the XAV939 at 3 - 6 μM; and further, the following: (1) Activin A or NODAL at a final concentration of 10 - 25 ng / ml; Y27632, Thiazovivin or Hydroxyfasudil at a final concentration of 0.5 - 2 μM; and the extracellular matrix in an amount of 0.1% - 0.5% (v / v); or (2) The Activin A or NODAL at a final concentration of 10 - 25 ng / ml; the Y27632, Thiazovivin or Hydroxyfasudil at a final concentration of 0.5 - 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.

[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 mixture of advanced DMEM / F12 and Neurobasal Medium in a 1:1 (v / v) ratio. In one or more embodiments, the culture medium is further supplemented with one or more components selected from the group consisting of serum replacement, alternative carbon sources, non-essential amino acids, L-glutamine or substitutes thereof, and antibiotics. In one or more embodiments, the serum replacement is selected from the group consisting of Knockout™ Serum Replacement (KOSR), N2, and B27, and combinations thereof, and is a mixture of N2 and B27 mixed in a 1:1 (w / w) ratio; An alternative carbon source is pyruvate, such as sodium pyruvate; The L-glutamine or substitute thereof 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.

[0018] On the other hand, the present disclosure discloses a method for converting primate PSCs into pre-implantation ICM-like cells (ICLCs) and / or 8-cell embryo-like cells (8CLCs), which involves culturing primate PSCs or ICLCs in the presence of an S-adenosylhomocysteine hydrolase (SAH) / polycomb repressive complex (PRC) / EZH2 inhibitor, a histone deacetylase (HDAC) inhibitor, and a WNT / β-catenin signaling inhibitor. The present disclosure further discloses a method for converting ICLCs into 8CLCs, which involves culturing ICLCs in the presence of an SAH / PRC / EZH2 inhibitor, a histone deacetylase (HDAC) inhibitor, and a WNT / β-catenin signaling inhibitor. In one or more embodiments, the method involves culturing primate PSCs or ICLCs in the presence of the SAH / PRC / EZH2 inhibitor, the HDAC inhibitor, and the WNT / β-catenin signaling inhibitor, and in the presence of one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, and a MAPK / ERK signaling inhibitor, and optionally, 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. 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, the WNT / β-catenin signaling inhibitor is a tankyrase inhibitor. In one or more embodiments, the tankyrase inhibitor is IWR1 or XAV939. In one or more embodiments, the final concentration of the WNT / β-catenin signaling inhibitor or the tankyrase inhibitor is 2-8 μM. In one or more embodiments, the primate PSC or ICLC is 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 the 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 the VPA at a final concentration of 0.25 to 2 mM, preferably 0.5 to 1.5 mM, or in the presence of the NaB at a final concentration of 0.25 to 2 mM, preferably 0.5 to 1.5 mM, and; in the presence of IWR1 or XAV939 at a final concentration of 2 to 8 μM, preferably 3 to 6 μM.

[0019] In one or more embodiments, L-ascorbic acid is present at a final concentration of 40 to 70 μg / ml. In one or more embodiments, the final concentration of the JAK / STAT3 signaling activator is 10 to 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 MAPK / ERK signaling inhibitor is 0.5 to 3 μM. In one or more embodiments, the MAPK / ERK signaling inhibitor is PD0325901. In one or more embodiments, the final concentration of the activin / NODAL signaling activator is 10 to 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 to 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™.

[0020] In another aspect, the present disclosure further provides a method for converting primate PSCs into ICLCs, which includes culturing primate PSCs in the culture medium disclosed herein, wherein the basal medium of the culture medium is selected from the group consisting of Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI1640, 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, wherein the basal medium is a mixture of Advanced DMEM / F12 and Neurobasal Medium at a ratio of 1:1 (v / v). In a further aspect, the present disclosure provides a method for converting primate PSCs or ICLCs into 8CLCs, which includes culturing primate PSCs or ICLCs in the culture medium disclosed herein, wherein the basal medium of the culture medium is selected from the group consisting of Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI1640, F10, F12, Alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (GMEM), Iscove's Modified Dulbecco's Medium, Neurobasal Medium, DMEM / F12, Advanced DMEM / F12, and combinations thereof, wherein the basal medium is a mixture of Advanced DMEM / F12 and Neurobasal Medium at a ratio of 1:1 (v / v).

[0021] In one or more embodiments, the primate PSCs are selected from the following: (i) cells derived from ESC lines and / or ECC lines; (ii) cells derived from iPSC lines; (iii) cells derived from the ICM of pre-implantation blastocysts cultured in vitro; (iv) cells derived from the ICM of post-implantation blastocysts cultured in vitro; (v) cells from the 8-cell stage embryo to the morula stage cultured in vitro; and are selected from the group consisting of. In one or more embodiments, primate PSCs or ICLCs are grown under one or more of the following conditions: (i) on feeder cells; (ii) on extracellular matrix without feeder cells; (iii) in suspension without feeder cells; (iv) at a temperature of about 37 °C in a hypoxic or normoxic state; (v) passaged as single cells every 3 to 4 days at a split ratio of 1:4 to 1:8; and (vi) the culture medium is changed daily; and cultured under one or more conditions selected from the group consisting of: In a further aspect, the disclosure provides isolated primate ICLCs having a transcriptome, transposon profile, DNA methylome, chromatin landscape, and metabolic state that are similar to the corresponding primate pre-implantation ICM. In one or more embodiments, the primate ICLCs are as follows: 1) capable of self-renewing in culture and maintaining pluripotency; 2) maintaining genomic stability in culture according to the karyotype; 3) capable of generating cells of the three germ layers; 4) capable of generating primordial germ cell-like cells; 5) integrating into mouse embryos and contributing to the embryo and extraembryonic tissues; 6) capable of transitioning to extraembryonic cell dynamics in vitro; and 7) capable of forming blastocyst-like structures in vitro; and are characterized by one or more selected from the group consisting of: In one or more embodiments, the ICLCs are obtained by any of the methods described herein for generating ICLCs.

[0022] In a further aspect, the disclosure provides isolated primate 8CLCs that express 8C embryo-specific markers at a substantially higher level than the priming PSCs from which the ICLCs and / or 8CLCs are produced. The cells have a transcriptome, transposon profile, and chromatin landscape that are similar to the corresponding primate 8C-stage embryo.

[0023] In one or more embodiments, further, the cells are as follows: 1) Maintain the genomic stability in culture according to the karyotype; 2) Be able to produce cells of the three germ layers; 3) Be able to produce primordial germ cell-like cells; 4) Integrate into mouse embryos and be able to contribute to embryos and extra-embryonic tissues; 5) Be able to migrate to extra-embryonic cell dynamics in vitro; and 6) Be able to form blastocyst-like structures in vitro; Characterized by one or more selected from the group consisting of. 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 culture medium for culturing, comprising the primate ICLC and / or 8CLC described in any of the embodiments described herein, preferably comprising any culture medium specified herein.

[0024] The present disclosure also provides a kit comprising an SAH / PRC / EZH2 inhibitor, a histone deacetylase (HDAC) inhibitor, and a WNT / β-catenin signaling inhibitor, optionally the following: (1) One or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, and a MAPK / ERK signaling inhibitor; (2) One or more components selected from the group consisting of an activin / NODAL signaling activator, a ROCK inhibitor, and an extracellular matrix; (3) One or more components selected from the group consisting of a basal medium, a serum replacement, an alternative carbon source, non-essential amino acids, L-glutamine or its substitute, and an antibiotic. In one or more embodiments, the kit comprises any culture medium specified herein. The present disclosure also provides a composition comprising an SAH / PRC / EZH2 inhibitor, a histone deacetylase (HDAC) inhibitor, and a WNT / β-catenin signaling inhibitor, and further optionally the following: (1) One or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, and a MAPK / ERK signaling inhibitor; and, (2) One or more components selected from the group consisting of an activin / NODAL signaling activator, a ROCK inhibitor, and an extracellular matrix; are included. In one or more embodiments, the composition described herein is the following: A composition comprising DZNep or CPI-1205, TSA or VPA or NaB, IWR1 or XAV939, LIF, PD0325901, and optionally L-ascorbic acid, preferably, each of the foregoing components is such that the culture medium comprising the composition is the following: (a) 5-15 nM, preferably 10 nM of DZNep, or 0.5-2 mM, preferably 1 mM of CPI-1205; (b) 4-6 nM, preferably 5 nM of TSA, or 0.25-1 mM, preferably 0.5 mM of VPA;, or 0.25-1 mM, preferably 0.5 mM of NaB; (c) 3-6 μM, preferably 5 μM of IWR1 or XAV939; (d) 10-30 ng / mL, preferably 20 ng / mL of LIF; (e) 0.5-1.5 μM, preferably 1 μM of PD0325901; and, optionally, (f) 40-90 μg / mL, preferably 50 μg / mL of L-ascorbic acid. The composition may further be present in an amount comprising activin A or NODAL, and / or Y27632, thiazovivin or hydroxyfasudil, and / or an extracellular matrix, wherein each of the foregoing components is such that the culture medium comprising the composition comprises 10-25 ng / mL, preferably 20 ng / mL of activin A or NODAL, and / or 0.5-2 μM, preferably 1 μM of Y27632, thiazovivin or hydroxyfasudil, and / or 0.1%-0.5% (v / v) of an extracellular matrix in an amount that can be included. It contains DZNep or CPI-1205, TSA or VPA or NaB, LIF, PD0325901, IWR1 or XAV939, and optionally L-ascorbic acid; preferably, each of the above components is such that the culture medium containing the composition is as follows: DZNep at 40 - 70 nM, preferably 50 nM, or CPI-1205 at 2 - 4 mM, preferably 3 mM; TSA at 10 - 30 nM, preferably 20 nM, or VPA at 0.5 - 1.5 mM, preferably 1 mM, or NaB at 0.5 - 1.5 mM, preferably 1 mM; IWR1 or XAV939 at 2 - 8 μM, preferably 3 - 6 μM, preferably 5 μM; LIF at 10 - 30 ng / mL, preferably 20 ng / mL; PD0325901 at 0.5 - 1.5 μM, preferably 1 μM; and optionally L-ascorbic acid at 40 - 90 μg / mL, preferably 50 μg / mL; are present in an amount that can contain them. The composition contains activin A or NODAL, and / or Y27632, thiazovivin or hydroxyfasudil, and / or extracellular matrix, where each of the above components is present in the culture medium containing the above in an amount that can contain activin A or NODAL at 10 - 25 ng / mL, preferably 20 ng / mL, and / or Y27632, thiazovivin or hydroxyfasudil at 0.5 - 2 μM, preferably 1 μM, and / or extracellular matrix at 0.1% - 0.5% (v / v).

[0025] 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, culture 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 agents that can promote the expression of STELLA or improve the activity of STELLA are inhibitors of SAH / PRC / EZH2, including but not limited to DZNep and CPI-1205. Preferably, inhibitors of SAH / PRC / EZH2 such as DZNep and CPI-1205 are used in the above applications in the amounts described in any of the embodiments described in the present application. The present disclosure further provides for the use of an agent that can promote the expression of a gene belonging to the eutherian totipotency cell homeobox (ETCHbox) family, including KHDC1L, TRIM60, and / or TPRX1 and ARGFX, or can improve the activity of a protein belonging to the ETCHbox family, including KHDC1L, TRIM60, and / or a protein containing TPRX1 and ARGFX, in the manufacture of a reagent, culture medium, or kit for promoting the conversion of primate PSCs or ICLCs to 8CLCs. Also provided is the use of an agent that can promote the expression of a gene belonging to the ETCHbox family, including KHDC1L, TRIM60, and / or TPRX1 and ARGFX, or can improve the activity of a protein belonging to the ETCHbox family, including KHDC1L, TRIM60, and / or a protein containing TPRX1 and ARGFX, for promoting the conversion of primate PSCs and / or ICLCs to 8CLCs.

[0026] In one or more embodiments, the agents that can promote the expression of KHDC1L, TRIM60, and / or a gene belong to the ETCHbox family including TPRX1 and ARGFX, or the agents that can improve the activity of KHDC1L, TRIM60, and / or a protein belong to the ETCHbox family including TPRX1 and ARGFX, are inhibitors of SAH / PRC / EZH2, including DZNep and CPI-1205. Inhibitors of SAH / PRC / EZH2 such as DZNep and CPI-1205 are used in the above applications in the amounts described in any of the embodiments described in the present application.

