WO2023173370A1

WO2023173370A1 - Media and methods for producing human cells and tissues from teratoma, organoids and embryoid bodies

Info

Publication number: WO2023173370A1
Application number: PCT/CN2022/081499
Authority: WO
Inventors: Miguel A. ESTEBAN; Wenjuan Li; Mazid MD. ABDUL; Yu Jiang; Yiwei LAI; Zhiwei Luo; Jinxiu LI
Original assignee: Guangzhou Institutes Of Biomedicine And Health, Chinese Academy Of Sciences
Priority date: 2022-03-17
Filing date: 2022-03-17
Publication date: 2023-09-21
Also published as: CN116769695A

Abstract

Provided are media and methods for producing human cells and tissues from teratoma, organoids and embryoid bodies. The culture media can be used to culture mammalian pluripotent stem cells (PSCs), are chemically defined, and comprise basal media for culturing stem cells supplemented with a S-adeosylhomocysteine hydrolase (SAH)/Polycomb repressive complexes (PRC)/EZH2 inhibitor and a histone deacetylase (HDAC) inhibitor. With the culture media, primate (human and non-human) PSCs can be converted to preimplantation ICM-like cells (naïve PSCs/ICLCs) or 8-cell embryo-like cells (8CLCs). These naïve PSCs/ICLCs and 8CLCs can generate human tissues and cells through teratomas, organoids and EBs differentiation. These tissues and cells can be purified and function as a good source for transplantation and clinical or scientific research.

Description

Media and Methods for Producing Human Cells and Tissues from Teratoma, Organoids and Embryoid Bodies

Technical Field

The subject invention relates to media and methods for producing human cells and tissues from teratoma, organoids and embryoid bodies.

Background

Human cells of potential therapeutic application include fully differentiated cells such as T and B lymphocytes, somatic stem/progenitor cells such as hematopoietic stem cells (HSC) and liver progenitor cells (LPC) . These cells may be obtained by isolation from human body or differentiation from pluripotent/totipotent stem cells. Generally, isolated cells are functional and will not cause immune rejection either used autologously or used analogously when a perfect HLA-matched donor is found. However, some of them are not accessible, and, for those accessible, the cell number is limited. Furthermore, the cells to be isolated are functionally abnormal and the cells are not easy to find a perfect HLA-match. Pluripotent/totipotent stem cells can be expanded indefinitely and readily engineered, and both autologous and analogous (HLA-universal, remove surface proteins to lower immunogenicity) pluripotent/totipotent stem cells can be used to generate target derivatives. However, target derivatives generated from pluripotent/totipotent stem cells might not be properly differentiated or functional.

To make pluripotent/totipotent stem cell derivatives applicable, cells need to achieve the right cell state and functionality over the differentiation process. In this regard, scientists have been putting efforts on developing various in vitro lineage-specific differentiation protocols for pluripotent stem cells (PSC) . Some protocols have been more effective than the others. For instance, neural stem cells can be produced readily for transplantation with partial success. Numbers of pre-clinical tests have been performed in animal models including non-human primates, and several clinical trials have started recently. However, other protocols such as HSC and LPC are still challenging due to limited efficiency and poor functionality. There are two major reasons: 1) starting PSCs have epigenetic abnormalities gained from either prolonged culture or reprogramming, or a chromatin state with limited flexibility; 2) the in vitro differentiation protocol fails to capture in vivo cues. Because of being at the top of the epigenetic landscape,

stem cells or totipotent-like cells may have more flexibility that allows greater differentiation potential compared to primed PSC. In addition, an in vivo environment can induce PSC differentiation that is not achievable in vitro. Combination of these two features might be instrumental for generating functional differentiated cells.

Summary of the Invention

In the first aspect, the present application provide a method for producing a teratoma, comprising the step of transplanting

PSCs/ICLCs, 8CLCs, or reprimed PSCs into different organ or position of recipient immunocompromised animals of interest and culturing the animals; wherein the

PSCs/ICLCs are obtained by a method comprising a step of culturing primate PSCs in a culture medium containing a SAH/PRC/EZH2 inhibitor, a HDAC inhibitor and a WNT/β-catenin signaling inhibitor, the 8CLCs are obtained by a method comprising a step of culturing the primate PSCs or the

PSCs/ICLCs in a culture medium containing dose optimized SAH/PRC/EZH2 inhibitor, dose optimized HDAC inhibitor, and WNT/β-catenin signaling inhibitor, the reprimed PSC are obtained through guided differentiation of

PSCs/ICLCs or 8CLCs.

In the second aspect, the present application provide a method for producing an organoid, comprising the step of culturing

PSCs/ICLCs, 8CLCs or reprimed PSCs as an aggregate or in a 3D scaffold in the presence of a culture medium allowing targeted organ differentiation; wherein the

PSCs/ICLCs or 8CLCs.

In the third aspect, the present application provide a method for producing an embryoid body, comprising the step of culturing

PSCs/ICLCs, 8CLCs or reprimed PSCs as aggregates and in suspension in the presence of a culture medium allowing automatic differentiation; wherein the

PSCs/ICLCs are obtained by a method comprising a step of culturing primate PSCs in a culture medium containing a SAH/PRC/EZH2 inhibitor, a HDAC inhibitor and a WNT/β-catenin signaling inhibitor, the 8CLCs are obtained by a method comprising a step of culturing the primate PSCs or

PSCs/ICLCs or 8CLCs.

In one or more embodiments of the above aspects, the culture medium is further supplemented with one or more components selected from a group consisting of L-ascorbic acid or a derivative thereof, an activator of JAK/STAT3 signaling, and an inhibitor of MAPK/ERK signaling; optionally, the culture medium is further supplemented with one or more components selected from a group consisting of an activator of ACTIVIN/NODAL signaling, a ROCK inhibitor, and an extracellular matrix.

In one or more embodiments of the above aspects, the PRC/EZH2 inhibitor or the SAH inhibitor is selected from a group consisting of DZNep and CPI-1205; preferably, the final concentration of DZNep in the culture medium is from 5 to 80 nM, preferably 5 to 50 nM; preferably, the final concentration of CPI-1205 in the culture medium is from 0.5 to 5 mM, preferably 1 to 3 mM.

In one or more embodiments of the above aspects, the HDAC inhibitor is selected from a group consisting of TSA, VPA and NaB; preferably, the final concentration of TSA in the culture medium is from 3 to 30 nM, preferably 3 to 25 nM; preferably, the final concentration of VPA in the culture medium is from 0.25 to 2 mM, preferably 0.5 to 1.5 mM; preferably, the final concentration of NaB in the culture medium is from 0.25 to 2 mM, preferably 0.5 to 1.5 mM; and/or the final concentration of the WNT/β-catenin signaling inhibitor in the culture medium is from 2 to 8 μM; preferably, the WNT/β-catenin signaling inhibitor is selected from a group consisting of IWR1 and XAV939.

In one or more embodiments of the above aspects, the final concentration of L-ascorbic acid in the culture medium is 40 to 70 μg/mL.

In one or more embodiments of the above aspects, the final concentration of the activator of JAK/STAT3 signaling in the culture medium is 10 to 50 ng/mL; preferably, the activator of JAK/STAT3 signaling is LIF.

In one or more embodiments of the above aspects, the final concentration of the inhibitor of MAPK/ERK signaling in the culture medium is 0.5 μM to 3 μM; preferably, the inhibitor of MAPK/ERK signaling is PD0325901.

In one or more embodiments of the above aspects, the final concentration of the activator of ACTIVIN/NODAL signaling is from 10 to 25 ng/mL; preferably, the activator of ACTIVIN/NODAL signaling is selected from a group consisting of ACTIVIN A and NODAL.

In one or more embodiments of the above aspects, the final concentration of the ROCK inhibitor in the culture medium is from 0.5 to 2 μM; preferably, the ROCK inhibitor is selected from a group consisting of Y27632, thiazovivin and hydroxyfasudil.

In one or more embodiments of the above aspects, the amount of the extracellular matrix in the culture medium is 0.1 to 0.5% (v/v) ; preferably, the extracellular matrix is selected from a group consisting of Matrigel ^TM, Geltrex ^TM and ECM ^TM.

In one or more embodiments of the above aspects, the culture medium for producing the

PSCs/ICLCs comprises:

(A) DZNep at a final concentration of 5 to 15 nM or CPI-1205 at a final concentration of 0.5 to 2 mM, and TSA at a final concentration of 3 to 30 nM, or VPA at a final concentration of 0.25 to 2 mM, or NaB at a final concentration of 0.25 to 2 mM, preferably TSA at a final concentration of 3 to 10 nM, or VPA at a final concentration of 0.25 to 1 mM, or NaB at a final concentration of 0.25 to 1 mM; or DZNep at a final concentration of 5 to 80 nM, preferably 5 to 50 nM or CPI-1205 at a final concentration of 0.5 to 5 mM, preferably 0.5 to 3 mM, and TSA at a final concentration of 3 to 10 nM, or VPA at a final concentration of 0.25 to 0.5 mM, or NaB at a final concentration of 0.25 to 0.5 mM;

(B) L-ascorbic acid at a final concentration of 40 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;

(E) IWR1 or XAV939 at a final concentration of 3 to 6 μM;

and the culture medium is further supplemented with:

(1) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; and an 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; and Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; or

(3) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; and an 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 an 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; or Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 to 2 μM; or an extracellular matrix in an amount of 0.1%to 0.5% (v/v) .

PSCs/ICLCs comprises 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 is further supplemented with (1) 20 ng/mL of ACTIVIN A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an extracellular matrix; or (2) 20 ng/mL of ACTIVIN A or NODAL, and 1 μM of Y27632, thiazovivin or hydroxyfasudil; (3) 20 ng/mL of ACTIVIN A or NODAL, and 0.2% (v/v) of an extracellular matrix; or (4) 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an 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 an extracellular matrix.

In one or more embodiments of the above aspects, the culture medium for producing the 8CLCs comprises DZNep at a final concentration of 40 to 70 nM or CPI-1205 at a final concentration of 2 to 4 mM; TSA at a final concentration of 10 to 30 nM, or VPA at a final concentration of 0.5 to 1.5 mM or NaB at a final concentration of 0.5 to 1.5 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 each at a final concentration of 3 to 6 μM; and is further supplemented with:

(2) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; and Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 to 2 μM; or

(5) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; or Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; or an extracellular matrix in an amount of 0.1%to 0.5% (v/v) .

In one or more embodiments of the above aspects, the culture medium for producing 8CLCs comprises 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 is further supplemented with (1) 20 ng/mL of ACTIVIN A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an extracellular matrix; or (2) 20 ng/mL of ACTIVIN A or NODAL, and 1 μM of Y27632, thiazovivin or hydroxyfasudil; (3) 20 ng/mL of ACTIVIN A or NODAL, and 0.2% (v/v) of an extracellular matrix; or (4) 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an 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 an extracellular matrix.

In one or more embodiments of the above aspects, the basal medium of the culture medium for producing the

PSCs/ICLCs and the 8CLCs is selected from a group consisting of Dulbecco's modified eagle's medium (DMEM) , minimal essential medium (MEM) , basal medium Eagle (BME) , RPMI1640, F10, F12, α minimal essential medium (α MEM) , Glasgow's minimal essential medium (GMEM) , Iscove's modified Dulbecco's medium, Neurobasal Medium, DMEM/F12 and Advanced DMEM/F12 and a combination thereof; preferably, the basal medium is a mixture of Advanced DMEM/F12 and Neurobasal Medium in a ratio of 1: 1 (v/v) .

In one or more embodiments of the above aspects, the culture medium is further supplemented with one or more components selected from a group consisting of serum replacement, alternative carbon source, non-essential amino acid, L-glutamine or its alternative and antibiotic.

In one or more embodiments of the above aspects, the serum replacement is selected from a group consisting of KOSR, N2 and B27, and combinations thereof; preferably, the serum replacement is a mixture of N2 and B27 in a ratio of 1: 1 (w/w) .