Brief Description of the Drawings

[0027]

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Mode for Carrying Out the Invention

[0028] Current methods for the induction and maintenance of naive human PSCs exhibit some characteristics of mouse ESCs that are similar to the pre-implantation ICM of mice (Chan, Goke et al. 2013; Takashima, Guo et al. 2014; Theunissen, Powell et al. 2014). Induction of naive human PSCs using current methods has problems such as long length, transgene-dependence for naive induction with variable levels of naive-specific genes, genomic instability and loss of imprinting, inability to differentiate into multiple lineages, lack of proper chimera formation ability, or low efficiency. None of these studies have reported the generation of cells close to the 8C stage. To overcome the above problems, the inventors first performed screening using a panel of epigenetic regulators and inhibitors targeting different signaling pathways related to the development of human pre-implantation ICM, and found that combinations of SAH / PRC / EZH2 inhibitors, HDAC inhibitors, and WNT / β-catenin signaling inhibitors, as well as combinations of other regulators such as JAK / STAT3 activators and MAPK / ERK inhibitors, were superior to other published culture conditions, enabling the epigenetic landscape of cultured PSCs to be brought significantly closer to that of human pre-implantation ICM. As a result, conventional human PSCs are converted into ICLCs that possess all the major characteristics of human pre-implantation ICM as described in the prior art section. Notably, the inventors also found that activation of WNT / β-catenin signaling inhibits the transition from primed PSCs to ICLCs. Therefore, modulators such as the GSK inhibitor CHIR99021 (an inhibitor widely used in published naive and extended PSC culture conditions) that activate WNT / β-catenin signaling should be excluded from the culture medium, while modulators such as IWR1 and XAV939 that suppress WNT / β-catenin signaling are required. Accordingly, in preferred embodiments of the various aspects of the subject applications including the culture media, kits, compositions, and methods described herein, CHIR99021, preferably a GSK inhibitor, more preferably any agent that activates the WNT / β-catenin signal, is not included in the culture medium, kit, or composition, or used in the method of culturing cells.

[0029] Thus, provided in the present application are multiple methods and chemically defined culture media that promote robust induction of primate ICLCs. The methods described herein can be applied to a number of human and non-human primate PSC lines that are in either a primed state as verified by the presence of pluripotency surface markers such as SSEA-3, SSEA-4, TRA-1-81, and TRA-1-60, or a pre-implantation ICM-like state as verified by the expression of genes such as DNMT3L, STELLA, DPPA5, and KLF17. The 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 pre-implantation ICMs of primates. The described methods are non-transgenic and direct, provided that primate PSCs can be converted to ICLCs in one culture condition in about two weeks.

[0030] As far as we know, there is currently no appropriate method to induce 8CLC in primates in vitro. To achieve this, the inventors further optimized the formulation for inducing ICLC and found that primed human PSCs and / or ICLCs can be converted into 8CLCs by only increasing the dosages of SAH / PRC / EZH2 inhibitors and HDAC inhibitors in the medium. Therefore, a chemically defined culture medium that facilitates the induction of primate 8CLC is provided in this application. The methods described herein can be applied to a number of human and non-human primate PSC lines, either in a primed state verified by the presence of pluripotency surface markers such as SSEA-3, SSEA-4, TRA-1-81, TRA-1-60, etc., or in a pre-implantation ICM-like state verified by the expression of genes such as DNMT3L, STELLA, DPPA5, KLF17, etc. Primate PSC lines used in this application include, but are not limited to, primed primate PSCs and ICM-like PSCs. The methods described herein can also be applied to the isolation of 8CLC from primate 8C embryos. The provided primate PSCs can be converted into 8CLCs in one culture condition in about one week, so the described method is transgene-free and direct. In fact, the activation of WNT / β-catenin signaling also inhibits the formation of 8CLC. Therefore, modulators such as the GSK inhibitor CHIR99021 (an inhibitor widely used in published naive and expanded PSC culture conditions) that activate WNT / β-catenin signaling should be excluded from the culture conditions, while modulators such as IWR1 and XAV939 that suppress WNT / β-catenin signaling are required. The following is a detailed description of the present invention. It should be understood that the features described in the various examples can be combined with each other to form preferred technical solutions, which are also considered within the scope described herein.

[0031] I. Terms Unless otherwise specified, all terms used in this specification have the meanings generally understood by those skilled in the art. To facilitate understanding of the invention, some of the terms used in this specification are defined as follows. The singular forms "a", "an", and "the" as used in the specification and claims include plural references unless the context clearly dictates otherwise. For example, the term "(1) cell" includes a plurality of cells including mixtures thereof. All digital indicators such as pH, temperature, time, concentration, and molecular weight including ranges are approximate. Although not always explicitly stated, it is important to understand that the term "about" is present before all digital indicators. Also, although not always explicitly described, the reagents described in this specification are only examples, and it should also be understood that their equivalents 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, potassium, etc., as well as vitamins, glucose, a buffering system, and essential amino acids. Examples of basal media used in this application include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI1640, F10, F12, Alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (GMEM), Iscove's Modified Dulbecco Medium, Neurobasal Medium, and DMEM / F12. Those skilled in the art know how to select a basal medium suitable for culturing cells. In a preferred embodiment, the basal medium used in this application is a mixture of high-glucose DMEM / F12 and Neurobasal Medium in a ratio of 1:1 (v / v).

[0032] The term "serum-free" means the absence of any serum of any kind, 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 supplements used in a basal medium to partially or completely replace serum to support cell survival and growth. Serum replacement generally includes factors such as insulin, metal proteins, trace elements, vitamins, etc. These factors are generally not included in the basal medium but are provided by the serum commonly used in cell culture. Serum replacement includes at least one or more of the following components that support cell growth: one or more insulins and insulin substitutes, one or more metal proteins and metal protein 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, including KOSR, N2, B27, insulin-transferrin-selenium supplement (ITS), G5, etc., which are readily available to those skilled in the art, are known in the art, and since each replacement has a specific composition, the concentration of each component can be determined according to each ratio in the medium. Those skilled in the art can easily set the 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 at a certain ratio. More preferably, the serum replacement used herein is a mixture of N2 and B27 at a ratio of 1:1 (w / w).

[0033] As used herein, the term "primate" or "primate animal" refers to animals belonging to the primates, including humans and non-human primates. Non-human primates include animals of the suborder Prosimii and the suborder Simiiformes. Specific non-human primates include, but are not limited to, macaques, cynomolgus monkeys, rhesus monkeys, orangutans, and baboons.

[0034] As used herein, the term "pluripotent stem cell" (PSC) refers to pluripotent cells derived from an embryo at any point prior to gastrulation and iPSCs generated from the reprogramming of somatic cells. Depending on their source and culture method, PSCs may be in alternative states including primed PSCs, naive PSCs, extended PSCs, and extended potential stem cells (Gafni et al., 2013; Takashima et al., 2014; Theunissen et al., 2014). PSCs also have the property that, under appropriate conditions, they are capable of generating progeny of different cell types that are all derivatives of the three germ layers (endoderm, mesoderm, ectoderm), such as the ability to form teratomas in 8- to 12-week-old SCID mice, and may also generate placentas of different cell types under appropriate conditions. PSC cultures are described as "undifferentiated" when a substantial proportion of the stem cells and their derivatives within the population exhibit morphological features of undifferentiated cells and are distinguishable from differentiated cells of embryonic or adult origin. It is understood that colonies of undifferentiated cells within the population may be surrounded by differentiated adjacent cells.

[0035] This application can be carried out using various types of stem cells. Particularly suitable for use in this application are primate PSCs. Non-limiting examples are primary cultures or established lines of ESCs and iPSCs. PSCs of mammals other than primates can also be used to practice the subject application. In one or more embodiments, the primate PSCs that can be used in this application are the following: (i) cells derived from an ESC line and / or an ECC line; (ii) cells derived from an iPSC line; (iii) cells derived from the ICM of a pre-implantation blastocyst cultured in vitro; (iv) cells derived from the ICM of a post-implantation blastocyst cultured in vitro; (v) cells from an 8C-stage embryo to the morula stage cultured in vitro; selected from the group consisting of. Non-limiting PSCs include 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), etc. Established cell lines in the art can be mentioned, but are not limited thereto.

[0036] II. Culture Medium The culture medium (medium) disclosed herein is a chemically defined culture medium that can efficiently convert primate PSCs from a primed state to a pre-implantation ICM-like state and produce ICLCs within two weeks without picking colonies. Also, the culture medium described herein can convert primate PSCs from a primed state and / or a pre-implantation ICM-like state to an 8C-like state and produce 8CLCs in about one week. Therefore, this type of medium can also be referred to as a "conversion medium" in this application. In one embodiment, the culture medium described herein can also support the survival, self-renewal, and proliferation of cells in a pre-implantation ICM-like state after induction, passage, and / or regeneration. In other embodiments, the culture medium described herein can support the survival, self-renewal, and proliferation of cells in a pre-implantation ICM-like state on an extracellular matrix without the need for feeder cells or conditioned medium after passage and / or regeneration. In one embodiment, the culture medium described herein can support the passage and / or regeneration, self-renewal, and proliferation of cells in a pre-implantation ICM-like state in suspension without the need for feeder cells or conditioned medium. In some other embodiments, the culture medium described herein can support the passage and / or regeneration, self-renewal, and proliferation of cells in a pre-implantation ICM-like state on feeder cells. The chemically defined culture medium described herein may be serum-free.

[0037] The culture medium used in the present application is a basal medium having the ability to support cell growth, particularly the growth of human and non-human primate PSCs, supplemented with an SAH inhibitor, an HDAC inhibitor, and a WNT / β-catenin signaling inhibitor, and optionally contains one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, and a MAPK / ERK signaling inhibitor. The basal medium used in the present application is a mixture of Advanced DMEM / F12 and Neurobasal medium at a ratio of 1:1 (v / v). It should be understood that SAH can also be inhibited by inhibiting PRC and / or EZH2. Thus, in certain embodiments, a PRC inhibitor and / or an 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.

[0038] The presence of SAH / PRC / EZH2 inhibitors under the culture conditions described in this specification is crucial for inducing multiple regulators including STELLA, DNMT3L, and MAEL that govern the human naive pluripotency network. STELLA is a DNA methylation regulator. Ectopic overexpression of it in somatic cells can induce global DNA demethylation by inhibiting the function of UHRF1, a DNA methylation regulator. The dysfunction of UHRF1 caused by STELLA deletion will result in the accumulation of abnormal DNA methylation during oogenesis (Li et al., 2018). The induction of STELLA was dose-dependent. The inventors further clarified the functional role of STELLA and found that STELLA knockout hinders the induction of ICLC and 8CLC. During the conversion from primed PSCs to ICLCs, pre-implantation ICM markers including KLF17, DPPA5, DNMT3L, TFCP2L1, and MAEL are not induced upon STELLA deletion. During the conversion from 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, the global DNA methylation level is significantly higher in STELLA knockout cells compared to wild-type cells. Therefore, STELLA is required for controlled DNA demethylation during the conversion to ICLC and 8CLC. Overall, this application discovers that the addition of SAH / PRC / EZH2 inhibitors promotes the induction of human ICLC and 8CLC through the rewiring of histone modifications and the DNA methylation landscape. Substances that act as inhibitors of SAH / PRC / EZH2 include, but are not limited to, DZNep (CAS NO: 102052-95-9: an inhibitor that acts on SAH) and CPI-1205 (CAS NO: 1621862-70-1: an inhibitor that acts on SAH / PRC / EZH2), and can be used in the culture medium of the present application. The SAH / PRC / EZH2 inhibitor can be used alone or in combination with the culture medium of the present application, and is generally used in an ordinary amount that does not lead to cell death. For example, the final concentration of DZNep in the culture medium is 5-80 nM, preferably 5-50 nM, and the final concentration of CPI-1205 in the culture medium is 0.5-5 mM, preferably 1-3 mM. In one or more embodiments, the SAH / PRC / EZH2 inhibitor is a PRC inhibitor. Substances that can act as inhibitors of HDAC include, but are not limited to, TSA, VPA, and NaB, and can be used in the medium of the present application. The HDAC inhibitor can be used alone or in combination with the culture medium of the present application, and is generally used in each ordinary amount that does not cause cell death. For example, in the culture medium, TSA with a final concentration of 3-30 nM, preferably 3-25 nM can be used, VPA with a final concentration of 0.25-2 mM, preferably 0.5-1.5 mM can be used, and NaB with a final concentration of 0.25-2 mM, preferably 0.5-1.5 mM can be used.

[0039] Furthermore, the inventors of the present invention have discovered that when both SAH / PRC / EZH2 inhibitors and HDAC inhibitors are used at higher concentrations, 8CLC can be obtained from primed PSCs and / or ICLCs in the culture medium of the present application. Specifically, in certain embodiments, to produce 8CLC, when used individually, DZNep can be used at a concentration of 40 to 80 nM, preferably about 50 nM or the like, at a concentration of 40 nM or higher; CPI-1205 can be used at a concentration of 2 to 5 mM, preferably about 3 mM or the like, at a concentration of 2 mM or higher; TSA can be used at a concentration of 10 to 30 nM, preferably about 20 nM or the like, at a concentration of 10 nM or higher; VPA can be used at a concentration of 1.0 to 2.0 mM, preferably about 1.5 mM or the like, at a concentration of 1.0 mM or higher; NaB can be used at a concentration of 1.0 to 2.0 mM, preferably about 1.5 mM or the like, at a concentration of 1.0 mM or higher. However, when using two or more SAH / PRC / EZH2 inhibitors or two or more HDAC inhibitors, it is necessary to understand that the final concentration of each SAH / PRC / EZH2 inhibitor or each HDAC inhibitor needs to be reduced to an amount sufficient to induce 8CLC by the combined use of these SAH / PRC / EZH2 inhibitors or HDAC inhibitors. These amounts can be easily determined by those skilled in the art based on the disclosure of the present application and the conventional knowledge of the art.

[0040] Furthermore, it should be understood that excessive amounts of SAH / PRC / EZH2 inhibitors and HDAC inhibitors may cause cell death. Therefore, in order to induce iCLCs while reducing cell death as much as possible, it is conceivable to use one or both of the SAH / PRC / EZH2 inhibitor and the HDAC inhibitor at relatively low concentrations. Specifically, when used alone, DZNep at a final concentration of 5-15 nM, preferably about 10 nM; CPI-1205 at a final concentration of 0.5-3 mM, preferably about 1 mM; TSA at a final concentration of 3-10 nM, preferably 4-6 nM, more preferably about 5 nM; VPA at a final concentration of 0.25-1 mM, preferably 0.5 mM; and NaB at a final concentration of 0.25-1 mM can be used. In certain embodiments, the SAH / PRC / EZH2 inhibitor can be used at a relatively high concentration. For example, DZNep can be used at a final concentration of 5-80 nM, preferably 5-50 nM, and CPI-1205 can be used in the medium at a final concentration of 0.5-5 mM, preferably 1-3 mM. On the other hand, the HDAC inhibitor is used at a relatively low concentration. For example, TSA is used at a final concentration of 3-10 nM, preferably 4-6 nM, more preferably about 5 nM, VPA is used at a final concentration of 0.25-0.5 mM, and NaB is used at a final concentration of 0.25-0.5 mM. In certain embodiments, the SAH / PRC / EZH2 inhibitor is used at a relatively low concentration. For example, DZNep is used at a final concentration of 5-15 nM in the culture medium, and CPI-1205 is used at a final concentration of 0.5-2 mM. However, the HDAC inhibitor can be used at a relatively high concentration. For example, TSA can be used at a final concentration of 3-30 nM, preferably 3-25 nM, VPA can be used at a final concentration of 0.25-2 mM, preferably 0.5-1.5 mM, and NaB can be used at a final concentration of 0.25-2 mM. The medium can convert primate PSCs into iCLCs.

[0041] One or more WNT / β-catenin signaling inhibitors that inhibit canonical WNT signaling can be supplemented to the culture medium of the test drug. Known WNT / β-catenin signaling inhibitors, particularly those commonly used in the culture of stem cells, can be used, including but not limited to tankyrase inhibitors such as IWR1 (CAS number: 1127442-82-3) and XAV939 (CAS number: 284028-89-3). The WNT / β-catenin signaling inhibitor can be used in an amount commonly used in the culture of stem cells. The normal final concentration of the WNT / β-catenin signaling inhibitor is in the range of 2-8 μM, preferably 3-6 μM. For example, for IWR1 and XAV939, the respective final concentrations in the culture medium of the test drug are in the range of 2-8 μM, preferably 3-6 μM, more preferably about 5 μM. Two or more WNT / β-catenin signaling inhibitors can be used in combination, and the amount of each inhibitor can be reduced.

[0042] As described in Chinese Patent Application No. 200910041331.9, L-ascorbic acid has been found to improve the generation and maintenance of mouse iPSCs (similar to mouse ESCs) from somatic cells by enhancing ten-eleven translocation domain-containing histone demethylase, the content of which is incorporated herein by reference. Therefore, the inventors have hypothesized that L-ascorbic acid has a similar effect on the formation of the pre-implantation ICM-like state in primates. Through appropriate tests, the inventors have found that the expression levels of ICM-specific genes such as DNMT3L, STELLA, DPPA5, and KLF17 are strongly increased 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 about 50 μg / ml. Derivatives of L-ascorbic acid can also be used in the present application, which refers to similar compounds having a structure and antioxidant activity similar to L-ascorbic acid. The derivative is more stable or more easily absorbed by cells while maintaining the biological activity of L-ascorbic acid. Derivatives of L-ascorbic acid include, but are not limited to, L-ascorbic acid organic esters such as L-ascorbic acid phosphate and L-ascorbic acid palmitate. The amount of the derivative in the culture medium of the present application is not limited, but generally should be sufficient to produce a sufficient amount of L-ascorbic acid specified above.

[0043] One or more JAK / STAT3 signal transduction activators can be added to a medium that can support the induction of a subset of the early embryo-specific genes of the present application. Any known JAK / STAT3 signal transduction activator can be used, and those commonly used in the culture of stem cells are particularly preferred. The normal final concentration of the JAK / STAT3 signal transduction activator is in the range of 10-50 ng / mL. One of the JAK / STAT3 signal transduction activators is LIF. As used herein, the term LIF refers to leukemia inhibitory factor, which is a growth factor commonly supplemented in the culture of stem cells. LIF may be human LIF. The JAK / STAT3 activator is used in an amount commonly used in the culture of stem cells. For example, in the case of LIF, particularly human LIF, the final concentration in the medium of the test drug may be in the range of 10-50 ng / mL, preferably 10-30 ng / mL, and more preferably about 20 ng / mL. One or more inhibitors of MAPK / ERK signaling can be added to the medium and help reduce DNA methylation in cooperation with other components in the medium of the test drug. Any known MAPK / ERK inhibitor can be used, and those commonly used in the culture of stem cells are particularly preferred. There is PD0325901 (CAS number: 391210-10-9), which is one of such MAPK / ERK inhibitors. The MAPK / ERK inhibitor is used in an amount commonly used in the culture of stem cells. The normal final concentration of the MAPK / ERK inhibitor is in the range of 0.5 - 3 μM, preferably 0.5 - 1.5 μM. For example, in the case of PD0325901, the final concentration in the medium of the test drug is in the range of 0.5 - 3 μM, preferably 0.5 - 1.5 μM, more preferably about 1 μM.

[0044] In one or more preferred embodiments, the culture 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; and IWR1 or XAV939 at a final concentration of 2-8 μM, preferably 3-6 μM; L-ascorbic acid at a final concentration of 40-70 μg / ml; LIF at a final concentration of 10-30 ng / mL; and PD0325901 at a final concentration of 0.5-1.5 μM. In one or more embodiments, the culture medium described herein comprises DZNep at a final concentration of 5-80 nM, preferably 5-50 nM or CPI-1205 at a final concentration of 0.5-5 mM, preferably 0.5-3 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; and IWR1 or XAV939 at a final concentration of 2-8 μM, preferably 3-6 μM; L-ascorbic acid at a final concentration of 40-70 μg / ml; LIF at a final concentration of 10-30 ng / mL; and PD0325901 at a final concentration of 0.5-1.5 μM. More preferably, the culture medium described herein comprises 10 nM DZNep or 1 mM CPI-1205; 5 nM TSA or 0.5 mM VPA or 0.5 mM NaB; and 5 μM IWR1 or 5 μM XAV939; 50 μg / ml L-ascorbic acid; 20 ng / mL LIF; 1 μM PD0325901. The medium is preferably used for converting primate PSCs into iCLCs.

[0045] In one or more preferred embodiments, the culture medium described herein contains DZNep at a final concentration of 40 - 70 nM or CPI-1205 at a final concentration of 2 - 4 mM; 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; L-ascorbic acid at a final concentration of 40 - 70 μg / ml; IWR1 or XAV939 at a final concentration of 2 - 8 μM, preferably 3 - 6 μM; LIF at a final concentration of 10 - 30 ng / mL; and PD0325901 at a final concentration of 0.5 - 1.5 μM. More preferably, the culture medium described herein contains 50 nM DZNep or 3 mM CPI-1205; 20 nM TSA or 1 mM VPA or 1 mM NaB; 5 μM IWR1 or 5 μM XAV939; 50 μg / ml L-ascorbic acid; 20 ng / mL LIF; 1 μM PD0325901. The medium is preferably used to convert primate PSCs or ICLCs into 8CLCs. The culture medium described herein can further contain at least one or more supplements selected from the group consisting of an extracellular matrix, an activin / NODAL signaling activator, and a ROCK inhibitor.

[0046] Compared with primed human PSCs, the expression levels of NODAL (an activin / NODAL signaling activator) increase in ICLCs and 8CLCs induced by the methods described herein. This observation indicates that activin / NODAL signaling is endogenously / autonomously activated during the conversion process and self-renewal. Thus, in certain embodiments of the present application, the culture medium further contains an activin / NODAL signaling activator to accelerate the conversion 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, which are amino acid sequences 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 be used in combination. Generally, the total concentration of human activin A and human NODAL in the medium ranges from 10-25 ng / ml, about 20 ng / ml.

[0047] When converted to ICLCs and / or 8CLCs, inhibition of ROCK signaling is not required for survival after subculture as single cells. Nevertheless, supplying a low concentration of a ROCK inhibitor increases the yield of ICLCs and 8CLCs and is beneficial for scale-up of the culture. Thus, in certain embodiments of the present invention, the medium further contains a ROCK inhibitor. Known ROCK inhibitors include, but are not limited to, Y27632 (CAS number: 146986-50-7), thiazovivin (CAS number: 1226056-71-8), and hydroxyfasudil (CAS number: 105628-72-6), and can be used in the culture media described herein. The ROCK inhibitor can be used at a final concentration in the range of 0.5-2 μM, preferably about 1 μM. Two or more ROCK inhibitors can be used in combination, and the total concentration in the medium is in the range of 0.5-2 μM, preferably about 1 μM. The inventors have discovered that when PSCs are cultured in the culture medium of the present application, they can be converted and maintained in suspension culture without feeder cells, and the converted cells can self-renew and grow as colonies such as spheres. Therefore, in certain embodiments of the present application, the described methods, culture conditions, and media are feeder-free.

[0048] In other certain embodiments, the inventors have found that supplying an extracellular matrix during conversion and maintenance promotes spherical colony formation. Under these conditions, more than 90% of the PSCs can be converted into dome-shaped colonies during conversion and express ICM markers such as DNMT3L and KLF17. Therefore, in certain embodiments, an extracellular matrix is used in the medium for culturing ICLCs and 8CLCs. The extracellular matrix is a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma (Matrigel™ or 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 generally present in the culture medium described herein in an amount of 0.1% - 0.5% (v / v). Optionally, combinations of different types of extracellular matrices can be used, and the total amount must also be in the range of 0.1% - 0.5% (v / v) in the medium. Preferably, the extracellular matrix is generally present in the culture medium described herein in an amount of about 0.2% (v / v). Thus, in one or more preferred embodiments, the culture medium described herein comprises (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 3-30 nM, or VPA at a final concentration of 0.25-3 mM or NaB at a final concentration of 0.25-3 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; or DZNep at a final concentration of 5-80 nM, preferably 5-50 nM, or at a final concentration of 0.5-5 mM, preferably 0.5-3 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, IWR1 or XAV939 at a final concentration of 2-8 μM, preferably 3-6 μM; (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 further comprising the following: (1) Activin A or Nodal at a final concentration of 10-25 ng / ml; Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2 μM; and an extracellular matrix in an amount of 0.1%-0.5% (v / v); or (2) the Activin A or Nodal at a final concentration of 10-25 ng / ml; the Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2 μM; or (3) the Activin A or Nodal at a final concentration of 10-25 ng / ml; and an extracellular matrix in an amount of 0.1%-0.5% (v / v); or (4) the Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2 μM; and an extracellular matrix in an amount of 0.1%-0.5% (v / v); or (5) the Activin A or Nodal at a final concentration of 10-25 ng / ml; or the Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2 μM; or the extracellular matrix at 0.1%-0.5% (v / v); is supplemented. The medium is preferably used for converting primate PSCs into iCLCs. More preferably, the culture 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 PD0325901; and 5 μM IWR1 or 5 μM XAV939; and further, the following: (1) 20 ng / mL activin A or NODAL, 1 μM Y27632, thiazovivin or hydroxyfasudil, 0.2% (v / v) extracellular matrix; or (2) 20 ng / mL activin A or NODAL, 1 μM Y27632, thiazovivin or hydroxyfasudil; or (3) activin A or NODAL at a final concentration of 10 - 25 ng / ml; and extracellular matrix in an amount of 0.1% - 0.5% (v / v); or (4) Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 - 2 μM; and extracellular matrix in an amount of 0.1% - 0.5% (v / v); or (5) activin A or NODAL at a final concentration of 10 - 25 ng / ml; Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 - 2 μM; or extracellular matrix in an amount of 0.1% - 0.5% (v / v); is supplemented. The medium is preferably used for converting primate PSCs into iCLCs.

[0049] In one or more preferred embodiments, the culture medium described herein contains DZNep at a final concentration of 40 - 70 nM or CPI-1205 at a final concentration of 2 - 4 mM; 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; 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; IWR1 or XAV939 at a final concentration of 2 - 8 μM, preferably 3 - 6 μM each; and further, the following: (1) Activin A or NODAL at a final concentration of 10 - 25 ng / ml; Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 - 2 μM; and an extracellular matrix in an amount of 0.1% - 0.5% (v / v); or (2) Activin A or NODAL at a final concentration of 10 - 25 ng / ml; Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 - 2 μM; or (3) Activin A or NODAL at a final concentration of 10 - 25 ng / ml; and an extracellular matrix in an amount of 0.1% - 0.5% (v / v); or (4) Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 - 2 μM; and an extracellular matrix in an amount of 0.1% - 0.5% (v / v); or (5) The above-mentioned Activin A or NODAL at a final concentration of 10 - 25 ng / ml; or the above-mentioned Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 - 2 μM; or the above-mentioned extracellular matrix in an amount of 0.1% - 0.5% (v / v); is supplemented. The medium is preferably used for converting primate PSCs or ICLCs into 8CLCs. More preferably, the culture 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 PD0325901; and 5 μM IWR1 or 5 μM XAV939; and further contains the following: (1) 20 ng / mL of activin A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v / v) of extracellular matrix; or (2) 20 ng / mL of activin A or NODAL, and 1 μM of Y27632, thiazovivin or hydroxyfasudil; or (3) 20 ng / mL of activin A or NODAL, and 0.2% (v / v) of extracellular matrix; or (4) 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v / v) of 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 extracellular matrix is supplemented. The medium is preferably used to convert primate PSCs or ICLCs to 8CLCs.