In one or more embodiments of the above aspects, the alternative carbon source is pyruvate, such as sodium pyruvate.

In one or more embodiments of the above aspects, the L-glutamine or its alternative is Glutamax ^TM supplement comprising L-alanyl-L-glutamine dipeptide in 0.85%NaCl.

In one or more embodiments of the above aspects, the antibiotic is selected from a group consisting of penicillin, streptomycin, or a mixture of penicillin and streptomycin.

In one or more embodiments of the above aspects, the

PSCs/ICLCs are obtained by a method comprising:

(a) genetically engineering the primate PSCs to reduce the activity of SAH, PRC and/or EZH2 of the PSCs by knockdown and/or knockout of one or more relevant genes in the cells; and

(b) culturing the genetically engineered cells obtained in step (a) in a culture medium comprising: TSA at a final concentration of 3 to 30 nM, or VPA at a final concentration of 0.25 to 2 mM, or NaB at a final concentration of 0.25 to 2 mM, preferably TSA at a final concentration of 3 to 10 nM, or VPA at a final concentration of 0.25 to 1 mM, or NaB at a final concentration of 0.25 to 1 mM, and optionally DZNep at a final concentration of 5 to 15 nM or CPI-1205 at a final concentration of 0.5 to 2 mM, or TSA at a final concentration of 3 to 10 nM, or VPA at a final concentration of 0.25 to 0.5 mM, or NaB at a final concentration of 0.25 to 0.5 mM and optionally DZNep at a final concentration of 5 to 80 nM, preferably 5 to 50 nM or CPI-1205 at a final concentration of 0.5 to 5 mM; 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; IWR1 or XAV939 at a final concentration of 3 to 6 μM; wherein the culture medium is further supplemented with:

(5) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; or Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 to 2 μM; or an extracellular matrix in an amount of 0.1%to 0.5% (v/v) ;

preferably the culture medium comprises: 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; 5 μM IWR1 or 5 μM XAV939; and optionally 10 nM DZNep or 1 mM CPI-1205; and wherein the culture medium is further supplemented with (1) 20 ng/mL of ACTIVIN A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an extracellular matrix; or (2) 20 ng/mL of ACTIVIN A or NODAL, and 1 μM of Y27632, thiazovivin or hydroxyfasudil; (3) 20 ng/mL of ACTIVIN A or NODAL, and 0.2% (v/v) of an extracellular matrix; or (4) 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an 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 an extracellular matrix.

In one or more embodiments of the above aspects, the 8CLCs are obtained by a method comprising:

(a) genetically engineering the primate PSCs or

PSCs/ICLCs to reduce the activity of SAH, PRC and/or EZH2 of the PSCs or

PSCs/ICLCs by knockdown and/or knockout of one or more relevant genes in the cells;

(b) culturing the genetically engineered cells obtained in step (a) in a culture medium comprising: TSA at a final concentration of 10 to 30 nM, or VPA at a final concentration of 0.5 to 1.5 mM or NaB at a final concentration of 0.5 to 1.5 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; IWR1 or XAV939 each at a final concentration of 3 to 6 μM;and optionally DZNep at a final concentration of 40 to 70 nM or CPI-1205 at a final concentration of 2 to 4 mM; and wherein the culture medium is further supplemented with:

(5) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; or Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; or an extracellular matrix in an amount of 0.1%to 0.5% (v/v) ;

preferably, the culture medium comprises: 20 nM TSA, or 1 mM VPA, or 1 mM NaB; 50 μg/mL L-ascorbic acid; 20 ng/mL LIF; 1 μM PD0325901; 5 μM IWR1 or 5 μM XAV939; and optionally 50 nM DZNep or 3 mM CPI-1205; and wherein the culture medium is further supplemented with (1) 20 ng/mL of ACTIVIN A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an extracellular matrix; or (2) 20 ng/mL of ACTIVIN A or NODAL, and 1 μM of Y27632, thiazovivin or hydroxyfasudil; (3) 20 ng/mL of ACTIVIN A or NODAL, and 0.2% (v/v) of an extracellular matrix; or (4) 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an 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 an extracellular matrix.

In one or more embodiments of the above aspects, the primate PSCs are selected from a group consisting of:

(i) cells from an ESC line and/or an ECC line;

(ii) cells from an iPSC line;

(iii) cells from ICM of a preimplantation blastocyst cultured in vitro;

(iv) cells from ICM of a post-implantation blastocyst cultured in vitro;

(v) cells from an embryo of 8C stage to morula stage cultured in vitro.

In one or more embodiments of the above aspects, the primate PSCs or the

PSCs/ICLCs are cultured under one or more conditions selected from a group consisting of: (i) on feeder cells; (ii) on an extracellular matrix devoid of feeders; (iii) in suspension devoid of feeder cells; (iv) in hypoxic or normoxic condition at about 37℃ temperature; (v) passaging as single cells every 3 to 4 days with a split ratio of 1: 4 to 1: 8; (vi) changing medium daily.

In one or more embodiments of the above aspects, the method further comprises a step of culturing somatic cells in the presence of the SAH/PRC/EZH2 inhibitor, the HDAC inhibitor and the WNT/β-catenin signaling inhibitor to reprogram the somatic cells to produce the primate

PSCs/ICLCs.

In the fourth aspect, the present application provides a teratoma produced by the method described in any one of the embodiments as described herein, and cells dissociated from the teratoma.

In the fifth aspect, the present application provides an organoid produced by the method described in any one of the embodiments as described herein, and cells dissociated from the organoid.

In the sixth aspect, the present application provides an embryoid body produced by the method described in any one of the embodiments as described herein, and cells dissociated from the embryoid body.

Description of Drawings

Fig. 1. (a) Schematic depicting the process of inducing

PSC and 8CLC from primed PSC with 4CL, the combination of 4CL and e4CL media (stepwise e4CL) , or e4CL alone (direct e4CL) . D, day. (b) Immunostaining images for KLF17 and TPRX1 of primed H9 ESC untreated or converted by 4CL (day 12) or stepwise e4CL (day 5) . Scale, 20 μm. (c) Heatmap showing the expression of preimplantation ICM-enriched genes in human

PSC cultured in NHSM,

5iLAF and 4CL, and human ICM cells. H9 ESC were used to generate our dataset. (d) Heatmap showing the expression of totipotency genes in

ESC cultured in

5iLAF or stepwise e4CL (day 5) , EPSC, and human 8C-embryo cells. H9 ESC were used to generate our dataset. (e) RT-qPCR validation of totipotency genes in primed H9 ESC cultured in direct e4CL (day 7) . Data are the mean values ± SEM of fold-change compared to primed ESC. n=3 biological replicates. P value was calculated using two-tailed unpaired Student’s t-test, ***P <0.001.

Fig. 2. (a) UMAP comparing the developmental rolling back from human E7 to E3 embryonic stages in stepwise or direct e4CL induction scRNA-seq time courses. H9 ESC were used to generate our dataset. (b) UMAP visualization of stepwise e4CL-day 5 cells show seven clusters. The encircled cluster 5 (8CLC) comprises 11.9 %of the whole population. (c) Bubble plot representing the frequency of expression and average expression of representative pluripotency and totipotency genes in early human embryonic stages and primed ESC untreated or converted by 4CL (days 8 [passage 2] and 12 [passage 3] ) and e4CL (day 5 C5 [8CLC] and non-8CLC [all other clusters summed] ) . D, day. (d) Violin plot showing the log normalized expression of representative early human embryo-enriched TE in early human embryonic stages and human ESC passage 10 compared to primed ESC, 4CL-day 12

ESC and 8CLC.

Fig. 3. (a) Representative images of G-banding karyotype of primed H9 ESC and primed iPSC-4 clone cultured in 4CL for 15 passages. Twenty metaphases were counted for each. (b) Representative images of G-banding karyotype of primed H9 ESC and iPSC-4 cultured in stepwise e4CL (day 5) . Twenty metaphases were counted for each.

Fig. 4. (a) Violin plot showing global CpG methylation levels measured by RRBS of human PSC cultured in primed conditions, 4CL (day 12) , 5iLAF,

NHSM, stepwise e4CL (day 5) and direct e4CL (day 7) , human 8C-embryo and ICM. Our dataset was generated using H9 ESC. (b) Heatmap showing CpG methylation levels at a panel of imprinting control regions of human PSC cultured in primed conditions, 4CL (day 12) , 5iLAF,

NHSM and stepwise e4CL (day 5) , ICM and post-implantation embryo. (c) Genome browser tracks showing CpG methylation levels at the indicated

pluripotency (blue) and totipotency (red) loci of PSC cultured in primed conditions, 4CL (day 12) , 5iLAF,

NHSM, stepwise e4CL (day 5) and direct e4CL (day 7) , human 8C-embryo and ICM. Each bar represents a single CpG and the height indicates the percentage of methylation.

Fig. 5. (a) UMAP visualization of gene score for all genes in the scATAC-seq of primed ESC untreated (red) or converted by 4CL (day 12; blue) or stepwise e4CL (day 5; green) . (b and c) UMAP visualization based on panel a highlighting the gene score for primed (ZIC2) , shared

pluripotency/8CLC (DPPA3) and totipotency (ZSCAN5B, ZNF280A, ARGFX) genes projected onto each individual cell in the scATAC-seq of primed ESC untreated or converted by 4CL (day 12) and stepwise e4CL (day 5) . (d) Genome browser tracks showing chromatin accessibility, H3K27ac level and transcription factor DNA binding motif location at the

pluripotency KLF17 and totipotency ZSCAN4 loci.

Fig. 6. (a) Upper panel: schematic representing the insertion of EGFP into the TPRX1 locus, and the donor constructs used for generating the TPRX1-EGFP reporter cell lines. Lower panels: TPRX1-EGFP knock-in cells were validated by culture in stepwise e4CL (day 5) and immunostaining with anti-TPRX1, which coincided with the GFP+ signal (left panel) . Scale, 10 μm.FACS analysis of TPRX1-EGFP cells in stepwise e4CL (day 5) showing the percentage of GFP+ cells (right panels) . (b) Bubble plot representing the frequency of expression and average expression of representative pluripotency and totipotency genes in early human embryonic stages and human ESC passage 10 compared to primed ESC, 4CL-day 12

ESC and sorted 8CLC.

Fig. 7. Bar chart showing expression levels of ICM and primed markers in H9, H1, HUES1 and WIBR3 human ESC lines which had been converted to

PSCs/ICLCs using 4CL medium 1.

Fig. 8. A bar chart of RT-qPCR data showing that a panel of preimplantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced in ICLCs converted on Geltrex ^TM coated plates using 4CL medium 1.

Fig. 9. A bar chart of RT-qPCR data showing that a panel of preimplantation epiblast markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced in

PSCs/ICLCs converted in suspension using 4CL medium 1. In the bar chart, the left column for each gene represents culture on a feeding cell and the right column represents culture in suspension.

Fig. 10. Bar charts of RT-qPCR data showing that a panel of preimplantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced in

PSCs/ICLCs converted by 4CL medium 2, 4CL medium 3, and 4CL medium 4, respectively.

Fig. 11. (A) Schematic diagram showing two methods to generate 8CLCs. Briefly, primed human PSCs culture media (e.g. mTeSR1) are changed to either e4CL medium or 4CL media. Cells are then either continually grown in e4CL or switched to e4CL medium after 2 passages in 4CL media. (B) Bar chart showing expression levels of selected

pluripotency markers in H9 primed and H9-e4CL cells. (C) Bar chart showing expression levels of selected

pluripotency markers in H9-e4CL cells and H9-4CL cells. (D) Induction of 8C specific genes in both methods is similar. (E) Immunofluorescence microscopy images showing expression of ZSCAN4 (green) or DAPI counterstain nuclei (blue) in primed H9, H9-4CL and H9-e4CL.

Fig. 12. A bar chart of RT-qPCR data showing 8C markers ZSCAN4, ARGFX, TPRX1, ZNF280A, and ZSCAN5B are significantly induced in 8CLCs converted in suspension using e4CL medium. In the bar chart, the left column for each gene represents culture on a feeding cell and the right column represents culture in suspension.

Fig. 13. A bar chart of RT-qPCR data showing 8C markers ZSCAN4, ARGFX, TPRX1, ZNF280A, ZSCAN5B, DUXA, DUXB, and MBD3L2 are significantly induced in 8CLCs converted from multiple hPSC lines. The columns for each of these genes in the primed HN10 and UH10 are basically absent, indicating that the expression of these genes in the primed HN10 and UH10 are extremely low.

Fig. 14. (a) Representative images of teratomas derived from primed ESC or primed ESC converted by 4CL (passage 15) and sorted 8CLC. H9 ESC were used for this assay. (b) Hematoxylin and eosin staining of teratomas derived from primed ESC converted by 4CL (passage 15) and sorted 8CLC. Representative images of tissues corresponding to the three germ layers are shown. Scale, 50 μm. H9 ESC were used for this assay. (c) UMAP visualization of the identified cell types in scRNA-seq of teratomas derived from sorted 8CLC, e4CL-day 5 cells, 4CL

ESC and primed ESC. H9 ESC were used for producing all these teratoma cell types.

Fig. 15. UMAP visualization based on Figure 7c highlighting the identified cell types in the scRNA-seq datasets generated from sorted 8CLC, stepwise e4CL-day 5 cells, 4CL

ESC and primed ESC, respectively.

Fig. 16. (a) Bar plot showing the contribution of the different teratomas to the identified cell types. (b) Bar plot showing the relative contribution of the different teratomas to each embryonic (ectoderm, mesoderm, and endoderm) and extraembryonic (trophoblast) lineage.

Fig. 17. (a) UMAP visualization of the identified trophoblast cell subtypes in scRNA-seq of teratomas derived from sorted 8CLC, e4CL-day 5 cells, 4CL

ESC and primed ESC. H9 ESC were used for producing all these teratoma cell types. (b) Bubble plot showing the frequency of expression and average expression of marker genes in cell subtypes of the extraembryonic trophoblast lineage. (c) Bar plot showing the relative contribution of sorted 8CLC, e4CL-day 5 cells, 4CL

ESC and primed ESC-derived teratomas to cell subtypes of the extraembryonic trophoblast lineage. (d) UMAP visualization based on Figure 7c highlighting the distribution and the expression of related markers in trophoblast cells from the indicated teratomas.

Fig. 18. (a) UMAP visualization showing the annotated cell subtypes of immune cells (left panel) and the contribution of primed PSC, 4CL

ESC, stepwise e4CL-day 5 cells and sorted 8CLC-derived teratoma cells (right panel) . (b) Bubble plot representing the frequency of expression and average expression of marker genes in different immune cell subtypes. (c) Bar plot showing the contribution of sorted 8CLC, stepwise e4CL-day 5, 4CL

and primed PSC-derived teratomas to different immune cell subtypes.

Fig. 19. (a) UMAP visualization showing the contribution of primed PSC, 4CL

PSC, e4CL cells and sorted 8CLC-derived teratoma cells. (b) UMAP visualization showing the annotated cell subtypes of neuronal cells derived from primed PSC, 4CL

PSC, e4CL cells and sorted 8CLC-derived teratomas. (c) Bar plot showing the relative contribution of the different neuronal cell types to the indicated teratoma. (d) Bar plot showing the contribution of the different teratomas to the identified cell types.

Fig. 20. (a) UMAP visualization showing the contribution of primed PSC and 4CL

PSC-derived brain organoid cells. (b) UMAP visualization showing the annotated sub-clusters of neuronal cells derived from primed PSC and 4CL

PSC-derived brain organoids. (c) Bar plot showing the relative contribution of the different neuronal cell types to the indicated brain organoid. (d) Bar plot showing the contribution of the different brain organoids to the identified cell types.

Fig. 21. (a) UMAP visualization showing the contribution of primed PSC and 4CL

PSC-derived EBs (embryoid bodies) . (b) UMAP visualization showing the annotated cell types derived from primed PSC and 4CL

PSC-derived EBs. (c) Bar plot showing the relative contribution of the different cell types to the indicated EBs. (d) Bar plot showing the contribution of the different EBs to the identified cell types.

Detailed Description of the Invention

The derivation of

human PSCs using existing methods from current studies is problematic, such as being lengthy, inducing variable levels of

specific genes, transgene dependency for

induction, genomic instability and imprinting loss, inability of multi-lineage differentiation and inefficient or no proper chimera formation competency. In addition, there is no report describing the generation of extraembryonic lineages in teratomas produced using human

or primed PSCs, nor the expansion of the differentiation capacity of

PSCs in vivo compared to the original unconverted primed PSCs.

To overcome the problems mentioned above, the inventors first conducted a screen with a panel of inhibitors that target epigenetic regulators and different signaling pathways relevant to human preimplantation ICM development, and found that combination of SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/β-catenin signaling inhibitor, and other modulators such as JAK/STAT3 activator and MAPK/ERK inhibitor outperforms other published culture conditions, permitting rewiring the epigenetic landscape of the cultured PSCs to be significantly closer to human preimplantation ICM, which transforms conventional human PSCs to ICM-liked

cells (

PSC/ICLC) that possess all major features of human preimplantation ICM. Of note, the inventors also found that activation of WNT/β-catenin signaling inhibits the transition from primed PSCs to

PSC/ICLC. Therefore, modulators such as GSK inhibitor CHIR99021 (an inhibitor widely used in published

and extended PSC culture conditions) that activates WNT/β-catenin signaling should be excluded from the culture mediums, whereas modulators such as IWR1 and XAV939 that suppress WNT/β-catenin signaling is required. Therefore, in the preferred embodiments of various aspects of the subject application, including the culture medium, the kit, the composition and the methods described herein, CHIR99021, preferably a GSK inhibitor, more preferably any agent that activates WNT/β-catenin signaling, is not contained in the culture medium, the kit or the composition, or is not used in the methods to culture cells.

Thus, methods and chemically defined culture mediums that facilitate robust derivation of primate

PSC/ICLC are provided in the subject application. The methods described herein can be applied to a number of human and non-human primate PSC lines, which are either at a primed state as validated by the presence of pluripotency surface markers such as SSEA-3, SSEA-4, TRA-1-81, and TRA-1-60, or at a preimplantation ICM-like state as validated by expression of genes such as DNMT3L, STELLA, DPPA5, and KLF17. The primate PSC lines that can be 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

PSC/ICLC from primate preimplantation ICM. The described methods are transgene free and straight forward as provided primate PSCs can be converted to

PSC/ICLC in one culture condition in approximately 2 weeks.

To our knowledge, so far there is no proper method for inducing primate 8-cell like cells (8CLCs) in vitro. To achieve this, the inventors further optimized the recipe for inducing

PSC/ICLC, and found that by increasing only the dosage of SAH/PRC/EZH2 inhibitor and HDAC inhibitor in the medium, primed human PSCs and/or

PSC/ICLC could be converted to 8CLCs. Thus, a chemically defined culture medium that facilitates derivation of primate 8CLCs is provided in the subject application. The method described herein can be applied to a number of human and non-human primate PSC lines, which are either at a primed state as validated by the presence of pluripotency surface markers such as SSEA-3, SSEA-4, TRA-1-81, and TRA-1-60, or at a preimplantation ICM-like state as validated by expression of genes such as DNMT3L, STELLA, DPPA5, and KLF17. The primate PSC lines that can be used in the present 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 8CLCs from primate 8C embryos. The described method is transgenic free and straight forward as the provided primate PSCs can be converted to 8CLCs in one culture condition in approximately 1 week. Indeed, activation of WNT/β-catenin signaling also inhibits the formation of 8CLC. Thus, modulators such as GSK inhibitor CHIR99021 (an inhibitor widely used in published

and extended PSC culture conditions) that activates WNT/β-catenin signaling should be excluded from the culture condition, whereas modulators such as IWR1, XAV939 that suppressing WNT/β-catenin signaling is required.

PSC and 8CLC capture characteristics of their in vivo counterparts, and have less epigenetic abnormalities, yet, in vitro differentiation protocols for

PSC and 8CLC are still lacking. Thus, we have applied these cells to in vivo environment for differentiation, which is a process of teratoma formation, brain organoid formation and EB formation.

Comparing to teratoma generated from primed PSC, those generated from

PSC and/or 8CLC contain high percentage of HSC, LPC and a significant percentage of extra-embryonic cells. By reconstructing differentiation trajectory of the HSC and LPC, we demonstrated that the HSC and LPC are capable to differentiate to functional cell types of hematopoietic lineage, liver lineage and extra-embryonic lineage, respectively. Brain organoids generated from

PSC contain significantly higher percentage of neuroepithelial cells and retinal progenitor cells, which can produce varies type of neuronal cells. The higher percentage of progenitor cells can potentially lead to a higher complexity and more functional brain organoid. In addition, EBs generated from

PSC contain more neuroepithelial, trophoblast and endoderm epithelial cells, which can be a good source for deriving human cells or tissues. Taken together, we have developed a method producing primate tissues, cells and biomolecules with therapeutic application potential.

Detailed descriptions of the invention will be described below. It should be understood that features described in various embodiments could be combined with each other to form preferred technical solutions, which are also contemplated in the scope of the subject application.

I. Terms

Unless otherwise specified, all terms used herein have the meanings generally understood by those skilled in the art. In order to facilitate the understanding of the invention, some terms used herein are defined as follows.

The singular "one" and "this" used in the description and claims include plural references, unless the context clearly states otherwise. For example, the term " (one) cell" includes a plurality of cells, including a mixture thereof.

All digital indicators, such as pH, temperature, time, concentration and molecular weight, including range, are approximate. It is important to understand that, although not always explicitly stated, all digital indicators are preceded by the term "about" . It is also to be understood that, although not always explicitly described, the reagents described herein are only examples and their equivalents are known in the art.

The term "basal medium" used herein refers to any medium capable of supporting cell growth. Basal media provide standard inorganic salts such as zinc, iron, magnesium, calcium, and potassium, as well as vitamins, glucose, buffer systems, and key amino acids. The basal medium which can be used for in the subject application includes but is not limited to Dulbecco's modified eagle's medium (DMEM) , minimal essential medium (MEM) , basal medium Eagle (BME) , RPMI1640, F10, F12, α minimal essential medium (α MEM) , Glasgow's minimal essential medium (GMEM) , Iscove's modified Dulbecco's medium, Neurobasal Medium, and DMEM/F12. Those skilled in the art know how to select a basal culture medium suitable for the cultured cells. In a preferred embodiment, the basal medium used in the subject application is a mixture of DMEM/F12 and Neurobasal Medium in a ratio of 1: 1 (w/w) .

The term "serum-free" means the absence of any blood serum of any species including, but not limited to, the absence of fetal bovine serum, calf bovine serum, human serum, or the like, or combinations thereof.

The term "serum replacement" as used herein refers to additives used in a basal culture medium to partially or completely replace serum to support cell survival and growth. A serum replacement generally includes factors such as insulin, metalloprotein, microelement, vitamin and the like. These factors are generally not contained in the basal culture medium, but are provided by a serum commonly used to culture cells. Serum replacement include at least one or more of the following components that support cell growth: one or more insulin and insulin substitutes, one or more metalloprotein and metalloprotein substitutes, one or more trace elements, one or more vitamins, one or more amino acids, one or more multiple hormones and hormone-like compounds, serum albumin or serum albumin substitutes, and one or more lipids, etc. A variety of commercial serum replacement are known in the art, including KOSR, N2, B27, Insulin-Transferrin-Selenium Supplement (ITS) , G5, etc., which are easily obtained by those skilled in the art. These replacements each have a defined composition, so the concentration of each component can be determined according to their respective proportions in the culture medium.