[0050] In addition to the above components, supplements that can be added to the culture medium of the present application include other supplements commonly used in media for culturing stem cells, such as N2 and / or B27; alternative carbon sources such as pyruvate, such as sodium pyruvate; non-essential amino acids; L-glutamine or its alternatives, such as Glutamax (trademark) supplements containing L-alanyl-L-glutamine dipeptide in 0.85% NaCl; and serum replacements such as antibiotics such as penicillin, streptomycin, or a mixture of penicillin and streptomycin, but are not limited thereto. These supplements can be used in amounts commonly used in cell culture, particularly in culturing stem cells.

[0051] III. Kits and Compositions Also provided is a kit containing the culture medium described herein, or all or part of the components of the culture medium described herein for preparing the medium.

[0052] In certain embodiments, the kits described herein include a ready-to-use medium, the components of which are described in any of the above-described embodiments of the medium. In certain embodiments, the kits described herein include a conversion medium for converting primate PSCs described in any of the embodiments described herein to iCLCs and / or a conversion medium for converting primate PSCs or iCLCs described in any of the embodiments described herein to 8CLCs.

[0053] In other embodiments, the kits described herein include at least an SAH / PRC / EZH2 inhibitor, an HDAC inhibitor, and a WNT / β-catenin signaling inhibitor, which may be individually packaged or provided as a mixture in one container. The kit may further include one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, and a MAPK / ERK signaling inhibitor. When these components are present, they may be individually packaged or provided as a mixture of any combination of the components. Preferably, the kit may include an SAH / PRC / EZH2 inhibitor, an HDAC inhibitor, L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a WNT / β-catenin signaling inhibitor. Additionally, the kit may sometimes include one or more components selected from the group consisting of an activin / NODAL signaling activator and a ROCK inhibitor. Matrigel (trademark) 、Conventional extracellular matrices such as Geltrex (trademark), ECM (trademark), etc. may be included in the kit. Preferably, the kit further includes one or more basal media such as DMEM / F12 (1:1), Neurobasal medium, etc. described herein, serum replacements such as N2 and B27, alternative carbon sources such as sodium pyruvate, non-essential amino acids, L-glutamine or its alternatives (such as Glutamax (trademark) supplement containing L-alanyl-L-glutamine dipeptide in 0.85% NaCl), antibiotics, and other components known to be used for culturing stem cells. The amounts of all these components should be sufficient to prepare the medium for the application in question. The kit may include instructions, and the kit may include text regarding the preparation and use of the medium.

[0054] In certain embodiments, compositions are also provided that include an SAH / PRC / EZH2 inhibitor, an HDAC inhibitor, and a WNT / β-catenin signaling inhibitor. The composition can further include one or more components selected from the group consisting of L-ascorbic acid, a JAK / STAT3 signaling activator, and a MAPK / ERK signaling inhibitor. Additionally, the composition can also include one or more components selected from the group consisting of an activin / NODAL signaling activator and a ROCK inhibitor. In preferred embodiments, the composition includes an SAH / PRC / EZH2 inhibitor, an HDAC inhibitor, L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, and a WNT / β-catenin signaling inhibitor, and optionally an activin / NODAL signaling activator and optionally a ROCK inhibitor. It should be understood that when each of the above components is present in the composition, each amount should be present in an acceptable amount in a culture medium containing a composition within the respective ranges of each of the media specified in any of the embodiments described herein. More preferably, by using the composition, a medium of any embodiment described in this application can be prepared.

[0055] In one or more preferred embodiments, the compositions described herein are compositions comprising DZNep or CPI-1205, and TSA or VPA or NaB, and optionally L-ascorbic acid, optionally LIF, optionally PD0325901 and optionally IWR1 or XAV939, preferably each of the components is present in an amount such that the medium containing the composition is as follows: 5 to 15 nM, preferably 10 nM of DZNep, or 0.5 to 2 mM, preferably 1 mM of CPI-1205, 2 to 8 nM, preferably 5 nM of TSA, or 0.25 to 1 mM, preferably 0.5 mM of VPA (VPA), or 0.25 to 1 mM, preferably 0.5 mM of NaB; 2 to 8 μM, preferably 3 to 6 μM, preferably 5 μM of IWR1 or XAV939; optionally 40 to 90 μg / mL, preferably 50 μg / mL of L-ascorbic acid, optionally 10 to 30 ng / mL, preferably 20 ng / mL of LIF, optionally 0.5 to 1.5 μM, preferably 1 μM of PD0325901. The composition can further comprise activin A or NODAL, and / or Y27632, thiazovivin or hydroxyfasudil, and / or extracellular matrix, and each component is present in an amount such that the culture 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.

[0056] 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, optionally PD0325901 and IWR1 or XAV939: preferably each of the components is present in an amount such that the medium containing the composition is as follows: 40 - 70 nM, preferably 50 nM of DZNep, or 2 - 4 mM, preferably 3 mM of CPI - 1205; 10 - 30 nM, preferably 20 nM of TSA, or 0.5 - 1.5 mM, preferably 1 mM of VPA, or 0.5 - 1.5 mM, preferably 1 mM of NaB; 2 - 8 μM, preferably 3 - 6 μM, preferably 5 μM of IWR1 or XAV939; optionally, 40 - 90 μg / mL, preferably 50 μg / mL of L - ascorbic acid; optionally, 10 - 30 ng / mL, preferably 20 ng / mL of LIF, and optionally 0.5 - 1.5 μM, preferably 1 μM of PD0325901, present in an amount that can be included. The composition can further include activin A or NODAL, and / or Y27632, thiazovivin or hydroxyfasudil, and / or extracellular matrix, and each component is such that the culture medium containing the composition can contain 10 - 25 ng / mL, preferably 20 ng / mL of activin A or NODAL, and / or 0.5 - 2 μM, preferably 1 μM of Y27632, thiazovivin or hydroxyfasudil, and / or 0.1% - 0.5% (v / v) of extracellular matrix, in an amount that can be included. In certain embodiments, the kit can include the above - described composition.

[0057] The kits described herein can further include a medium for the maintenance of 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 8 - 15 μM of Y27632 or without Y27632. Reagents commonly used for culturing stem cells can also be included in the kit. Such reagents include, but are not limited to, PBS, EDTA solution, and / or TrypLE: 0.5 mM EDTA (1:1). Feeder cells and / or extracellular matrix can also be provided in the kit.

[0058] IV. Methods and Uses The culture medium described in this specification can be used for reprogramming somatic cells of primates into iCLCs, converting PSCs of primates into iCLCs, and converting PSCs or iCLCs of primates into 8CLCs.

[0059] Accordingly, one aspect described in this specification discloses a method for reprogramming somatic cells of primates into iCLCs, which includes culturing somatic cells in a conversion medium containing, regardless of the presence or absence of an extracellular matrix, an SAH / PRC / EZH2 inhibitor, an HDAC inhibitor, L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, a WNT / β-catenin signaling inhibitor, optionally an activin / NODAL signaling activator, and optionally a ROCK inhibitor. The obtained iCLCs can be used in a method for converting iCLCs into 8CLCs. Preferably, the conversion culture is as described in any of the embodiments described in this specification.

[0060] Another aspect described in this specification discloses a method for converting PSCs of primates into iCLCs, or a method for converting PSCs or iCLCs of primates into 8CLCs, which includes culturing PSCs of primates in a conversion medium containing, regardless of the presence or absence of an extracellular matrix, an SAH / PRC / EZH2 inhibitor, an HDAC inhibitor, L-ascorbic acid, a JAK / STAT3 signaling activator, a MAPK / ERK signaling inhibitor, a WNT / β-catenin signaling inhibitor, optionally an activin / NODAL signaling activator, and optionally a ROCK inhibitor. In a preferred embodiment, the conversion medium is the medium specified in any of the above embodiments. In one or more preferred embodiments, the method is a method for converting PSCs of primates into iCLCs, and the conversion medium is the medium specified in any of the above embodiments with relatively low concentrations of an SAH / PRC / EZH2 inhibitor and an HDAC inhibitor. In some other preferred embodiments, the method is a method for converting a primate PSC or ICLC into an 8CLC, and the conversion medium is the medium specified in any of the above embodiments with a relatively high concentration of an SAH / PRC / EZH2 inhibitor and an HDAC inhibitor.

[0061] Conventional conditions for culturing stem cells can be used to convert PSCs into ICLCs or 8CLCs. For example, a single primed PSC can be seeded in a conventional medium such as mTeSR1 or E8, and optionally supplemented with a ROCK inhibitor such as Y27632 at 5 - 15 μM. After a culture time, for example, after 24 hours, the medium is switched to the culture medium of the present application, and cell culture is continued until the desired ICLC or 8CLC is generated. During the culture, the medium is replaced as needed, preferably daily. For subculture, after dissociating the cells into single cells in the usual way, they can be reseeded and cultured again in the culture medium of the present application until ICLCs or 8CLCs are formed. The cells are preferably subcultured as single cells every 3 - 4 days at a splitting ratio of 1:4 - 1:8, preferably 1:6 - 1:8. Generally, it takes about 2 weeks to convert primed PSCs into ICLCs and about 1 week to convert primed PSCs into 8CLCs. After culturing ICLCs in a culture medium containing a relatively high concentration of an SAH / PRC / EZH2 inhibitor and an HDAC inhibitor, they are converted into 8CLCs in 3 - 5 days. The ICLCs used for conversion into 8CLCs may be ICLCs obtained by culturing primate PSCs by any of the methods described herein, or may be known ICLCs or ICLCs prepared from any of the methods known in the art. Generally, the cells can be cultured at 37°C under normal oxygen conditions (5% CO2) or hypoxic conditions (5% CO2 and 5% O2). There is no particular limitation on the culture time, which 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 the common general knowledge of those skilled in the art and the actual manufacturing conditions.

[0062] In certain embodiments described herein, the cells are grown under one or more conditions selected from the group consisting of: (i) on feeder cells; (ii) on a feeder-free extracellular matrix; (iii) in suspension without feeder cells; (iv) at a temperature of about 37°C in a hypoxic or normoxic state; (v) passaged and grown as single cells every 3 to 4 days at a split ratio of 1:4 to 1:8; and (vi) changing the culture medium daily. and can be cultured under one or more conditions selected from the group consisting of.

[0063] In certain embodiments, to convert primate PSCs to ICLCs, a single primate PSC is seeded on a feeder with mTeSR1 or E8 medium supplemented with 5 μM to 15 μM of a ROCK inhibitor (such as Y27632), cultured for a period such as 24 hours, and then the mTeSR1 or E8 medium is switched to the conversion medium of the present application supplemented with a relatively low concentration of SAH / PRC / EZH2 inhibitor and HDAC inhibitor, and the cells are cultured at a temperature of about 37°C in a hypoxic or normoxic state while replacing the medium daily. During the 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 certain embodiments, a single primed primate PSC is cultured as described above, except that the cells are seeded on a DMEM-F12 coated plate instead of feeder cells in about 1% (v / v) of an extracellular matrix such as Geltrex (trademark).

[0064] In certain embodiments, to convert primate PSCs to ICLCs, a single primed primate PSC is seeded on a plate with mTeSR1 or E8 medium supplemented with 5 μM to 15 μM of a ROCK inhibitor (such as Y27632), cultured for a period such as 24 hours, and then the mTeSR1 or E8 medium is switched to the conversion medium of the present application containing a relatively low concentration of SAH / PRC / EZH2 inhibitor and HDAC inhibitor, and the cells are cultured under hypoxic conditions. After small spheres are formed, the spheres are transferred to a flask for suspension culture and the medium is replaced daily; herein, the cells are passaged as single cells at a split ratio of 1:4 to 1:8 every 4 to 5 days until ICLCs are obtained.

[0065] In certain embodiments, for the conversion from primate PSCs to 8CLCs, single priming PSCs are seeded on feeders for a period such as 24 hours using mTeSR1 or E8 medium supplemented with 5 - 15 μM of a ROCK inhibitor (such as Y27632), and then the medium is switched to the conversion medium of the present application with relatively high concentrations of SAH / PRC / EZH2 inhibitor and HDAC inhibitor, and the cells are cultured in a hypoxic or normoxic state while replacing the medium daily. Herein, the cells are passaged every 3 - 4 days as single cells with a split ratio of 1:4 - 1:8. In certain embodiments, for the conversion from iCLCs to 8CLCs, single cells are isolated from iCLCs and seeded on feeders using the conversion medium of the present application with relatively low concentrations of SAH / PRC / EZH2 inhibitor and HDAC inhibitor for a period such as 24 hours, and then the medium is switched to the conversion medium of the present application with relatively high concentrations of SAH / PRC / EZH2 inhibitor and HDAC inhibitor, and the cells are cultured for 3 - 5 days without passaging while replacing the medium daily. In certain embodiments, for the conversion from iCLCs to 8CLCs, single cells are isolated from iCLCs and cultured in suspension for a certain period in the conversion medium of the present application with relatively low concentrations of SAH / PRC / EZH2 inhibitor and HDAC inhibitor for suspension culture, and 5 - 15 μM of a ROCK inhibitor (such as Y27632) is supplemented to the conversion medium. After small aggregates are formed, the medium is changed to the conversion medium of the present application with relatively high concentrations of SAH / PRC / EZH2 inhibitor and HDAC inhibitor without supplementing an extra ROCK inhibitor (such as Y27632) for conversion for several days without passaging, and the medium is replaced daily. Also included in the present application is the use of the conversion medium described in any of the embodiments described herein when reprogramming primate somatic cells to iCLCs, when converting primate PSCs to iCLCs, or when converting primate PSCs or iCLCs to 8CLCs, or in the manufacture of a medium or kit for reprogramming primate somatic cells to iCLCs, or when converting primate PSCs to iCLCs, or when converting primate PSCs or iCLCs to 8CLCs. In certain embodiments, the subject application also includes the use of SAH / PRC / EZH2 inhibitors, HDAC inhibitors, and WNT / β-catenin signaling inhibitors in the manufacture of a medium or kit for reprogramming a primate somatic cell into an iPSC, or for converting a primate PSC into an iCLC, or for converting a primate PSC or iCLC into an 8CLC. 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, an activin / NODAL signaling activator, a ROCK inhibitor (such as Y27632), and an extracellular matrix.

[0066] In some other embodiments, the method of converting primate PSCs to iCLCs and converting primate PSCs or iCLCs to 8CLCs may include a genetic engineering step of reducing the activities of SAH, PRC, and / or EZH2 of PSCs and / or reducing the activity of HDAC in the cells by knocking down and / or knocking out one or more relevant genes in the cells before culturing the primate PSCs in the culture medium of the present application. To reduce the activities of SAH, PRC, and / or EZH2 of PSCs, it may be possible to knock down the expression of any of the SAH, PRC, and EZH2 regulators by means such as siRNA technology, or to knock out any gene, such as by CRISPR / Cas9 technology. Similarly, the expression of the HDAC regulator can also be knocked down or knocked out by the same means as described above. After knockdown or knockout, the resulting cells can be cultured in any medium of the test drug according to the above method. In one embodiment, when the activities of SAH, PRC, and / or EZH2 of PSCs are reduced, the medium used for culturing the recombinant primate PSCs may or may not contain an SAH / PRC / EZH2 inhibitor. Similarly, when the activity of HDAC of PSCs is reduced, the medium may or may not contain an HDAC inhibitor. When both the activities of SAH, PRC, and / or EZH2 and the activity of HDAC are reduced, the medium may or may not contain both an SAH / PRC / EZH2 inhibitor and an HDAC inhibitor. Thus, in certain embodiments, the present application further provides a culture medium that does not contain either a SAH / PRC / EZH2 inhibitor or an HDAC inhibitor, or contains either a SAH / PRC / EZH2 inhibitor or an HDAC inhibitor, and the other components and amounts are the same as any of the above embodiments regarding the medium in Part II. In certain embodiments, the medium can contain reagents for liposomal transfection. For example, in the above method, primate PSCs are cultured in a culture medium containing, in addition to the other components described in the medium described in Part II, a vector for shRNA expression such as any of SAH, PRC, and EZH2 regulators, and a reagent for liposomal transfection for introducing the vector into PSCs for genetic engineering, and the medium may or may not contain a SAH / PRC / EZH2 inhibitor.

[0067] V. Biological functions of STELLA STELLA is a regulator of DNA methylation. Ectopic overexpression of it in somatic cells can induce global DNA demethylation by inhibiting the function of UHRF1, a DNA methylation regulator. Dysfunction of UHRF1 caused by STELLA deletion will lead to the accumulation of abnormal DNA methylation during oogenesis (Li et al., 2018). Also, STELLA has been reported to maintain maternal imprinting by preventing the conversion of 5mC to 5hmC via Tet3 at specific loci (Nakamura et al., 2012). In the present application, the inventors first discovered that STELLA knockout hinders the induction of ICLCs and 8CLCs. That is, STELLA is required for controlled DNA demethylation in the conversion process. Also, the inventors found that supplementation with a SAH / PRC / EZH2 inhibitor promotes the induction of ICLCs and 8CLCs through rewiring of histone modifications and the DNA methylation landscape. Thus, in certain embodiments, the present application further includes the use of an agent that can promote the expression of STELLA or improve the activity of STELLA in the manufacture of an agent, medium, or kit for promoting the conversion of primate PSCs to iCLCs, or the use of an agent that can promote the expression of STELLA or improve 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, including culturing primate PSCs in the presence of an effective amount of an agent that can promote the expression of STELLA or improve the activity of STELLA. The effective amount of the agent can be readily determined by those skilled in the art based on the disclosure of the subject application and the teachings of the prior art.

[0068] In some preferred embodiments, the agent that can promote the expression of STELLA or improve the activity of STELLA is an inhibitor of SAH / PRC / EZH2, including but not limited to DZNep and CPI-1205. The SAH / PRC / EZH2 inhibitors can be used alone or in combination, and are generally used at normal doses that do not lead to cell death. For example, DZNep can be used in a medium with a final concentration of 5-80 nM, preferably 5-50 nM, and CPI-1205 can be used in a medium with a final concentration of 0.5-5 mM, preferably 1-3 mM. In one or more embodiments, the SAH / PRC / EZH2 inhibitor is generally known as a PRC inhibitor.

[0069] In some more preferred embodiments, a method for promoting the conversion of primate PSCs to ICLCs includes culturing the primate PSCs in the presence of 5 - 15 nM, preferably 10 nM, of DZNep, or 0.5 - 2 mM, preferably 1 mM, of CPI-1205. In some other preferred embodiments, a method for promoting the conversion of primate PSCs or ICLCs to 8CLCs includes culturing the primate PSCs in the presence of 40 - 70 nM, preferably 50 nM, of DZNep, or 2 - 4 mM, preferably 3 mM, of CPI-1205. Also included in this application are SAH / PRC / EZH2 inhibitors for use in methods for promoting the conversion of primate PSCs to ICLCs, or the conversion of primate PSCs or ICLCs to 8CLCs.

[0070] VI. Cells This application also provides isolated primate ICLCs. The ICLCs described herein have a transcriptome similar to that of human pre-implantation ICMs, have a transposon profile similar to that of human pre-implantation ICMs, have a DNA methylome similar to that of human pre-implantation ICMs, have a chromatin landscape similar to that of human pre-implantation ICMs, and have a metabolic state similar to that of human pre-implantation ICMs. As used herein, the term "similar (proximate)" is intended to mean "substantially identical" or "without substantial difference". One of ordinary skill in the art, based on general knowledge in the art, can recognize that the cells of the subject application, including cells from the ICLCs or 8CLCs described herein, are substantially identical to natural ICM cells or 8C embryo cells, even if there are some minor differences. Preferably, the ICLCs described herein exhibit significantly high expression levels of pre-implantation ICM markers, including KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, MAEL, and REX1. More preferably, the expression level of at least one of the above pre-implantation ICM markers in the ICLCs described herein is at least 10-fold higher than the expression level of the corresponding pre-implantation ICM marker in primed human PSCs. Even more preferably, the expression levels of all of the above pre-implantation ICM markers in the ICLCs described herein are at least 10-fold higher than the expression levels of the corresponding pre-implantation ICM markers in primed human PSCs.

[0071] Even more preferably, the ICLCs described herein are as follows: 1) Can self-renew in culture and maintain pluripotency; 2) Maintain genomic stability in culture according to the karyotype; 3) Can generate cells of the three germ layers; 4) Can generate primordial germ cell-like cells; 5) Can integrate into mouse embryos and contribute to the embryo and extra-embryonic tissues; 6) Can migrate into extra-embryonic cell dynamics in vitro; and 7) Can form blastocyst-like structures in vitro; are 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 obtained by any of the methods described herein, specifically ICLCs. In addition, the present application provides isolated 8CLCs that express 8C state-specific markers including ZSCAN4, TPRX1, ZIM3, ZSCAN5B, ZNF280A, and ARGFX at substantially higher levels than cells in the pre-implantation ICM-like state or primed state. Preferably, at least one specific marker exhibits an expression level that is at least 5-fold 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 at least 5-fold 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 those of human 8C-stage embryos. More preferably, the 8CLCs described herein have the following: 1) Maintain genomic stability in culture according to karyotype; 2) Can generate cells of the three germ layers; 3) Can generate primordial germ cell-like cells; 4) Integrate into mouse embryos and can contribute to embryos and extra-embryonic tissues; 5) Can transition to extra-embryonic cell dynamics in vitro; and 6) Can form blastocyst-like structures in vitro; are 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 the present application. Cell cultures containing the cells described herein, particularly ICLCs and / or 8CLCs described herein, are also contemplated in the present application. Any of the media described herein can also be included in cell cultures. 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 various ways. Within the scope of the spirit described herein, various modifications and alterations can be made. The techniques referred to in this specification are, unless otherwise specified, conventional techniques in various fields such as molecular biology, cell biology, biochemistry, etc., and are well-known to those skilled in the art.

Example

[0072] Materials and Methods 4CL Basal Medium Neurobasal Medium (Gibco) and Advanced DMEM / F12 (Gibco) were mixed at a ratio of 1:1 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 (trademark) (1X, Gibco), penicillin-streptomycin (1X, Gibco).

[0073] 4CL Supplement The 4CL basal medium was supplemented with the following: SAH / PRC / EZH2 inhibitor (10 nM DZNep), HDAC inhibitor (5 nM TSA), WNT / β-catenin signaling inhibitor (5 μM IWR1), L-ascorbic acid (50 μg / ml), JAK / STAT3 activator (20 ng / ml human LIF), MAPK / ERK inhibitor (1 μM PD0325901), activin A / NODAL activator (20 ng / ml human activin A), extracellular matrix (0.2% (v / v) Geltrex (trademark)), and optionally, a ROCK inhibitor (1 μM Y27632) to obtain 4CL Medium 1. The catalogs of these reagents and their alternatives are shown in Table 1.

[0074]

Table 1

[0075] Experimental results Figure 1(A) is a schematic diagram of ICLC induction from primed human PSCs. Figure 1(B) is a representative image of the colony morphology of primed human PSCs (left panel) and ICLCs (right panel) under phase-contrast microscopy. Flat primed human PSCs become dome-shaped ICLCs after conversion. Figure 1(C) shows RT-qPCR and immunostaining data indicating that in ICLCs, a panel of pre-implantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, REX1 is significantly induced compared to primed human PSC cells. Figure 1(D) shows that ICLCs induced under normoxic or hypoxic conditions have similar pre-implantation ICM marker gene expression levels. To characterize the gene expression profile of ICLCs at the single-cell level, the inventors applied single-cell RNA-Seq (scRNA-seq) to cells at the priming 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) is a 2D scatter plot of UMAP analysis for cells at different time points, together with publicly available scRNA-seq data of in vivo human embryos at days 3, 4, 5, 6, and 7 (E3 / 4 / 5 / 6 / 7) from embryo (E-MTAB-3929). This shows that conventional human PSCs in 4CL medium gradually acquire a gene expression profile similar to that of human embryo day 5 cells, which corresponds to the early stage of pre-implantation blastocysts. Figure 2(B) is a heatmap using bulk RNA-seq of primed human PSCs, ICLCs, and human pre-implantation ICM cells (GSE101571), indicating that the expression levels of known ICM markers in ICLCs are upregulated to the level of ICM cells. Subgroups of TEs such as SVA_D are known to be specifically activated from the 8-cell stage to the pre-implantation ICM stage of the human embryo. To investigate activated TEs in ICLCs, the inventors extracted TE profiles from the scRNA-seq data described in Figure 2(A).Figure 3(A) is a 2D scatter plot of the UMAP analysis of the TE expression profiles in cells at the priming stage (Primed-D0), and cells at days 1, 2, 3, 5, 8, and 12 (D1 / 2 / 3 / 5 / 8) after culturing in 4CL medium 1 with human embryonic cells (from E-MTAB-3929) on 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 on days 4 (morula stage) and 5 (blastocyst stage) of the embryo. Figure 3(B) further shows that the expression levels of multiple TE subgroups of ICLCs are induced to those of pre-implantation human embryos (from GSE101571). Figure 4 shows that ICLCs maintain a normal karyotype even after long-term culture (tested in passage 15 in 4CL medium 1 for about 60 days). 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 pre-implantation ICM-like gene expression profile and maintain a stable genome even after extended culture. In the epigenetic landscape, the genome of the pre-implantation ICM is hypomethylated and the chromatin is more open compared to the post-implantation ICM. To determine the effect of 4CL medium 1 on the DNA methylation status, the inventors performed reduced representation bisulfite sequencing (RRBS) for ICLCs and primed human PSCs. Figure 5 is a box plot showing CpG DNA methylation across the entire genome (upper left panel). In ICLCs, it was significantly reduced compared to primed human PSCs, but the methylation status of the TSS was slightly different (upper right panel). Notably, the global decrease in DNA methylation levels is prevented by knocking out STELLA (lower left panel). Figure 6 shows that the imprinting status of ICLCs is maintained similar to that of the ICM. To determine the chromatin accessibility of ICLCs, the inventors 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 pre-implantation ICM-specific genes such as KLF17, STELLA, DPPA5, and CD70 are largely open in ICLCs. Figure 7(C) shows that shared pluripotency genes such as POU5F1 maintain a similar open chromatin state between primed human PSCs and ICLCs while post-implantation-specific genes such as THY1 (Figure 7D) become closer. The time-course bulk ATAC-seq in Figure 8 shows that chromatin accessibility changes stepwise during the conversion from primed human PSCs to ICLCs. Pre-implantation-specific gene loci (TFAP2C, KLF5, TFE3, etc.) that are close in primed human PSCs gradually open during the conversion, while post-implantation-specific gene loci (ZIC3, FOXA2, etc.) that are open in primed human PSCs gradually close (Figure 8A). According to motif enrichment analysis, regions close to the open regions are likely to bind to pre-implantation ICM-specific transcription factors such as DUX, TFAP2C, and KLF5 (upper panel of Figure 8B), while regions close to the closed regions are likely to bind to post-implantation-related transcription factors such as SOX3, NKX6.1, and NEUROD1 (lower panel of Figure 8). Figure 8(C) shows the correlation between gene expression and chromatin accessibility. These results indicate that 4CL medium 1 has successfully rewired the epigenetic landscape towards the pre-implantation ICM.