Those skilled in the art can easily configure serum replacement according to the prior art, the type of cells to be cultured and other aspects. Preferably, the serum replacement used herein is a mixed additive obtained by mixing KOSR, N2 and/or B27 in a certain proportion. More preferably, the serum replacement used herein is a mixture of N2 and B27 in a ratio of 1: 1 (w/w) .

The term “primate” or “primate animal” used herein refers to animals belonging to Primates, including human and non-human primates. The non-human primates include animals of Prosimian and Simiae. Specific non-human primates include but are not limited to Macaques, lemurs, gibbons, orangutans, and baboons.

The term “Pluripotent Stem cells” (PSCs) used herein refers to pluripotent cells derived from embryo at any time before gastrulation and iPSCs generated from somatic cell reprogramming. Depending on their source and method of culture, the PSCs may be at alternative states, including primed PSCs,

PSCs, extended PSCs and expanded potential stem cells (Gafni et al., 2013; Gao et al., 2019; Takashima et al., 2014; Theunissen et al., 2014; Yang et al., 2017) . PSCs have the characteristic of being capable under appropriate conditions of producing progeny of different cell types that are derivatives of all of the three germinal layers (endoderm, mesoderm, and ectoderm) , according to a standard art-accepted test, such as the ability to form a teratoma in 8-12 week-old SCID mice, and may also be capable under appropriate conditions of producing different cell types of placenta. PSC cultures are described as “undifferentiated” when a substantial proportion of stem cells and their derivatives in the population display morphological characteristics of undifferentiated cells, distinguishing them from differentiated cells of embryo or adult origin. It is understood that colonies of undifferentiated cells within the population may be surrounded by neighboring cells that are differentiated.

The subject application can be practiced using stem cells of various types. Particularly suitable for use in the subject application are primate PSCs. Non-limiting examples are primary cultures or established lines of ESCs and iPSCs. PSCs of any non-primate mammals can also be used to practice the subject application.

In one or more embodiments, the primate PSCs that may be used in the present application can be selected from a group consisting of:

(i) cells from an ESC line and/or an ECC line;

(ii) cells from an iPSC line;

(iii) cells from ICM of a preimplantation blastocyst cultured in vitro;

(iv) cells from ICM of a post-implantation blastocyst cultured in vitro; and

(v) cells from an embryo of 8C stage to morula stage cultured in vitro.

Non-limiting PSCs include but are not limited to any established cell lines in the art, such as human ESC lines, such as H1 (male) , H9 (female) , HN10 (female) , HUES1 (female) and WIBR3 (female) ; human iPSC lines, such as CBC14 (female) , C11 (female) , Phoenix (female) , DiPS 1016SevA (male) , STiPS O-XX1 (female) , and UH10 (male) .

The term “teratoma” as used herein is characterized as a model for multi-lineage human or other animals of interest developed with cell types represented across all three germ layers. The teratoma can be utilized to enable assaying of effect of genetic perturbations simultaneously across multiple cell types, and can be sculpted molecularly via microRNA (miRNA) -regulated suicide gene expression to enrich for specific tissues. Teratoma can be used as a platform for modeling multi-lineage development, pan-tissue functional genetic screening, and tissue engineering.

The term “organoid” as used herein refers to a 3D “mini-organ” derived from PSC,

PSC/ICLC or 8CLC in which the cells spontaneously self-assemble into differentiated, functional cell types that structurally and functionally mimic their in vivo counterparts. Organoids may be used to model specific development of human being or animals of interest in a 3D context, which is especially beneficial for modeling rare genetic diseases or cancers.

The term “embryoid body (EB) ” as used herein refers to aggregates of pluripotent cells that are induced to differentiate by a combination of a change in culture medium (removal of factors that support pluripotency) and allow the cells to interact in 3D structures. EB form a 3D structure under differentiation condition and is commonly used as a starting material for directed differentiation. EBs contain different germ layer cell types, and can be used as a quick model to generate target cell types in vitro.

II. Culture Media

The culture media disclosed herein are chemically defined media, which can efficiently convert primate PSCs from a primed state to a preimplantation ICM-like state to produce

PSC/ICLC within 2 weeks without picking colonies. The culture media of the subject application can also convert primate PSCs from a primed state and/or a preimplantation ICM-like state to an 8C like state to produce 8CLCs in approximately one week. Therefore, this kind of culture media can also be called as “conversion culture media” in the subject application. In some embodiments, the culture medium of the present application can also support derivation, survival after passage and/or revival, self-renewal, and proliferation of cells in a preimplantation ICM-like state. In some other embodiments, the culture medium of the present application can also support survival after passage and/or revival, self-renewal and proliferation of cells in a preimplantation ICM-like state on an extracellular matrix without the need for feeder cells or conditioned medium. In some embodiments, the culture medium of the present application can support survival after passage and/or revival, self-renewal and proliferation of cells in a preimplantation ICM-like state in suspension without the need for feeder cells or conditioned medium. In some other embodiments, the culture medium of the present application can support survival after passage and/or revival, self-renewal, and proliferation of cells in a preimplantation ICM-like state on feeder cells. Preferably, the chemically defined culture media of the subject application are serum-free.

The culture media of the subject application contain a basal medium capable of supporting cell growth, especially capable of supporting growth of human and non-human primate PSCs, supplemented with a PRC inhibitor and/or an EZH2 inhibitor, a HDAC inhibitor and a WNT/β-catenin signaling inhibitor, and optionally one or more components selected from a group consisting of L-ascorbic acid, an activator of JAK/STAT3 signaling, and an inhibitor of MAPK/ERK signaling. Preferred basal medium used in the subject application is a mixture of Advanced DMEM/F12 and Neurobasal Medium in a ratio of 1: 1 (v/v) . It should be understood that the inhibitor of SAH can also inhibit PRC and EZH2. Therefore, in some embodiments, the PRC inhibitor and/or EZH2 inhibitor (PRC/EZH2 inhibitor) is a SAH inhibitor. In the context of the subject application, the term “SAH/PRC/EZH2 inhibitor” refers to an inhibitor of SAH, PRC and/or EZH2.

The presence of SAH/PRC/EZH2 inhibitor in the described culture conditions is critical for inducing multiple regulators including STELLA, DNMT3L, and MAEL that govern the human

pluripotency network. STELLA is a DNA methylation regulator. Its ectopic over-expression in somatic cells can induce comprehensive DNA demethylation by interfering with the function of UHRF1, a DNA methylation regulator. The dysfunction of UHRF1 caused by STELLA deletion would lead to the accumulation of abnormal DNA methylation during oogenesis (Li et al., 2018) . The induction of STELLA was found to be dose dependent. The inventors further uncovered the functional role of STELLA and found that STELLA knock out hinders the induction of

PSCs/ICLCs and 8CLCs. During primed PSCs to

PSCs/ICLCs conversion, preimplantation ICM markers including KLF17, DPPA5, DNMT3L, TFCP2L1, and MAEL fail to be induced upon STELLA deletion. During primed PSCs and

PSCs/ICLCs to 8CLCs conversion, 8C markers including TPRX1, ZSCAN4, YPEL2, ZNF280A fail to be induced upon STELLA deletion. As demonstrated in the subject application, global DNA methylation levels are significantly higher in STELLA knock out cells compared to wild-type cells during 4CL or e4CL conversion. Thus, STELLA is necessary for controlled DNA demethylation during conversion to

PSCs/ICLCs and 8CLCs. Altogether, the subject application discovers that adding SAH/PRC/EZH2 inhibitors promotes induction of human

PSCs/ICLCs and 8CLCs through rewiring histone modification and DNA methylation landscape.

Any substances that can act as an inhibitor of SAH/PRC/EZH2 can be used in the culture media of the subject application, which include but are not limited to DZNep (CAS NO: 102052-95-9, a SAH inhibitor) and CPI-1205 (CAS NO: 1621862-70-1, a PRC/EZH2 inhibitor) . The SAH/PRC/EZH2 inhibitors can be used alone or in combination in the culture media of the subject application, generally in their respective conventional amounts which will not lead to cell death. For example, DZNep can be used in the media at a final concentration of 5 to 80 nM, preferably 5 to 50 nM, and CPI-1205 can be used in the media at a final concentration of 0.5 to 5 mM, preferably 1 to 3 mM. In one or more embodiments, the SAH/PRC/EZH2 inhibitor is a PRC inhibitor.

Any substances that can act as an inhibitor of HDAC can be used in the culture media of the subject application, which include but are not limited to TSA, VPA and NaB. The HDAC inhibitors can be used alone or in combination in the culture media of the subject application, generally in their respective conventional amounts which will not lead to cell death. For example, TSA can be used in the media at a final concentration of 3 to 30 nM, preferably 3 to 25 nM, VPA can be used in the media at a final concentration of 0.25 to 2 mM, preferably 0.5 to 1.5 mM, and NaB can be used in the media at a final concentration of 0.25 to 2 mM, preferably 0.5 to 1.5 mM.

The inventors also find that 8CLCs can be obtained with the culture media of the subject application from primed PSCs and/or

PSCs/ICLCs when both the SAH/PRC/EZH2 inhibitor and the HDAC inhibitor are used in a higher concentration (also called dose optimized SAH/PRC/EZH2 inhibitor and HDAC inhibitor) . Specifically, in some embodiments, in order to produce 8CLCs, DZNep may be used at a concentration of 40 nM or higher, such as 40 to 80 nM, preferably about 50 nM; CPI-1205 may be used at a concentration of 2 mM or higher, such as 2 to 5 mM, preferably about 3 mM; TSA may be used at a concentration of 10 nM or higher, such as 10 to 30 nM, preferably about 20 nM; VPA may be used at a concentration of 1.0 mM or higher, such as 1.0 to 2.0 mM, preferably about 1.5 mM; and NaB may be used at a concentration of 1.0 mM or higher, such as 1.0 to 2.0 mM, preferably about 1.5 mM, when each of them is used alone. It should be understood when two or more of the SAH/PRC/EZH2 inhibitors or two or more of the HDAC inhibitors are used, the final concentration of each SAH/PRC/EZH2 inhibitor or each HDAC inhibitor should be reduced to an amount sufficient to induce 8CLCs by combination of these SAH/PRC/EZH2 inhibitors or HDAC inhibitors. These amounts could readily be determined by the skilled artisan based on the disclosure of the subject application and the conventional knowledge of the art.

Furthermore, it should also be understood that excessive amount of SAH/PRC/EZH2 inhibitor and HDAC inhibitor may cause cell death. Thus, in order to induce

PSCs/ICLCs while reducing cell death as much as possible, either one of or both the SAH/PRC/EZH2 inhibitor and HDAC inhibitor may be used in a relatively low concentration. Specifically, DZNep can be used at a final concentration of 5 to 15 nM, preferably about 10 nM, CPI-1205 can be used at a final concentration of 0.5 to 3 mM, preferably about 1 mM, TSA can be used at a final concentration of 3 to 10 nM, preferably 4 to 6 nM, more preferably about 5 nM, VPA can be used at a final concentration of 0.25 to 1 mM, preferably 0.5 mM, and NaB can be used at a final concentration of 0.25 to 1 mM, preferably 0.5 mM, when each of them is used alone. In some embodiments, the SAH/PRC/EZH2 inhibitor can be used in a relatively high concentration, for example, DZNep can be used at a final concentration of 5 to 80 nM, preferably 5 to 50 nM, CPI-1205 can be used in the media at a final concentration of 0.5 to 5 mM, preferably 1 to 3 mM, while the HDAC inhibitor is used in a relatively low concentration, for example, TSA is used at a final concentration of 3 to 10 nM, preferably 4 to 6 nM, VPA is used at a final concentration of 0.25 to 0.5mM, and NaB is used at a final concentration of 0.25 to 0.5 mM. In some embodiments, the SAH/PRC/EZH2 inhibitor is used in a relatively low concentration, for example, DZNep is used at a final concentration of 5 to 15 nM, CPI-1205 is used at a final concentration of 0.5 to 2 mM, while the HDAC inhibitor can be used in a relatively high concentration, for example, TSA can be used in the media at a final concentration of 3 to 30 nM, preferably 3 to 25 nM, VPA can be used in the media at a final concentration of 0.25 to 2 mM, and NaB can be used in the media at a final concentration of 0.25 to 2 mM. Such culture media can convert primate PSCs to

PSCs/ICLCs.