[0076] The inventors further investigated the metabolic state of ICLCs induced with 4CL medium 1. Pre-implantation ICM mainly uses oxidative phosphorylation (OxPhos) as an energy source, and post-implantation mainly depends 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.

[0077] To determine the differentiation potential of ICLCs, the inventors performed a teratoma formation assay 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 1 million ICLCs. They showed the presence of cells from all three germ layers: mesoderm (left panel), endoderm (central panel), and ectoderm (right panel). It is known in the art that human ICLCs can generate trophoblast ectoderm. Therefore, the inventors induced trophoblast stem cells (TSCs) from ICLCs using a previously published protocol. As shown in Figure 11(A), multiple TSC markers such as GATA3, CGA, ELF5, TP63, KRT18, KRT8, PSG6, and CCR7 were significantly upregulated in TSCs compared to undifferentiated ICLCs. Figure 11(B) is an immunofluorescence image 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 human placental choriocarcinoma cell lines JEG3 and BeWo compared to ICLCs and placental cells (EGFR and HLAG). Figure 11(D) shows the DNA methylation status in the ELF5 promoter region of ICLC-derived TSCs and other cell types. These results indicate that ICLCs acquire developmental potential equivalent to that of human pre-implantation embryos.

[0078] Due to ethical issues, the ability of ICLCs to generate cannot be tested using human embryos. Therefore, the inventors performed interspecies chimera experiments by aggregating ICLCs with mouse 8C-stage blastomeres. When human ICLCs were examined after 24 hours of in vitro culture, it was found that they were successfully incorporated into most mouse embryos and formed chimeric blastocysts (Figs. 12A-C). At this stage, human ICLCs were located in both the ICM and TE parts of the chimeric blastocysts. Fig. 12(A) is an overview of the chimera assay using DsRed-labeled human PSCs and DsRed-labeled ICLCs at the blastocyst stage. Fig. 12(B) is a representative image showing the phase contrast (left) or red fluorescence channel (right) of blastocysts generated from mouse 8C blastomeres aggregated with DsRed-labeled human PSCs (top) or DsRed-labeled ICLCs (bottom). Fig. 12(C) is the immunofluorescence of chimeric blastocysts stained with anti-OCT4 (ICM, green) and anti-CDX2 (TE, gray), where the red signal is from integrated DsRed-labeled ICLCs and DAPI (blue) is used as nuclear counterstaining. When these chimeric blastocysts were transplanted into the uterus of pseudopregnant mice and allowed to develop until embryonic day 10.5 (E10.5), as shown in the microscopic images of Fig. 13, human cells developed with the mouse embryos and could contribute to various tissues including embryonic tissues, extraembryonic placenta, and yolk sac. Fig. 13(A) is a representative image showing the phase contrast (top) or red fluorescence channel (bottom) of an E10.5 chimeric embryo (left), placenta (middle), or yolk sac (right). Fig. 13(B) is an immunofluorescence image showing that hN (green) differentiated human cells into GATA6 (red)-positive endodermal tissue. Fig. 13(C) is an immunofluorescence image showing that DsRed-labeled human cells (red) differentiated into placental tissue indicated by GATA3 (green). Collectively, these results indicate that ICLCs can be strongly integrated into mouse blastocysts and contribute to mouse E10.5 embryos and extraembryonic tissues in vivo.

[0079] Recently, blastoid (embryoid-like structure) was generated from extended pluripotent stem cells (EPSCs) of mouse (Non-Patent Document 11). However, this model using human cells has not been fully studied yet. The inventors observed that when ICLCs were applied to a medium rich in extracellular matrix, structures such as blastocysts developed from ICLCs alone, but not from primed human PSCs (Fig. 14A-B). Fig. 14(A) shows the morphology of blastocysts developed from ICLCs in REM medium. Fig. 14(B) is an immunofluorescence image 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 WNT / β-catenin signaling pathway) are widely used in published naive or extended PSC media. However, it inhibits the formation of ICLCs containing it (Fig. 36). Fig. 36(A) shows that the colony morphology is flatter, indicating a state similar to that of primed human PSCs. Figs. 36(B-D) show that when the GSK inhibitor is added to 4CL (4CL+CHIR), the activation of human pre-implantation ICM-enhancing genes is blocked. Consistently, when the WNT / β-catenin signaling inhibitor IWR1 (suppression of WNT / β-catenin signaling pathway) is removed from 4CL (4CL-IWR1), ICLC formation is inhibited (Fig. 37). These results indicate that the activation of the WNT / β-catenin signaling pathway by GSK inhibitors is harmful to ICLC formation.

Example

[0080] Materials and Methods 4CL Basal Medium Same as Example 1. 4CL Supplement Same as Example 1. Cells 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 gifted from the laboratory of Ulrich Martin), DiPS 1016SevA (purchased from the Harvard Stem Cell Institute, male), STiPS O-XX1 (purchased from the Harvard Stem Cell Institute, female), 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 pre-implantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced in ICLCs converted from multiple primed human PSC lines. This indicates that 4CL medium 1 can be generally applied to human PSCs.

Example

[0081] Materials and methods 4CL basal medium The same as in Example 1. 4CL supplement The same as in Example 1. Cells H9 human ESC line. Procedure The same procedure as in Example 1 was used, except that the cells were seeded in 1% (v / v) Geltrex™ -coated plates of DMEM-F12 (cat#) instead of feeder cells. Experimental results Figure 16 is a bar graph of RT-qPCR data showing that in ICLCs converted on Geltrex™ -coated plates using 4CL medium 1, pre-implantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced, similar to ICLCs on feeder cells. This indicates that 4CL medium 1 is effective even without feeder cells.

Example

[0082] Materials and Methods 4CL basal medium The same as in Example 1 4CL supplement The same as in Example 1 Cells H9 human ESC line Procedure The primed human PSCs were cultured in the same procedure as in Example 1. One day before the start of conversion, the 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 in 3 days. Then, the spheres were lifted and transferred to a flask (Greiner Bio-One, 658190) for suspension culture. The medium was replaced daily. The cells were passaged every 4 - 5 days. For passage, the cells were dissociated into single cells using TrypLE:0.5 mM EDTA (1:1), and then the cells were resuspended in 4CL medium 1 at a density of 150,000 cells / ml. The resuspended cells were added to a flask (Greiner Bio-One, 658190) for suspension culture. The cells formed small aggregates in 24 hours. Generally, the cells were converted to iCLCs approximately 3 weeks after the start Experimental Results Figure 17 is a bar graph of RT-qPCR data, showing that pre-implantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 were significantly induced in iCLCs converted in suspension using 4CL medium 1. 4CL medium 1 is shown to be effective for suspension culture

Example

[0083] Materials and Methods 4CL basal medium The same as in Example 1 4CL supplement 4CL medium 2 (excluding extracellular matrix) was added to the 4CL basal medium as follows: Supplemented with SAH / PRC / EZH2 inhibitor (10 nM DZNep), HDAC inhibitor (5 nM TSA), WNT / β-catenin signaling inhibitor (5 μM IWR1), L-ascorbic acid (50 μg / ml), JAK / STAT3 activator (20 ng / ml human LIF), MAPK / ERK inhibitor (1 μM PD0325901), activin A / NODAL activator (20 ng / ml human activin A), ROCK inhibitor (1 μM Y27632). 4CL medium 3 (minus ROCK inhibitor) was prepared by supplementing 4CL basal medium with the following: SAH / PRC / EZH2 inhibitor (10 nM DZNep), HDAC inhibitor (5 nM TSA), WNT / β-catenin signaling inhibitor (5 μM IWR1), L-ascorbic acid (50 μg / ml), JAK / STAT3 activator (20 ng / ml human LIF), MAPK / ERK inhibitor (1 μM PD0325901), activin A / NODAL activator (20 ng / ml human activin A), extracellular matrix (0.2% (v / v) Geltrex™). 4CL medium 4 (minus activin / NODAL activator) was prepared by supplementing 4CL basal medium with the following: SAH / PRC / EZH2 inhibitor (10 nM DZNep), HDAC inhibitor (5 nM TSA), WNT / β-catenin signaling inhibitor (5 μM IWR1), L-ascorbic acid (50 μg / ml), JAK / STAT3 activator (20 ng / ml human LIF), MAPK / ERK inhibitor (1 μM PD0325901), extracellular matrix (0.2% (v / v) Geltrex™), ROCK inhibitor (1 μM Y27632). Cells 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 pre-implantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced in ICLCs converted using 4CL medium 2, 4CL medium 3, and 4CL medium 4, respectively. These results indicate that 4CL medium lacking any of Geltrex™, ROCK inhibitor, or activin / NODAL activator is also effective.

Example

[0084] Materials and Methods 4CL Basal Medium Same as Example 1. e4CL Supplement e4CL medium, the following were added to 4CL basal medium: SAH / PRC / EZH2 inhibitor (50 nM DZNep, or 3 mM CPI-1205), HDAC inhibitor (20 nM TSA, or 1 mM VPA, or 1 mM NaB), WNT / β-catenin signaling inhibitor (5 μM IWR1 or 5 μM XAV939), L-ascorbic acid (50 μg / ml), JAK / STAT3 activator (20 ng / ml human LIF), MAPK / ERK inhibitor (1 μM PD0325901), activin A / NODAL activator (20 ng / ml human activin A, 20 ng / ml human NODAL), ROCK inhibitor (1 μM of Y27632, 1 μM of thiazovivin, or 1 μM of hydroxyfasudil) and extracellular matrix (0.2% (v / v) Geltrex™ 又は Matrigel™) was supplemented. Cells H9, H1, UH10 human ESC lines. Procedure: 1) Conversion from primed human PSCs to 8CLCs The primed human PSCs were cultured in the same procedure as in Example 1. One day before the start of conversion, the primed human PSCs were dissociated into single cells and seeded on feeders at 2,000 - 3,000 cells / cm using mTeSR1 or E8 medium supplemented with 10 μM of Y27632 2It was seeded. After 24 hours, the medium was changed to e4CL medium, and the cells were cultured in an incubator at 37 °C, 5% CO2, under hypoxic or normoxic conditions. The medium was replaced daily. The cells were passaged every 3 - 4 days. For passage, TrypLE:0.5 mM EDTA (1:1) seeded at 2,000 - 3,000 cells / cm 2 on the feeder-coated plate was used to dissociate the cells into single cells. Generally, the cells were converted to 8CLC in about one week. 2) Conversion from ICLC to 8CLC One day before the start of the conversion, the ICLCs were dissociated into single cells and seeded at 2,000 - 3,000 cells / cm 2 on the feeder using 4CL medium. After 24 hours, the medium was changed to e4CL medium. The medium was replaced daily. The cells were converted to 8CLC in 3 - 5 days without passage. 3) Blastoid formation The same procedure as in Example 1 was used.

[0085] Experimental results Figure 19(A) shows the scheme of the 8CLC induction procedure, one is the direct induction from primed human PSCs, and the other is the induction from ICLCs. Figures 19(B-C) are bar graphs of RT-qPCR data showing that the 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 methods of conversion (Figure 19D). Figure 19(E) is an immunofluorescence image showing the expression of ZSCAN4 in 8CLCs. To characterize the gene expression profile of 8CLCs at the single-cell level, the inventors performed scRNA-Seq on ICLCs cultured in e4CL medium and then on cells at the primed stage (primed-D0) and days 1, 2, 3, 5 (e4CL-D1 / 2 / 3 / 5). Figure 20(A) is a 2D scatter plot of UMAP analysis for cells at different time points, shown together with published scRNA-seq data of in vivo human embryos at embryonic days 3, 4, 5, 6, 7 (left panel of E3 / 4 / 5 / 6 / 7) (from E-MTAB-3929). Cells in e4CL medium gradually acquire a gene expression profile similar to that of human embryo cells at embryonic day 3 (8C stage) and day 4 (morula stage). Figure 20(B) shows that the expression levels of human 8C-stage-specific markers in 8CLCs are upregulated compared to those in human 8C-stage embryos (GSE101571). Collectively, these results indicate that 8CLCs derived from e4CL medium acquire the gene expression profiles of in vivo human morula embryos and 8C-stage embryo-like.