As used herein, WNT/β-catenin signaling inhibitors include tankyrase inhibitors which inhibit canonical WNT signaling. Any known tankyrase inhibitors can be used, especially those generally used in culture of stem cells, are preferred, which include but are not limited to IWR1 (CAS No.: 1127442-82-3) and XAV939 (CAS No.: 284028-89-3) . Tankyrase inhibitors can be used in an amount commonly used in culturing stem cells. Exemplary final concentration of the tankyrase inhibitors may be in a range of from 2 to 8 μM, preferably 3 to 6 μM. For example, for IWR1 and XAV939, their respective final concentration in the culture media of the subject application may be in a range of from 2 to 8 μM, preferably 3 to 6 μM, more preferably about 5 μM. Two or more tankyrase inhibitors can be used in combination, with reduced amount for each of the inhibitors.

L-ascorbic acid is found to improve generation and maintenance of mouse iPSCs (close to mouse ESCs) from somatic cells through enhancing Jumonji-domain-containing histone demethylase, as described in Application number CN 200910041331.9, the content of which is incorporated herein by reference. Therefore, the inventors hypothesize that L-ascorbic acid has similar effects on formation of primate preimplantation ICM-like state. With proper testing, the inventors found that it potently increases expression levels of ICM specific genes such as DNMT3L, STELLA, DPPA5, and KLF17, when used at a final concentration of 40 to 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 subject application, which refer to similar compounds with similar structure and antioxidant activity to L-ascorbic acid. The derivatives are more stable or easy to be absorbed by cells while maintaining the biological activity of L-ascorbic acid. The derivatives of L-ascorbic acid include but are not limited to L-ascorbic acid phosphate and L-ascorbic acid organic ester, such as L-ascorbic acid palmitate. Amount of the derivative in the subject media is not limited, but it generally should be sufficient to produce sufficient amount of L-ascorbic acid as defined above.

One or more activators of JAK/STAT3 signaling can be added into the culture media that can cooperate to induce subset of early embryo specific gene of the subject application. Any known JAK/STAT3 activators can be used, especially those generally used in culture of stem cells, are preferred. Exemplary final concentration of the JAK/STAT3 activators may be in a range of 10 to 50 ng/mL. One kind of such JAK/STAT3 activators is LIF. LIF used herein refers to leukemia inhibitory factor, which is a growth factor commonly added to culture stem cells. Preferably LIF is a human LIF. JAK/STAT3 activator can be used in an amount commonly used in culturing stem cells. For example, for LIF, especially human LIF, its final concentration in the culture media of the subject application may be in a range of from 10 to 50 ng/mL, preferably 10 to 30 ng/mL, more preferably about 20 ng/mL.

One or more inhibitors of MAPK/ERK signaling can be added into the culture media which help to reduce DNA methylation in cooperation with other components in the media of the subject application. Any known MAPK/ERK inhibitors can be used, especially those generally used in culture of stem cells, are preferred. One kind of such MAPK/ERK inhibitors is PD0325901 (CAS No.: 391210-10-9) . MAPK/ERK inhibitors can be used in an amount commonly used in culturing stem cells. Exemplary final concentration of the MAPK/ERK inhibitors may be in a range of 0.5 to 3 μM, preferably 0.5 to 1.5 μM. For example, for PD0325901, its final concentration in the culture media of the subject application may be in a range of 0.5 to 3 μM, preferably 0.5 to 1.5 μM, more preferably about 1 μM.

In one or more preferred embodiments, the culture medium of the present application comprises DZNep at a final concentration of 5 to 15 nM or CPI-1205 at a final concentration of 0.5 to 2 mM; TSA at a final concentration of from 3 to 30 nM, or VPA at a final concentration of 0.25 to 2 mM or NaB at a final concentration of 0.25 to 2 mM, preferably, TSA at a final concentration of from 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 each at a final concentration of 3 to 6 μM. In one or more embodiments, the culture medium of the present application comprises DZNep at a final concentration of 5 to 80 nM, preferably 5 to 50 nM or CPI-1205 at a final concentration of 0.5 to 5 mM, preferably 0.5 to 3 mM; TSA at a final concentration of from 3 to 10 nM, or VPA at a final concentration of 0.25 to 0.5 mM or NaB at a final concentration of 0.25 to 0.5 mM; 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 each at a final concentration of 3 to 6 μM. More preferably, the culture medium of the present application comprises 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. These culture media are preferably used to convert primate PSCs to

PSCs/ICLCs.

In one or more preferred embodiments, the culture medium of the present application comprises DZNep at a final concentration of 40 to 70 nM or CPI-1205 at a final concentration of 2 to 4 mM; TSA at a final concentration of from 10 to 30 nM, or VPA at a final concentration of 0.5 to 1.5 mM or NaB at a final concentration of 0.5 to 1.5 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 each at a final concentration of 3 to 6 μM. More preferably, the culture medium of the present application comprises 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. These culture media are preferably used to convert primate PSCs or

PSCs/ICLCs to 8CLCs.

The culture media of the subject application can further comprise at least one or more additives selected from a group consisting of an extracellular matrix, an activator of ACTIVIN/NODAL signaling and a ROCK inhibitor.

Compared to primed human PSCs, expression levels of NODAL (an activator of ACTIVIN/NODAL signaling) are increased in

PSCs/ICLCs and 8CLCs derived by methods described herein. This observation indicates that ACTIVIN/NODAL signaling is endogenously/automatically activated in the conversion process and during self-renewal. Therefore, in some embodiments of the subject application, the culture medium further comprises an activator of ACTIVIN/NODAL signaling to accelerate the conversion process. Any known activators of ACTIVIN/NODAL signaling can be added to the culture medium of the subject application, which include but are not limited to human ACTIVIN A and human NODAL, the amino acid sequences of which are well 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 to 25 ng/mL, preferably about 20 ng/mL. A combination of human ACTIVIN A and human NODAL can also be used. Generally, the total concentration of human ACTIVIN A and human NODAL in the culture medium is in a range of 10 to 25 ng/mL, preferably about 20 ng/mL.

Upon conversion to

PSCs/ICLCs and/or 8CLCs, inhibition of ROCK signaling is no longer required for survival after passaging as single cells. Nevertheless, supplying with a ROCK inhibitor at a low concentration increases the yield of

PSCs/ICLCs and 8CLCs, which will be beneficial for scaling up the culture. Thus, in some embodiments of the invention, the culture medium further includes a ROCK inhibitor. Any known ROCK inhibitors can be used in the culture medium of the present application, which include but are not limited to Y27632 (CAS No.: 146986-50-7) , thiazovivin (CAS No.: 1226056-71-8) and hydroxyfasudil (CAS No.: 105628-72-6) . ROCK inhibitor can be used at a final concentration in a range of 0.5 to 2 μM, preferably about 1 μM. Two or more ROCK inhibitors can be used in combination, with their total concentration in the culture medium in a range of 0.5 to 2 μM, preferably about 1 μM.

The inventors find that when the PSCs are cultured in the culture medium of the subject application, they can be converted and maintained in a suspension culture without feeder cells, and the converted cells can self-renew and propagate as sphere-like colonies. Therefore, in some embodiments of the present application, the described methods, culture conditions and culture media are feeder-free.

In some other embodiments, the inventors find that during conversion and maintenance, supplying an extracellular matrix will promote sphere-shape colonization. In this condition, 90%or more of PSCs could be transformed into dome-shape colonies during conversion, which express ICM marker such as DNMT3L and KLF17. Therefore, in some embodiments, an extracellular matrix is used in the medium for culturing the

PSCs/ICLCs and 8CLCs. Extracellular matrix is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma (Matrigel ^TM or Geltrex ^TM or ECM ^TM) or a matrix that includes human matrix proteins collagen IV and at least one member selected from fibronectin, laminin, and vitronectin. The extracellular matrix generally is present in an amount of 0.1%to 0.5% (v/v) in the culture medium of the present application. A combination of different kinds of extracellular matrices can be used, if necessary, and the total amount thereof should also be in the range of 0.1%to 0.5% (v/v) in the culture medium. Preferably, the extracellular matrix generally is present in an amount of about 0.2% (v/v) in the culture medium of the present application.

Therefore, in one or more preferred embodiments, the culture medium of the present application comprises:

(A) DZNep at a final concentration of 5 to 15 nM or CPI-1205 at a final concentration of 0.5 to 2 mM, and TSA at a final concentration of from 3 to 30 nM, or VPA at a final concentration of 0.25 to 3 mM or NaB at a final concentration of 0.25 to 3 mM, preferably TSA at a final concentration of from 3 to 10 nM, or VPA at a final concentration of 0.25 to 1 mM or NaB at a final concentration of 0.25 to 1 mM; or DZNep at a final concentration of 5 to 80 nM, preferably 5 to 50 nM or CPI-1205 at a final concentration of 0.5 to 5 mM, preferably 0.5 to 3 mM, and TSA at a final concentration of from 3 to 10 nM, or VPA at a final concentration of 0.25 to 0.5 mM or NaB at a final concentration of 0.25 to 0.5 mM;

(B) L-ascorbic acid at a final concentration of 40 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;

(E) IWR1 or XAV939 at a final concentration of 3 to 6 μM; and is further supplemented with:

(4) Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; and an 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; or Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; or an extracellular matrix in an amount of 0.1%to 0.5% (v/v) . These culture media are preferably used to convert primate PSCs to

PSCs/ICLCs.

More preferably, the culture medium of the present application comprises 10 nM of DZNep or 1 mM of CPI-1205; 5 nM of TSA, or 0.5 mM of VPA, or 0.5 mM of NaB; 50 μg/mL of L-ascorbic acid; 20 ng/mL of LIF; 1 μM of PD0325901; and 5 μM of IWR1 or 5 μM of XAV939; and is further supplemented with (1) 20 ng/mL of ACTIVIN A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an extracellular matrix; or (2) 20 ng/mL of ACTIVIN A or NODAL, and 1 μM of Y27632, thiazovivin or hydroxyfasudil; (3) 20 ng/mL of ACTIVIN A or NODAL, and 0.2% (v/v) of an extracellular matrix; or (4) 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an 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 an extracellular matrix. These culture media are preferably used to convert primate PSCs to

PSCs/ICLCs.

In one or more preferred embodiments, the culture medium of the present application comprises DZNep at a final concentration of 40 to 70 nM or CPI-1205 at a final concentration of 2 to 4 mM; TSA at a final concentration of from 10 to 30 nM, or VPA at a final concentration of 0.5 to 1.5 mM or NaB at a final concentration of 0.5 to 1.5 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 each at a final concentration of 3 to 6 μM; and is further supplemented with:

(5) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; or Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; or an extracellular matrix in an amount of 0.1%to 0.5% (v/v) . These culture media contain dose optimized SAH/PRC/EZH2 inhibitor (s) and HDAC inhibitor (s) and thus are preferably used to convert primate PSCs or

PSCs/ICLCs to 8CLCs.

More preferably, the culture medium of the present application comprises 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 is further supplemented with (1) 20 ng/mL of ACTIVIN A or NODAL, 1 μM of Y27632, thiazovivin or Hydroxyfasudil, and 0.2% (v/v) of an extracellular matrix; or (2) 20 ng/mL of ACTIVIN A or NODAL, and 1 μM of Y27632, thiazovivin or hydroxyfasudil; (3) 20 ng/mL of ACTIVIN A or NODAL, and 0.2%(v/v) of an extracellular matrix; or (4) 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2%(v/v) of an 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 an extracellular matrix. These culture media are preferably used to convert primate PSCs or

PSCs/ICLCs to 8CLCs.

In addition to the above-mentioned components, other additives commonly used in a culture medium for culturing stem cells can also be added in the culture medium of the subject application, which include but are not limited to serum replacement, such as N2 and/or B27; alternative carbon source, such as pyruvate, such as sodium pyruvate; non-essential amino acid; L-glutamine or its alternative, such as Glutamax ^TM supplement comprising L-alanyl-L-glutamine dipeptide in 0.85%NaCl; and antibiotic, such as penicillin, streptomycin, or a mixture of penicillin and streptomycin. These additives can be used in an amount commonly used in cell culture, especially culture of stem cells.

III. Methods

Culture media of the subject application can be used to reprogram primate somatic cells to

PSCs/ICLCs, to convert primate PSCs to

PSCs/ICLCs, and to convert primate PSCs or

PSCs/ICLCs to 8CLCs.

Therefore, one aspect of the present application discloses a method for reprogramming primate somatic cells to

PSCs/ICLCs, comprising culturing the somatic cells in a conversion culture medium comprising a SAH/PRC/EZH2 inhibitor, a HDAC inhibitor, L-ascorbic acid, an activator of JAK/STAT3 signaling, an inhibitor of MAPK/ERK signaling, and a tankyrase inhibitor, and optionally an activator of ACTIVIN/NODAL signaling and/or an ROCK inhibitor, with or without an extracellular matrix. The resultant

PSCs/ICLCs can be used in the method of converting

PSCs/ICLCs to 8CLCs. Preferably, the conversion cultures are those described in any embodiments of the present application.

Another aspect of the present application discloses a method for converting primate PSCs to

PSCs/ICLCs, or for converting primate PSCs or

PSCs/ICLCs to 8CLCs, comprising culturing the primate PSCs in a conversion culture medium comprising a SAH/PRC/EZH2 inhibitor, a HDAC inhibitor, L-ascorbic acid, an activator of JAK/STAT3 signaling, an inhibitor of MAPK/ERK signaling, and a tankyrase inhibitor, and optionally an activator of ACTIVIN/NODAL signaling and/or an ROCK inhibitor, with or without an extracellular matrix. In the preferred embodiments, the conversion culture medium is the culture medium as defined in any of the above-mentioned embodiments.

In one or more preferred embodiments, the method is a method for converting primate PSCs to

PSCs/ICLCs, and the conversion culture medium is the culture medium as defined in any of the above-mentioned embodiments with a relative lower concentration of the SAH/PRC/EZH2 inhibitor and the HDAC inhibitor.

In some other preferred embodiments, the method is a method for converting primate PSCs or

PSCs/ICLCs to 8CLCs and the conversion culture medium is the culture medium as defined in any of the above-mentioned embodiments with a relative higher concentration of the SAH/PRC/EZH2 inhibitor and the HDAC inhibitor.

Conventional conditions for culturing stem cells may be used to convert PSCs to

PSCs/ICLCs or 8CLCs. For example, single primed PSC may be plated in conventional culture medium, such as mTeSR1 or E8, optionally supplemented with 5 to 15 μM ROCK inhibitor, such as Y27632. After a period of culture, for example, 24 hours later, the medium is switched to the culture medium of the subject application and the cells are continued to be cultured until the desired

PSCs/ICLCs or 8CLCs are produced. During culture, the culture medium may be refreshed as necessary, preferably refreshed daily. For passaging, cells may be dissociated into single cells with conventional methods and then plated and cultured again with the culture medium of the subject application until

PSCs/ICLCs or 8CLCs are formed. It is preferred that cells are passaged as single cells every 3 to 4 days with a split ratio of 1: 4 to 1: 8, preferably 1:6 to 1: 8, and generally, cells will be converted to

PSCs/ICLCs from primed PSC in approximately 2 weeks, will be converted to 8CLCs from primed PSC in about one week, and will be converted to 8CLCs from

PSCs/ICLCs in 3 to 5 days after culturing

PSCs/ICLCs with the culture medium containing a relatively higher concentration of the SAH/PRC/EZH2 inhibitor and the HDAC inhibitor. It should be understood that,

PSCs/ICLCs used for converting to 8CLC may be the

PSCs/ICLCs obtained by culturing primate PSC with any of the methods described herein, or may be the known

PSCs/ICLCs or the

PSCs/ICLCs prepared from any methods known in the art.

In general, cells may be cultured at 37℃ under a normoxic condition (5%CO ₂) or a hypoxic condition (5%CO ₂ and 5%O ₂) . There is no specific limitation on the time of culture, which can readily be determined by the skilled artisan based on the subject disclosure and the conventional techniques of the art. Plating concentration could be determined by the skilled artisan according to the common knowledge of the art and the actual production condition.

In some embodiments of the present application, cells may be cultured under one or more conditions selected from a group consisting of: (i) on feeder cells; (ii) on an extracellular matrix devoid of feeders; (iii) in suspension devoid of feeder cells; (iv) propagation in hypoxic or normoxic condition at about 37℃ temperature; (v) passaging as single cells every 3-4 days with a split ratio of 1: 4 to 1: 8; (vi) changing medium daily.

In some embodiments, for conversion primate PSCs to

PSCs/ICLCs, the single primed primate PSCs are plated on feeder in mTeSR1 or E8 medium supplemented with 5 to 15 μM ROCK inhibitor (such as Y27632) and cultured for a period of time, such as 24 hours, then the mTeSR1 or E8 medium is switched to the conversion culture medium of the subject application with relatively lower concentration of the SAH/PRC/EZH2 inhibitor and the HDAC inhibitor and cells are cultured under hypoxic or normoxic condition at about 37℃ temperature with the medium being refreshed daily. During culture, cells are passaged every 3 to 4 days as single cells with a split ratio of 1: 4 to 1: 8 until

PSCs/ICLCs are obtained. In some embodiments, the single primed primate PSCs are cultured as described above except plating the cells on about 1% (v/v) of an extracellular matrix, such as Geltrex ^TM, in DMEM-F12 coated plates instead of on feeder cell.

In some embodiments, for conversion primate PSCs to

PSCs/ICLCs, the single primed primate PSCs are plated on plate using mTeSR1 or E8 medium supplemented with 5 to 15 μM ROCK inhibitor (such as Y27632) and cultured for a period of time, such as 24 hours, then the mTeSR1 or E8 medium is switched to the conversion culture medium of the subject application with relatively lower concentration of the SAH/PRC/EZH2 inhibitor and the HDAC inhibitor and cells are cultured under hypoxic condition; after forming small spheres, the spheres are transferred to flasks for suspension culture with medium being refreshed daily; wherein cells are passaged every 4 to 5 days as single cells with a split ratio of 1: 4 to 1: 8 until

PSCs/ICLCs are obtained.

In some embodiments, for conversion to 8CLCs from primate PSCs, single primed PSCs are plated on feeder using mTeSR1or E8 medium supplemented with 5 to 15 μM ROCK inhibitor (such as Y27632) for a period of time, such as 24 hours, then the medium is switched to the conversion culture medium of the subject application which has a relatively higher concentration of SAH/PRC/EZH2 inhibitor and HDAC inhibitor and cells are cultured under hypoxic or normoxic condition with the medium being refreshed daily; wherein cells are passaged every 3 to 4 days as single cells with a split ratio of 1: 4 to 1: 8.

In some embodiments, for conversion to 8CLCs from

PSCs/ICLCs, single cells are dissociated from

PSCs/ICLCs and plated on feeders using the conversion culture medium of the subject application which has a relatively lower concentration of SAH/PRC/EZH2 inhibitor and HDAC inhibitor for a period of time, such as 24 hours, then the culture medium is switched to the conversion culture medium of the subject application which has a relatively higher concentration of SAH/PRC/EZH2 inhibitor and HDAC inhibitor and cells are cultured for 3 to 5 days without passaging, with the medium being refreshed daily.

In some embodiments, for conversion to 8CLCs from

PSCs/ICLCs, single cells are dissociated from

PSCs/ICLCs and suspended in conversion culture medium of the subject application which has a relatively lower concentration of SAH/PRC/EZH2 inhibitor and HDAC inhibitor for suspension culture for a period of time, wherein the conversion culture medium is supplemented with 5 to 15 μM ROCK inhibitor (such as Y27632) ; after forming small aggregates, medium is changed to the conversion culture medium of the subject application which has a relatively higher concentration of SAH/PRC/EZH2 inhibitor and HDAC inhibitor but without adding extra ROCK inhibitor (such as Y27632) for conversion for several days without passaging, with the medium being refreshed daily.

In some embodiments, teratomas are generated through injecting the naive PSCs/ICLCs, 8CLCs or reprimed cells as described in any embodiment of the present application subcutaneously into different organ or position of immune-deficient animals or recipient immunocompromised animals of interest and culturing the animals. The organ or position that can be injected includes but is not limited to back, neck, leg and testis. The immune-deficient animals or recipient immunocompromised animals include are but not limited to immune-deficient mice, including but are not limited to nude mice, SCID mice, NOD-SCID mice, NOD-scid-IL2Rg-/- (NSI) mice, CBA/N mice, Beige mice, Xid mice, NPG mice, URG mice, NPG-B2M mice, DK-NPg mice, hIL-3 NPG mice, hSCF1 NPG mice, hSCF2 NPG mice, NPG-Fah mice, and F344RG Rat. For example, for teratoma formation, cells were suspended with a prechilled 1: 1 mixture of DMEM/F12 and Matrigel. The cell suspension is injected subcutaneously into the immune-deficient animals of interest, such as 6 to 8-week-old male NOD-scid IL2Rg ^-/-mice. The animals are cultured under a normal condition. The teratoma can be seen within 3-4 weeks and can be collected for further use at 7-8 weeks.

In some embodiments, embryoid body is formed by culturing

PSCs/ICLCs, 8CLCs or reprimed PSCs as described in any embodiments disclosed herein as aggregates and in suspension in a culture medium that allows automatic differentiation. In some embodiments, the cells may be seeded in wells of plates to form aggregates. The plates for seeding the cells may be any known plates for producing EB, including but are not limited to ultralow attachment plate and AggreWell ^TM plate. Any known culture media allowing automatic differentiation can be used in the subject application. Culture conditions are well known in the art and generally culture is performed at 37 ℃ and 5%CO ₂.

In some embodiments, organoids are generated through first culturing primed PSC,

PSC/ICLC or 8CLC as described in any embodiment of the subject application to form uniform EBs and then culturing the EBs with a culture medium that allows targeted organ differentiation. The EB may be produced according to any of the methods for producing EB as described herein. The organ may be any organs of interest, including but is not limited to brain, liver, kidney, heart, lung, spleen and intestine. Generally, the primed PSC,

PSC/ICLC or 8CLC are cultured in a medium for forming EB, which may be changed every one or two days and transferred into the culture medium that allows targeted organ differentiation at about day 6 and then culture for another several days, such as another 2-6 days. In some embodiments, the cells are seeded in wells of an ultralow attachment plate in suspension in the presence of a culture medium that allows automatic differentiation (such as mTesR supplemented with Y-27632) to form EB; the culture medium may be changed to the same culture medium but without Y-27632 after culturing about 3 or 4 days; after culturing for one to three days, the EB is transferred to a low-attachment plate and cultured in the presence of a culture medium that allows targeted organ differentiation at about day 6 for further culture for another several days, such as another 2-6 days. The tissues thus formed may be cultured as an aggregate or in a 3D scaffold in a suitable differentiation medium for producing a desired organ, which may be performed on such as an orbital shaker installed in the incubator with a rotation speed at 50-100 RPM, to produce organoids of organ of interest. The 3D scaffold used herein may be any known materials in the art for forming an organoid. Exemplary 3D scaffold includes but is not limited to Matrigel and Geltrex, etc. The culture medium that allows targeted organ differentiation may be any known culture medium in the art allowing the EB to differentiate to an organ of interest. In some embodiments, for a brain organoid, a neural induction medium and a cerebral differentiation medium can be used. In some embodiments, The EB medium is changed every one or two days and then transferred into a neural induction medium at day 6 to continue to culture for another 4 days in the neural induction medium. EBs then will form neuroepithelial tissues. The tissues may be embedded into a 3D scaffold such as Matrigel droplets and cultured in a cerebral differentiation medium on orbital shaker installed in the incubator with a rotation speed at 50-100 RPM. The brain organoids can be collected for further use.