[0086] To investigate the activated TEs in 8CLCs, we extracted TE profiles from the scRNA-seq data described in Figure 20(A). Figure 21(A) is a 2D scatter plot of the UMAP analysis of TE expression in cells at the priming stage (Primed-D0), and then after culturing in e4CL medium and human embryonic cells on days 3, 4, 5, 6, and 7 (E3 / 4 / 5 / 6 / 7) of the embryo, TE is expressed on days 1, 2, 3, and 5 (D1 / 2 / 3 / 5) (from E-MTAB-3929). This indicates that cells in the e4CL medium gradually acquire a TE expression profile similar to that of human embryonic cells on days 3 (8C stage) and 4 (morula stage) of the embryo. Figure 21(B) further shows that the expression levels of multiple TE subgroups in 8CLCs are induced to those of human 8C-stage 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 indicate that ICLCs derived from 4CL medium 1 acquire human 8C-stage embryos such as gene expression and TE profiles and maintain a stable genome. To determine the DNA methylation status of 8CLCs, we applied RRBS to 8CLCs and primed human PSCs. Figure 23 is a box plot showing CpG DNA methylation across the entire genome (upper left panel), and in 8CLCs, it is substantially decreased compared to primed human PSCs, although the methylation status at TSS is slightly different (upper right panel). Notably, the global decrease in DNA methylation level is prevented by knocking out STELLA (lower left panel). Figure 24 compares the imprinting status of 8CLCs with in vivo human embryonic DNA methylation data. In addition to DNA methylation, chromatin accessibility also changes. The bulk ATAC-seq of Fig. 25 shows the differences in chromatin accessibility between primed human PSCs and 8CLCs. 8C-specific loci proximal to primed human PSCs are open in 8CLCs, while post-implantation-specific loci that are open in primed human PSCs are proximal. These results indicate that 4CL medium 1 successfully rewired the epigenetic landscape into an 8C-like state.

[0087] The inventors further investigated the metabolic state of 8CLCs induced in e4CL medium. Human 8C-stage embryos mainly rely on oxidative phosphorylation (OxPhos) as an energy source, while post-implantation embryos mainly rely on glycolysis. Fig. 26 shows that the expression levels of genes related to oxidative phosphorylation are significantly upregulated in 8CLCs compared to primed human PSCs. These results imply that oxidative phosphorylation is activated in 8CLCs. To determine the differentiation potential of 8CLCs, the inventors performed a teratoma formation assay using nude mice as recipient animals. Fig. 27 is an image of hematoxylin and eosin-stained teratoma tissue formed 8 weeks after injecting 1 million 8CLCs. These show the presence of all three germ layer structures: mesoderm (left panel), endoderm (central panel), and ectoderm (right panel). The inventors also used 8CLCs to induce trophoblast stem cell-like cells (TSCLCs) using a previously published protocol. As shown in Fig. 28, multiple TSC markers such as GATA3, CGA, KRT18, KRT8, PSG6, and CCR7 are significantly induced in TSCLCs compared to undifferentiated 8CLCs. These results indicate that 8CLCs have embryonic and extraembryonic developmental potential.

[0088] Due to ethical issues, it is not possible to test developmental ability using human embryos. Therefore, the inventors conducted interspecies chimera experiments by aggregating 8CLCs with mouse 8-cell stage blastomeres. It has been found that human 8CLCs are successfully incorporated into most mouse embryos and form chimeric blastocysts when confirmed after 24 hours of in vitro culture. At this stage, human 8CLCs are located in both the ICM and TE parts of the chimeric blastocysts. Figure 29(A) is a representative image showing the phase contrast (left) or red fluorescence channel (right) of blastocysts generated from mouse 8-cell blastomeres aggregated with primed human PSCs labeled with DsRed (top) or 8CLCs labeled with DsRed (bottom). Figure 29(B) is the immunofluorescence of chimeric blastocysts stained with anti-OCT4 (ICM, green) and anti-CDX2 (TE, gray), where the red signal is from the integrated DsRed-labeled 8CLCs, and DAPI (blue) is used as nuclear counterstaining. When these chimeric blastocysts are transplanted into the uterus of pseudopregnant mice and allowed to develop until embryonic day 10.5 (E10.5), as shown in the microscopic images of Figure 30, human cells can develop together with mouse embryos and contribute to various tissues including embryonic and extraembryonic placenta and yolk sac. Figure 30(A) is a representative image showing the phase contrast (top) or red fluorescence channel (bottom) of an E10.5 chimeric embryo (left), placenta (middle), or yolk sac (right). Figure 30(B) is an immunofluorescence image showing that hN (green) differentiates human cells into GATA6 (red) - positive endodermal tissue. Figure 30(C) is an immunofluorescence image showing that DsRed-labeled human cells (red) differentiate into placental tissue indicated by GATA3 (green). Collectively, these results indicate that 8CLCs can be strongly integrated into mouse blastocysts and contribute to mouse E10.5 embryos and extraembryonic tissues in vivo.

[0089] To determine the blastocyst-like structures that give rise to 8CLCs, the inventors applied 8CLCs to a matrix-rich medium and observed the blastocyst-like structures formed in 5 days, but did not observe primed human PSCs (Figure 31A). Figure 31(B) is an immunofluorescence image of self-formed blastocysts stained with anti-OCT4 (ICM, red), anti-GATA3 (TE, green) antibodies, or nuclear counterstain DAPI (blue). 8CLCs serve as a robust model for the functional study of 8C regulators. In a pilot study, the inventors identified three potential new regulators, TPRX1, KHDC1L, and TRIM60, that govern the 8C state. Figure 37 shows that the induction of 8C-specific genes during the conversion from ICLCs to 8CLCs is inhibited by knockdown of TPRX1, KHDC1L, or TRIM60. GSK inhibitors such as CHIR99021 (activating the WNT / β-catenin signaling pathway) are widely used in published naive or extended PSC media. However, when included, they inhibit the formation of 8CLCs (Figure 38). Figure 38 shows that adding a GSK inhibitor to e4CL (e4CL+CHIR) inhibits the activation of human 8C embryo-enriched genes. Consistently, removing the WNT / β-catenin signaling inhibitor IWR1 (suppressing 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

[0090] Materials and Methods e4CL basal medium Same as Example 1. Supplements for e4CL Same as Example 6. Cells H9 human ESC line Procedure: Conversion from ICLCs to 8CLCs in suspension The ICLCs were cultured following the same procedure as in Example 1. One day before the start of conversion, the 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 changed to e4CL without adding Y27632. The medium was replaced daily, and the cells were converted to 8CLCs in 3 - 5 days without subculture. Experimental results Figure 32 is a bar graph of RT-qPCR data showing that the 8C markers ZSCAN4, ARGFX, TPRX1, ZNF280A, and ZSCAN5B were significantly induced in 8CLCs converted to suspension using e4CL medium. This indicates that e4CL medium is also effective for suspension culture.

Example

[0091] Materials and methods e4CL basal medium Same as in Example 1. e4CL supplement Same as in Example 6. Cells Human ESC lines: HN10 and UH10 Procedure: Same as in 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 were significantly induced in 8CLCs converted from multiple hPSC lines. This indicates that the e4CL medium can be generally applied to human PSCs.

Example

[0092] Materials and methods e4CL basal medium Same as in Example 1. e4CL supplement Same as in Example 6. Cells Mouse ESC lines: 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 using serum / LIF medium. After 24 hours, the medium was changed to e4CL medium. The medium was replaced daily. The cells were converted to a mouse 2C-like state in 3 days without subculture. 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. This indicates that e4CL medium is also potent in inducing the mouse 2C-like state, and it is not cell line-specific.

Example

[0093] Materials and methods 4CL basal medium Same as Example 1. 4CL supplement Same as 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. Cells 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 converted using 4CL medium 1 supplemented with different doses of either PD0325901, DZNep, or TSA, compared to primed human PSCs cells.

[0094] References

[0095]

Table 2

[0096]

Table 3

[0097]

Table 4

Claims

1. A chemically defined culture medium for culturing stem cells, comprising a basal medium supplemented with an S-adenosylhomocysteine hydrolase (SAH) / polycomb repressive complex (PRC) / EZH2 inhibitor, a histone deacetylase (HDAC) inhibitor, and a WNT / β-catenin signaling inhibitor, L-ascorbic acid, an activator of JAK / STAT3 signaling, and a mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) signaling inhibitor, wherein the basal medium is a 1:1 (v / v) ratio mixture of high-glucose DMEM / F12 and Neurobasal medium, supplemented with N2 supplement, B27 supplement, sodium pyruvate, non-essential amino acids, Glutamax (registered trademark), and penicillin-streptomycin, and the SAH / PRC / EZH2 inhibitor is an SAH inhibitor, and the WNT / β-catenin signaling inhibitor is a tankyrase inhibitor, wherein the SAH inhibitor is selected from the group consisting of 3-deazaneplanocin A (DZNep) and CPI-1205, the HDAC inhibitor is selected from the group consisting of trichostatin A (TSA), valproic acid (VPA), and sodium butyrate (NaB), the WNT / β-catenin signaling inhibitor and the tankyrase inhibitor are selected from the group consisting of IWR1 and XAV939, the activator of JAK / STAT3 signaling is LIF, and the MAPK / ERK signaling inhibitor is PD0325901, a culture medium.

2. The culture medium according to claim 1, wherein the SAH inhibitor is DZNep, the HDAC inhibitor is TSA, the WNT / β-catenin signaling inhibitor and the tankyrase inhibitor are IWR1, the activator of JAK / STAT3 signaling is LIF, and the inhibitor of MAPK / ERK signaling is PD0325901.

3. The culture medium according to claim 1 or 2, 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.

4. The final concentration of DZNep in the culture medium is 5 - 80 nM or 5 - 50 nM; The final concentration of said CPI-1205 in said culture medium is 0.5 to 5 mM or 1 to 3 mM; and / or, The final concentration of said TSA in said culture medium is 3 to 30 nM or 3 to 25 nM; The final concentration of said VPA in said culture medium is 0.25 to 2 mM or 0.5 to 1.5 mM; The final concentration of said NaB in said culture medium is 0.25 to 2 mM or 0.5 to 1.5 mM; and / or, The final concentration of said WNT / β-catenin signaling inhibitor in said culture medium is 2 to 8 μM, The final concentration of L-ascorbic acid in said culture medium is 40 to 70 μg / ml; and / or, The final concentration of an activator of JAK / STAT3 signaling in said culture medium is 10 to 50 ng / mL; and / or, The final concentration of a MAPK / ERK signaling inhibitor in said culture medium is 0.5 μM to 3 μM; and / or, The final concentration of an activin / Nodal signaling activator is 10 to 25 ng / ml; Said activin / Nodal signaling activator is activin A or Nodal; and / or, The final concentration of a ROCK inhibitor in said culture medium is 0.5 to 2 μM; Said ROCK inhibitor is selected from the group consisting of Y27632, thiazovivin, and hydroxyfasudil; and / or, The amount of extracellular matrix in said culture medium is 0.1 to 0.5% (v / v); Said extracellular matrix is selected from the group consisting of Matrigel (registered trademark), Geltrex (registered trademark), ECM (registered trademark), the culture medium for culturing according to claim 1 or 2.

5. The following: (A) Said DZNep at a final concentration of 5 to 15 nM or said CPI-1205 at a final concentration of 0.5 to 2 mM; said TSA at a final concentration of 3 to 30 nM, or said VPA at a final concentration of 0.25 to 2 mM, or said NaB at a final concentration of 0.25 to 2 mM, or Said TSA at a final concentration of 3 to 10 nM, or said VPA at a final concentration of 0.25 to 1 mM, or said NaB at a final concentration of 0.25 to 1 mM; and IWR1 or XAV939 at a final concentration of 2 to 8 μM or 3 to 6 μM; or The DZNep at a final concentration of 5 to 80 nM or 5 to 50 nM, or the CPI-1205 at a final concentration of 0.5 to 5 mM or 0.5 to 3 mM; or the TSA at a final concentration of 3 to 10 nM, or the VPA at a final concentration of 0.25 to 0.5 mM, or the NaB at a final concentration of 0.25 to 0.5 mM; and the IWR1 or XAV939 at a final concentration of 2 to 8 μM or 3 to 6 μM; (B) L-ascorbic acid at a final concentration of 40 to 70 μg / ml; (C) LIF at a final concentration of 10 to 30 ng / mL; (D) PD0325901 at a final concentration of 0.5 to 1.5 μM; comprising, further, 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) 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 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 in an amount of 0.1% to 0.5% (v / v); is supplemented, or 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 5 μM of the IWR1 or 5 μM of the XAV939; 50 μg / ml of the L-ascorbic acid; 20 ng / mL of the LIF; 1 μM of the PD0325901; comprising, further, the following: (1) 20 ng / mL of the Activin A or Nodal, 1 μM of the Y27632, thiazovivin or hydroxyfasudil, 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) The culture medium according to claim 1, supplemented with 20 ng / mL of said activin A or Nodal, 0.2% (v / v) of said extracellular matrix; or (4) The culture medium according to claim 1, supplemented with 1 μM of said Y27632, thiazovivin or hydroxyfasudil, 0.2% (v / v) of said extracellular matrix; or (5) The culture medium according to claim 1, supplemented with 20 ng / mL of said activin A or Nodal, 1 μM of said Y27632, thiazovivin or hydroxyfasudil, 0.2% (v / v) of said extracellular matrix; The culture medium according to claim 1, supplemented with