The reprimed PSCs as used herein may be obtained through guided differentiation of

PSCs/ICLCs or 8CLCs with a method commonly used in the art. For examples, reprimed PSCs may be produced by culturing human ESCs or iPSCs in a commercial culture medium, such as mTeSR1or Essential 8.

Uses of any of the conversion culture medium described in any of the embodiments of the present application in reprogramming primate somatic cells to

PSCs/ICLCs, in conversion of primate PSCs to

PSCs/ICLCs, or in conversion of primate PSCs or

PSCs/ICLCs to 8CLCs, in the manufacture of a culture medium or a kit for reprogramming primate somatic cells to

PSCs/ICLCs, or for converting primate PSCs to

PSCs/ICLCs, or for converting primate PSCs or

PSCs/ICLCs to 8CLCs, and in the manufacture of a teratomas, a organoid or an embryoid body or in the manufacture of a culture medium or a kit for producing a teratomas, a organoid or an embryoid body, are also included in the subject application.

In some embodiments, the subject application also comprises use of a SAH/PRC/EZH2 inhibitor and a HDAC inhibitor in the manufacture of a culture medium or a kit reprogramming primate somatic cells to iPSCs, or for converting primate PSCs to

PSCs/ICLCs, or for converting primate PSCs or

PSCs/ICLCs to 8CLCs, or for producing a teratomas, an organoid or an embryoid body. Preferably, the culture medium or the kit may further comprise one or more components selected from a group consisting of L-ascorbic acid, an activator of JAK/STAT3 signaling, an inhibitor of MAPK/ERK signaling, and a tankyrase inhibitor, and an optional activator of ACTIVIN/NODAL signaling and an optional ROCK inhibitor (such as Y27632) , and optional an extracellular matrix.

In some other embodiments, the methods for converting primate PSCs to

PSCs/ICLCs, and converting primate PSCs or

PSCs/ICLCs to 8CLCs may comprise a genetically engineering step to reduce the activity of SAH, PRC and/or EZH2 of the PSCs, and/or to reduce the activity of HDAC of the cells, by knockdown and/or knockout of one or more relevant genes in the cells, before culturing the primate PSCs with the culture medium of the subject application. Preferably, to reduce the activity of SAH, PRC and/or EZH2 of the PSCs, expression of any of the SAH, PRC and EZH2 regulators can be knocked down by, such as a siRNA technique, or any of the genes can be knocked out by, such as CRISPR/Cas9 technique. Similarly, the expression of HDAC regulators can be knocked down or knocked out by the same above-mentioned means. After knocking down or knocking out, the resultant cells can be cultured in any of the culture media of the subject application in accordance with the aforementioned methods. In some embodiments, when the activity of SAH, PRC and/or EZH2 of the PSCs is reduced, the culture medium used for culturing the genetically engineered primate PSCs may or may not contain the SAH/PRC/EZH2 inhibitor. Similarly, if the activity of HDAC of the PSCs is reduced, the culture medium may or may not contain the HDAC inhibitor. In the case that both the activity of SAH, PRC and/or EZH2 and the activity of HDAC are reduced, the culture medium may contain neither the SAH/PRC/EZH2 inhibitor nor the HDAC inhibitor, or may contain either the SAH/PRC/EZH2 inhibitor or the HDAC inhibitor.

Therefore, in some embodiments, the subject application further provides culture mediums comprising neither the SAH/PRC/EZH2 inhibitor nor the HDAC inhibitor, or comprising either the SAH/PRC/EZH2 inhibitor or the HDAC inhibitor, with other components and amounts identical to any of the above-mentioned embodiments for culture medium in Part II. In some embodiments, the culture medium may contain reagents for liposome transfection. For example, in the above-mentioned methods, the primate PSCs are cultured in a culture medium containing vectors for expression shRNA directed to, such as any of the SAH, PRC and EZH2 regulators, and reagents for liposome transfection for transfecting the vector into the PSCs for genetically engineering, in addition to other components described in the culture medium described in Part II, and the culture medium may or may not contain the SAH/PRC/EZH2 inhibitor.

IV. Cells

The subject application provides teratomas, organoids and EBs obtained by any of the methods described herein, and cells/tissues from the teratomas, organoids and EBs. Preferably, the teratomas, organoids and EBs are derived from human cells and the cells/tissues from the teratomas, organoids and EBs are human cells and tissues. In the present application, the human cells/tissues have transcriptome close to human in vivo cell/tissue types, including but not limited to airway epithelial cell, epithelial cell, epithelial progenitor, radial glia, cycling radial glia, neuron, melanoblast, mesenchymal stem cell, cycling mesenchymal stem cell, adipogenic mesenchymal stem cell, vascular endothelial cell, smooth muscle cell, pericyte, fibroblast, fibroblast progenitor, hemogenic endothelial cell, immune cells, erythrocyte, primitive gut endodermal cell, trophoblast, cytotrophoblast, villous cytotrophoblast, placental endothelial cell, granulocyte-macrophage progenitor, hemogenic endothelial cell, mast cell, lymphocyte, dopaminergic neuron progenitor, dopaminergic neurons, GABAergic neurons, Glutamatergic neurons, neuroblasts, serotonergic neurons, cycling neural progenitor, granulocyte-macrophage progenitor, oligodendrocyte precursor cell, Schwann cell precursor, endothelial cell, arterial endothelial cell, midgut epithelial cell, hindgut epithelial cell, neural stem cell, neuroblast, neuroepithelial, retinal progenitor.

The subject application also provides isolated primate

PSCs/ICLCs. The

PSCs/ICLCs of the present application have transcriptome close to human preimplantation ICM, have transposable element profile close to human preimplantation ICM, have DNA methylome close to human preimplantation ICM, have chromatin landscape close to human preimplantation ICM, and have metabolic state close to human preimplantation ICM.

As used herein, the term “close to” is intended to mean “substantially identical” or “without substantial difference” . The skilled artisan of the art is able to acknowledge, based on the common knowledge of the art, that the cells of the subject application, including cells from ICLC or from 8CLC of the present application, are substantially identical to the native ICM cells or 8C embryo cells, even though there may have some minor differences.

Preferably, the

PSCs/ICLCs of the present application exhibit significantly higher expression level of preimplantation ICM markers, including KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, MAEL, and REX1. More preferably, the expression level of at least one of the above-mentioned preimplantation ICM markers in

PSCs/ICLCs of the present application is 10 or more times higher than the expression level of that corresponding preimplantation ICM markers in primed human PSCs; preferably the expression level of all the above-mentioned preimplantation ICM markers in

PSCs/ICLCs of the present application is 10 or more times of the expression level of the corresponding preimplantation ICM marker in primed human PSCs.

Preferably, the

PSCs/ICLCs of the present application are further characterized by one or more of the following characteristics:

1) being able to self-renew and maintain pluripotency in culture;

2) maintaining genomic stability in culture according to karyotype;

3) being able to give rise to cells of the 3 germ layers;

4) being able to give rise to primordial germ cell-like cells;

5) being able to integrate to mouse embryo and contribute to embryonic and extraembryonic tissues;

6) being able to transit to extraembryonic cell fate in vitro; and

7) being able to form blastocyst-like structures in vitro.

Such

PSCs/ICLCs can be obtained by culturing primate PSCs by any of the methods as described in any of the embodiments of the subject application. Therefore, in some embodiments, the subject application also includes cells, specifically,

PSCs/ICLCs, obtained by any of the methods described herein.

The subject application also provides isolated 8CLCs, which express 8C state specific markers, including ZSCAN4, TPRX1, ZIM3, ZSCAN5B, ZNF280A and ARGFX, at a level substantially higher than cells of preimplantation ICM-like state or primed state. Preferably, at least one of the specific markers exhibits an expression level which is 5 or more times higher than the expression level of the corresponding 8C specific marker in primed PSCs or

PSCs/ICLCs. Preferably, all the above-mentioned specific markers exhibit an expression level which is 5 or more times higher than that the expression level of the corresponding 8C specific markers in primed PSCs or

PSCs/ICLCs.

Preferably, the 8CLCs of the present application have transcriptome, transposable element profile, and chromatin landscape close to human 8C stage embryos. More preferably, the 8CLCs of the present application are further characterized by one or more of the following characteristics:

1) maintaining genomic stability in culture according to karyotype;

2) being able to give rise to cells of the 3 germ layers;

3) being able to give rise to primordial germ cell-like cells;

4) being able to integrate to mouse embryos and contribute to embryonic and extraembryonic tissues;

5) being able to transit to extraembryonic cell fate in vitro; and

6) being able to form blastocyst-like structures in vitro.

PSCs/ICLCs obtained by reprogramming of somatic cells with the conversion culture medium of the present application are also contemplated in the subject application.

Cell cultures containing the cells of the present application, especially the

PSCs/ICLCs and/or the 8CLCs of the present application, are also contemplated in the present application. Culture medium described in any of the subject application can also be included in the cell cultures.

The subject invention will be described in the following non-limiting examples. It should be understood that these examples are only for illustrative purpose, but not for limiting the scope of the invention in various manner. Various variations, and modifications may be made within the spirit of the present application. The technologies involved, unless otherwise specified, are conventional technologies in various fields, such as molecular biology, cell biology, biochemistry, etc., which are well known to those skilled in the art.

Example 1: Generation of

PSCs/ICLCs and 8CLC

1.

PSCs/ICLCs Generation

Materials and methods

4CL basal medium

1: 1 mix of Neurobasal Medium (Gibco) and Advanced DMEM/F12 (Gibco) , supplemented with N2 supplement (1X, Gibco) , B27 supplement (1X, Gibco) (homemade N2 and B27 can be used) , Sodium Pyruvate (1X, Hyclone) , Non-Essential Amino Acid (NEAA) (Gibco) , Glutamax ^TM (1X, Gibco) and Penicillin-Streptomycin (1X, Gibco) .

4CL supplements

4CL medium 1, supplemented in the 4CL basal medium with:

SAH/PRC/EZH2 inhibitor (10 nM DZNep) , HDAC inhibitor (5 nM TSA) , L-ascorbic acid (50 μg/mL) , JAK/STAT3 activator (20 ng/mL human LIF) , MAPK/ERK inhibitor (1 μM PD0325901) , tankyrase inhibitor (5 μM IWR1) , ACTIVIN A/NODAL activator (20 ng/mL human ACTIVIN A) , extracellular matrix (0.2% (v/v) Geltrex ^TM) , optional ROCK inhibitor (1 μM Y27632) . Catalogues for these reagents and their substitutes are listed in Table 1.

Table 1

Cells

H9 human ESC line.

Procedures:

1) Maintenance of primed human PSCs

All provided human PSCs were routinely maintained on Matrigel ^TM or Geltrex ^TM coated plates in mTeSR1or E8 medium. Generally, cells were passaged every 4 to 5 days with 0.5 mM EDTA. For passaging, cells were washed with PBS once and treated with 0.5 mM EDTA for 5 mins. Then, EDTA was removed and cells were detached as small clumps using a Pasteur pipette with mTeSR1 or E8 medium. Primed human PSCs were grown in an incubator under normoxic conditions (37 ℃, 5%CO ₂) .

2) Conversion to

PSCs/ICLCs on feeders

One day before initiation of conversion, primed human PSCs were washed with PBS once and dissociated into single cells and plated at a density of 1,000 to 1, 500 cells/cm ² on feeder in mTeSR1 or E8 medium supplemented with 10 μM Y27632. Twenty-four hours later, culture medium was switched to 4CL medium 1. The culture medium was refreshed with the same medium every 24 hours. Colonies became round and domed-shape in 24 to 48 hours. Cells were passaged every 3 to 4 days. For passaging, cells were dissociated into single cells using TrypLE: 0.5 mM EDTA (1: 1) and plated at a density of 1,000 to 1, 500 cells/cm ² on feeder (feeders were seeded on Geltrex ^TM /Matrigel ^TM pre-treated plate) . The

PSCs/ICLCs induction and maintenance can be conducted under hypoxic condition (37 ℃, 5%CO ₂, 5%O ₂) , or normoxic condition (37 ℃, 5%CO ₂, 21%O ₂) , preferably hypoxia condition.