6. Said DZNep at a final concentration of 40 - 70 nM or said CPI-1205 at a final concentration of 2 - 4 mM; Said TSA at a final concentration of 10 - 30 nM or said VPA at a final concentration of 0.5 - 1.5 mM or said NaB at a final concentration of 0.5 - 1.5 mM; and said IWR1 or XAV939 at a final concentration of 2 - 8 μM or 3 - 6 μM; 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 further comprising the following: (1) Said activin A or Nodal at a final concentration of 10 - 25 ng / ml; said Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 - 2 μM; and said extracellular matrix in an amount of 0.1% - 0.5% (v / v); or (2) Said activin A or Nodal at a final concentration of 10 - 25 ng / ml; said Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 - 2 μM; or (3) Said activin A or Nodal at a final concentration of 10 - 25 ng / ml; and said extracellular matrix in an amount of 0.1% - 0.5% (v / v); or (4) Said Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 - 2 μM; and said extracellular matrix in an amount of 0.1% - 0.5% (v / v); or (5) Said activin A or Nodal at a final concentration of 10 - 25 ng / ml; or said Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 - 2 μM; or said extracellular matrix in an amount of 0.1% - 0.5% (v / v); The culture medium according to claim 1, supplemented with

7. 50 nM of said DZNep or 3 mM of said CPI-1205; 20 nM of said TSA or 1 mM of said VPA or 1 mM of said NaB; 5 μM of said IWR1 or 5 μM of said XAV939; 50 μg / ml of said L-ascorbic acid; 20 ng / mL of said LIF; 1 μM of said PD0325901; and further comprising the following: (1) 20 ng / mL of said activin A or Nodal, 1 μM of said Y27632, thiazovivin or hydroxyfasudil, 0.2% (v / v) of said extracellular matrix; or, (2) 20 ng / mL of said activin A or Nodal, 1 μM of said Y27632, thiazovivin or hydroxyfasudil; or, (3) 20 ng / mL of said activin A or Nodal, 0.2% (v / v) of said extracellular matrix; or, (4) 1 μM of said Y27632, thiazovivin or hydroxyfasudil, 0.2% (v / v) of said extracellular matrix; or (5) 20 ng / mL of said activin A or Nodal, 1 μM of said Y27632, thiazovivin or hydroxyfasudil, 0.2% (v / v) of said extracellular matrix; The culture medium according to claim 6, supplemented with.

8. The basal medium is selected from the group consisting of Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), RPMI1640, F10, F12, Alpha Minimum Essential Medium (αMEM), Glasgow Minimum Essential Medium (GMEM), Iscove's Modified Dulbecco Medium, Neurobasal Medium, and DMEM / F12, and combinations thereof. The culture medium according to any one of claims 1 to 7.

9. Furthermore, one or more components selected from the group consisting of serum replacement, alternative carbon source, non-essential amino acids, L-glutamine or its substitute, and antibiotics are supplemented, The following: The serum replacement is selected from the group consisting of Knockout (trademark) Serum Replacement (KOSR), N2 and B27, and combinations thereof, and is a mixture in which N2 and B27 are mixed at a ratio of 1:1 (w / w); The alternative carbon source is pyruvic acid such as sodium pyruvate; L-glutamine or its substitute is Glutamax (registered trademark) 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. The culture medium according to any one of claims 1 to 8.

10. A method for converting primate PSCs into pre-implantation ICM-like cells (ICLCs) and / or 8-cell embryo-like cells (8CLCs), or a method for converting ICLCs into 8CLCs, comprising culturing primate PSCs or ICLCs in the presence of an S-adenosylhomocysteine hydrolase (SAH) / polycomb repressive complex (PRC) / EZH2 inhibitor, a histone deacetylase (HDAC) inhibitor, and a WNT / β-catenin signaling inhibitor, a basal medium, L-ascorbic acid, an activator of JAK / STAT3 signaling, and a mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) signaling inhibitor, wherein the basal medium is a mixture of advanced DMEM / F12 and Neurobasal medium at a ratio of 1:1 (v / v), supplemented with N2 supplement, B27 supplement, sodium pyruvate, non-essential amino acids, Glutamax (registered trademark), and penicillin-streptomycin, and the SAH / PRC / E, ZH2 inhibitor is an SAH inhibitor, and the WNT / β-catenin signaling inhibitor is a tankyrase inhibitor, wherein the SAH inhibitor is selected from the group consisting of 3-deazaneplanocin A (DZNep) and CPI-1205, the HDAC inhibitor is selected from the group consisting of trichostatin A (TSA), valproic acid (VPA), and sodium butyrate (NaB), the WNT / β-catenin signaling inhibitor and the tankyrase inhibitor are selected from the group consisting of IWR1 and XAV939, the activator of JAK / STAT3 signaling is LIF, and the inhibitor of MAPK / ERK signaling is PD0325901. Method.

11. The method according to claim 10, comprising culturing primate PSCs or ICLCs 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.

12. Cultivate primate PSCs or iCLCs in the presence of the DZNep at a final concentration of 5-80 nM or 5-50 nM, or in the presence of the CPI-1205 at a final concentration of 0.5-5 mM or 1.5-3 mM, and in the presence of the TSA at a final concentration of 3-30 nM or 3-25 nM, or in the presence of the VPA at a final concentration of 0.25-2 mM or 0.5-1.5 mM, or in the presence of the NaB at a final concentration of 0.25-2 mM or 0.5-1.5 mM, and; cultivate in the presence of the tankyrase inhibitor at a final concentration of 2-8 μM; The method according to claim 10 or 11.

13. L-ascorbic acid is present at a final concentration of 40-70 μg / ml; and / or The final concentration of the activator of JAK / STAT3 signaling is 10-50 ng / mL; and / or The final concentration of the MAPK / ERK signaling inhibitor is 0.5-3 μM; and / or The final concentration of the activin / Nodal signaling activator is 10-25 ng / ml; and / or The final concentration of the ROCK inhibitor is 0.5-2 μM, and the ROCK inhibitor is selected from the group consisting of Y27632, thiazovivin, and hydroxyfasudil; and / or The extracellular matrix is present in an amount of 0.1%-0.5% (v / v), and the extracellular matrix is selected from the group consisting of Matrigel (registered trademark), Geltrex (registered trademark), ECM (registered trademark); The method according to claim 11.

14. A method for converting primate PSCs into iCLCs, comprising culturing primate PSCs in the culture medium according to claim 5.

15. A method for converting primate PSCs or iCLCs into 8CLCs, comprising culturing primate PSCs or iCLCs in the culture medium according to claim 6 or 7.

16. Primate PSCs are the following: (i) Cells derived from the ESC line and / or the ECC line; (ii) Cells derived from the iPSC line; (iii) Cells derived from the ICM of a pre-implantation blastocyst cultured in vitro; (iv) Cells derived from the ICM of a post-implantation blastocyst cultured in vitro; (v) Cells from the 8-cell stage embryo to the morula stage cultured in vitro; The method according to any one of claims 10-15, selected from the group consisting of.

17. Furthermore, culturing somatic cells in the presence of the SAH / PRC / EZH2 inhibitor, the HDAC inhibitor, and the WNT / β-catenin signaling inhibitor, and reprogramming the somatic cells to produce primate iCLCs, the method according to claim 10, comprising.

18. PSCs have a transcriptome, transposon profile, DNA methylome, chromatin landscape, and metabolic state similar to the corresponding pre-implantation ICM of primates, and are isolated primate iCLCs obtained by the method according to claim 15.

19. An isolated primate 8CLC that expresses an 8C embryo-specific marker at a level substantially higher than that of the priming PSCs from which the 8CLC and / or iCLCs are produced, wherein the cells have a transcriptome, transposon profile, and chromatin landscape similar to the corresponding primate 8C-stage embryo, and wherein the 8CLC is an isolated primate 8CLC obtained by the method according to claim 15.

20. A kit comprising an SAH / PRC / EZH2 inhibitor, an HDAC inhibitor, a WNT / β-catenin signaling inhibitor, a basal medium, L-ascorbic acid, an activator of JAK / STAT3 signaling, and a mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) signaling inhibitor, wherein the basal medium is a mixture of high-glucose DMEM / F12 and Neurobasal medium at a ratio of 1:1 (v / v), supplemented with N2 supplement, B27 supplement, sodium pyruvate, non-essential amino acids, Glutamax (registered trademark), and penicillin-streptomycin, and the SAH / PRC / EZH2 inhibitor is an SAH inhibitor, and the WNT / β-catenin signaling inhibitor is a tankyrase inhibitor, wherein the SAH inhibitor is selected from the group consisting of 3-deazaneplanocin A (DZNep) and CPI-1205, the HDAC inhibitor is selected from the group consisting of trichostatin A (TSA), valproic acid (VPA), and sodium butyrate (NaB), the WNT / β-catenin signaling inhibitor and the tankyrase inhibitor are selected from the group consisting of IWR1 and XAV939, the activator of JAK / STAT3 signaling is LIF, the inhibitor of MAPK / ERK signaling is PD0325901, and One or more components selected from the group consisting of activin / Nodal signaling activators, ROCK inhibitors, and extracellular matrices A kit comprising the same. **Claim 21** A composition comprising an SAH / PRC / EZH2 inhibitor, an HDAC inhibitor, a WNT / β-catenin signaling inhibitor, a basal medium, L-ascorbic acid, an activator of JAK / STAT3 signaling, and a mitogen-activated protein kinase / extracellular signal-regulated kinase (MAPK / ERK) signaling inhibitor, wherein the basal medium is a mixture of high-glucose DMEM / F12 and Neurobasal medium at a ratio of 1:1 (v / v), supplemented with N2 supplement, B27 supplement, sodium pyruvate, non-essential amino acids, Glutamax (registered trademark), and penicillin-streptomycin, and the SAH / PRC / EZH2 inhibitor is an SAH inhibitor, and the WNT / β-catenin signaling inhibitor is a tankyrase inhibitor, wherein the SAH inhibitor is selected from the group consisting of 3-deazaneplanocin A (DZNep) and CPI-1205, the HDAC inhibitor is selected from the group consisting of trichostatin A (TSA), valproic acid (VPA), and sodium butyrate (NaB), the WNT / β-catenin signaling inhibitor and the tankyrase inhibitor are selected from the group consisting of IWR1 and XAV939, the activator of JAK / STAT3 signaling is LIF, the inhibitor of MAPK / ERK signaling is PD0325901, and One or more components selected from the group consisting of activin / Nodal signaling activators, ROCK inhibitors, and extracellular matrices; A composition comprising the same. **Claim 22** Each of the above components is such that the culture medium containing the composition has the following: 5-15 nM or 10 nM of DZNep, or 0.5-2 mM or 1 mM of CPI-1205; 2-8 nM or 3-6 nM or 5 nM of TSA, or 0.25-1 mM or 0.5 mM of VPA; or 0.25-1 mM or 0.5 mM of NaB; 2-8 μM or 3-6 μM or 5 μM of IWR1 or XAV939; 10-30 ng / mL or 20 ng / mL of LIF; 0.5-1.5 μM or 1 μM of PD0325901; and 40-90 μg / mL or 50 μg / mL of L-ascorbic acid; Present in an amount that can contain activin A or Nodal at 10 - 25 ng / mL or 20 ng / mL, and / or Y27632, thiazovivin, or hydroxyfasudil at 0.5 - 2 μM or 1 μM, and / or extracellular matrix at 0.1% - 0.5% (v / v); or, Present in an amount that can contain DZNep at 40 - 70 nM or 50 nM, or CPI - 1205 at 2 - 4 mM or 3 mM; TSA at 10 - 30 nM or 20 nM, or VPA at 0.5 - 1.5 mM or 1 mM, or NaB at 0.5 - 1.5 mM or 1 mM; IWR1 or XAV939 at 2 - 8 μM or 3 - 6 μM or 5 μM; LIF at 10 - 30 ng / mL or 20 ng / mL; PD0325901 at 0.5 - 1.5 μM or 1 μM; and L - ascorbic acid at 40 - 90 μg / mL or 50 μg / mL; and each of the above components is present in the culture medium in an amount that can contain activin A or Nodal at 10 - 25 ng / mL or 20 ng / mL, and / or Y27632, thiazovivin, or hydroxyfasudil at 0.5 - 2 μM or 1 μM, and / or extracellular matrix at 0.1% - 0.5% (v / v), the composition according to claim 21.

23. A method for converting mouse PSCs to 2CLCs, comprising culturing mouse PSCs in the presence of an SAH inhibitor, an HDAC inhibitor, a WNT / β - catenin signaling inhibitor, and an activator of JAK / STAT3 signaling, wherein the SAH inhibitor is an SAH / PRC / EZH2 inhibitor, and the WNT / β - catenin signaling inhibitor is a tankyrase inhibitor.

24. Culturing mouse PSCs in the presence of an SAH inhibitor, an HDAC inhibitor, a WNT / β - catenin signaling inhibitor, and an activator of JAK / STAT3 signaling, and Culturing one or more components selected from the group consisting of L - ascorbic acid and a MAPK / ERK signaling inhibitor, and 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, The method according to claim 23, comprising the above steps.

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