2. 8CLC Generation

Materials and methods

4CL basal medium

Same as example 1.

e4CL supplements

e4CL medium, supplemented in the 4CL basal medium with:

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, or 20 ng/ml human NODAL) , ROCK inhibitor (1 μM Y27632, or 1 μM thiazovivin , or 1 μM hydroxyfasudil) and extracellular matrix (0.2% (v/v) GeltrexTM or MatrigelTM) .

Cells

H9, H1, UH10 human ESC lines.

Procedures:

1) Conversion to 8CLCs from primed human PSCs

Primed human PSCs were cultured following the same procedures as described above. One day before initiation of the conversion, primed human PSCs were dissociated into single cells and plated at 2,000 to 3,000 cells/cm ² on feeder using mTeSR1 or E8 medium supplemented with 10 μM Y27632. Twenty-four hours later, culture medium was changed into e4CL medium, cells were cultured in incubator at 37℃, 5%CO ₂, hypoxic or normoxic condition. Medium was refreshed daily. Cells were passaged every 3 to 4 days. For passaging, cells were dissociated into single cells using TrypLE: 0.5 mM EDTA (1: 1) , plated at 2,000 to 3,000 cells/cm ² on feeder coated plates. Generally, cells were converted to 8CLCs in approximately one week.

2) Conversion to 8CLCs from

PSCs/ICLCs

One day before initiation of the conversion,

PSCs/ICLCs obtained in the above experiment were dissociated into single cells and plated at 2,000 to 3,000 cells/cm ² on feeders using 4CL medium 1 as described above. Twenty-four hours later, culture medium is changed into e4CL medium. Medium was refreshed daily. Cells were converted to 8CLCs in 3 to 5 days without passaging.

Experimental results

Results are shown in Figures 1-6. Fig. 1 shows induction of

PSCs/ICLCs and 8CLC that express

or 8C-specific genes, which are demonstrating by the immunostaining images for KLF17 and TPRX1 of primed H9 ESC untreated or converted by 4CL (day 12) or stepwise e4CL (day 5) as shown in Fig. 1 (b) , the heatmap showing the expression of preimplantation ICM-enriched genes in human

PSC cultured in NHSM,

5iLAF and 4CL, and human ICM cells as shown in Fig. 1 (c) , the heatmap showing the expression of totipotency genes in

ESC cultured in NHSM,

5iLAF or stepwise e4CL (day 5) , EPSC and human 8C-embryo cells as shown in Fig. 1 (d) , and the RT-qPCR validation of totipotency genes in primed H9 ESC cultured in direct e4CL (day 7) as shown in Fig. 1 (e) .

Fig. 2 describes transcriptome of

PSCs/ICLCs and 8CLC match with human embryo, which are demonstrated by the UMAP comparing the developmental rolling back from human E7 to E3 embryonic stages in stepwise or direct e4CL induction scRNA-seq time courses as shown in Fig. 2 (a) , the UMAP visualization of stepwise e4CL-day 5 cells as shown in Fig. 2 (b) which shows seven clusters with the encircled cluster 5 (8CLC) comprising 11.9 %of the whole population, the bubble plot representing the frequency of expression and average expression of representative pluripotency and totipotency genes in early human embryonic stages and primed ESC untreated or converted by 4CL (days 8 [passage 2] and 12 [passage 3] ) and e4CL (day 5 C5 [8CLC] and non-8CLC [all other clusters summed] ) as shown in Fig. 2 (c) , and the violin plot showing the log normalized expression of representative early human embryo-enriched TE in early human embryonic stages and human ESC passage 10 compared to primed ESC, 4CL-day 12

ESC and 8CLC, as shown in Fig. 2 (d) .

Karyotype of cells induced with

PSC medium (4CL) and 8CLC medium (e4CL) are normal after extended culture, as demonstrated by Fig. 3 (a) , which shows representative images of G-banding karyotype of primed H9 ESC and primed iPSC-4 clone cultured in 4CL for 15 passages, and Fig. 3 (b) , which shows representative images of G-banding karyotype of primed H9 ESC and iPSC-4 cultured in stepwise e4CL (day 5) .

Cells cultured in

PSC medium (4CL) and 8CLC medium (e4CL) have lower DNA methylation level compared with primed PSC, as demonstrated by Fig. 3 (a) , which shows violin plot showing global CpG methylation levels measured by RRBS of human PSC cultured in primed conditions, 4CL (day 12) , 5iLAF,

NHSM, stepwise e4CL (day 5) and direct e4CL (day 7) , human 8C-embryo and ICM, Fig. 3 (b) , which shows heatmap showing CpG methylation levels at a panel of imprinting control regions of human PSC cultured in primed conditions, 4CL (day 12) , 5iLAF,

NHSM and stepwise e4CL (day 5) , ICM and post-implantation embryo, and Fig 3 (c) , which shows genome browser tracks showing CpG methylation levels at the indicated

NHSM, stepwise e4CL (day 5) and direct e4CL (day 7) , human 8C-embryo and ICM.

Chromatin accessibility of

PSCs/ICLCs and 8CLC match with human embryo, as demonstrated by Fig. 5a to 5d, which respectively show UMAP visualization of gene score for all genes in the scATAC-seq of primed ESC untreated (red) or converted by 4CL (day 12; blue) or stepwise e4CL (day 5; green) , UMAP visualization based on panel a highlighting the gene score for primed (ZIC2) , shared

pluripotency/8CLC (DPPA3) and totipotency genes projected onto each individual cell in the scATAC-seq of primed ESC untreated or converted by 4CL (day 12) and stepwise e4CL (day 5) , and Genome browser tracks showing chromatin accessibility, H3K27ac level and transcription factor DNA binding motif location at the

pluripotency KLF17 and totipotency ZSCAN4 loci.

Fig. 6 shows that 8CLC can be enriched by sorting with TPRX1-GFP reporter. As shown in Fig. 6 (a) , EGFP was inserted into the TPRX1 locus of H9 ESC or HN10-DsRed ESC (used for the chimera experiments) , and the donor constructs used for generating the TPRX1-EGFP reporter cell lines. TPRX1-EGFP knock-in cells were validated by culture in stepwise e4CL (day 5) and immunostaining with anti-TPRX1, which coincided with the GFP ⁺ signal (left panel) . FACS analysis of TPRX1-EGFP cells in stepwise e4CL (day 5) shows the percentage of GFP ⁺cells (right panels) . Fig. 6 (b) shows bubble plot representing the frequency of expression and average expression of representative pluripotency and totipotency genes in early human embryonic stages and human ESC passage 10 compared to primed ESC, 4CL-day 12

ESC and sorted 8CLC.

Example 2: Generation of

PSCs/ICLCs using ESC or iPSCs

Materials and methods

4CL basal medium

Same as example 1.

4CL supplements

Same as example 1.

Cells

Human ESC lines: H1 (male) , HN10 (female) , HUES1 (male) , and WIBR3 (female) ; human iPSC lines: CBC14 (generated by the inventors, female) , C11 (generated by the inventors, female) , Phoenix (agift from Ulrich Martin’s lab, female) , DiPS 1016SevA (purchased from Harvard Stem Cell Institute, male) , STiPS O-XX1 (purchased from Harvard Stem Cell Institute, female) , UH10 (male) .

Procedures

Using the same procedures of example 1.

Experimental results

RT-qPCR data in Figure 7 showed that preimplantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced in

PSCs/ICLCs converted from multiple primed human PSC lines. It demonstrates that 4CL medium 1 is commonly applicable to human PSCs.

Example 3: Generation of

PSCs/ICLCs on extracellular matrix

Materials and methods

4CL basal medium

Same as example 1.

4CL supplements

Same as example 1.

Cells

H9 human ESC line.

Procedures

Using the same procedures of example 1, except plating cells on 1% (v/v) Geltrex ^TM in DMEM-F12 (cat#) coated plates instead of on feeder cells.

Experimental results

RT-qPCR data in Figure 8 showed that preimplantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced in

PSCs/ICLCs converted on Geltrex ^TM coated plates using 4CL medium 1, which is similar to

PSCs/ICLCs on feeder. It indicates that 4CL medium 1 is also effective without feeder cells.

Example 4: Generation of

PSCs/ICLCs in suspension

Materials and methods

4CL basal medium

Same as example 1.

4CL supplements

Same as example 1.

Cells

H9 human ESC line.

Procedures

Primed human PSCs were cultured following the same procedures of example 1. One day before initiation of the conversion, primed human PSCs were dissociated into single cells and plated 60,000 cells/well in Aggrewell ^TM800 plates using mTeSR1or E8 medium supplemented with 10 μM Y27632. Twenty-four hours later, culture medium was changed into 4CL medium 1, culture condition was then switched to hypoxic condition. Cells formed small spheres in 3 days. The spheres were then lifted and transferred to flasks (Greiner Bio-One, 658190) for suspension culture. Medium was refreshed daily. Cells were passaged every 4 to 5 days. For passaging, cells were dissociated into single cells using TrypLE: 0.5 mM EDTA (1: 1) , then cells were resuspended in 4CL medium 1 at a density of 150,000 cells/ml. The resuspended cells were added into flasks (Greiner Bio-One, 658190) for suspension culture. Cells formed small aggregates in 24 hours. Generally, cells were converted to

PSCs/ICLCs in approximately 3 weeks after initiation.

Experimental results

RT-qPCR data in Figure 9 showed that preimplantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced in

PSCs/ICLCs converted in suspension using 4CL medium 1. It indicates that 4CL medium 1 is also effective for suspension culture.

Example 5: Generation of

PSCs/ICLCs in 4CL without ECM/ROCK inhibitor/ACTIVIN/NODAL activator

Materials and methods

4CL basal medium

Same as example 1.

4CL supplements

4CL medium 2 (minus extracellular matrix) , supplemented in the 4CL basal medium with:

SAH/PRC/EZH2 inhibitor (10 nM DZNep) , HDAC inhibitor (5 nM TSA) , L-ascorbic acid (50 μg/mL) , JAK/STAT3 activator (20 ng/mL human LIF) , MAPK/ERK inhibitor (1 μM PD0325901) , tankyrase inhibitor (5 μM IWR1) , ACTIVIN A/NODAL activator (20 ng/mL human ACTIVIN A) , and ROCK inhibitor (1 μM Y27632) .

4CL medium 3 (minus ROCK inhibitor) , supplemented in the 4CL basal medium with:

SAH/PRC/EZH2 inhibitor (10 nM DZNep) , HDAC inhibitor (5 nM TSA) , L-ascorbic acid (50 μg/mL) , JAK/STAT3 activator (20 ng/mL human LIF) , MAPK/ERK inhibitor (1 μM PD0325901) , tankyrase inhibitor (5 μM IWR1) , ACTIVIN A/NODAL activator (20 ng/mL human ACTIVIN A) , extracellular matrix (0.2% (v/v) Geltrex ^TM) .

4CL medium 4 (minus ACTIVIN/NODAL activator) , supplemented in the 4CL basal medium with:

SAH/PRC/EZH2 inhibitor (10 nM DZNep) , HDAC inhibitor (5 nM TSA) , L-ascorbic acid (50 μg/mL) , JAK/STAT3 activator (20 ng/mL human LIF) , MAPK/ERK inhibitor (1 μM PD0325901) , tankyrase inhibitor (5 μM IWR1) , extracellular matrix (0.2% (v/v) Geltrex ^TM) , and ROCK inhibitor (1 μM Y27632) .

Cells

H9 human ESC line

Procedures:

Using the same procedures of example 1.

Experimental results

RT-qPCR data in Figure 10 showed that preimplantation ICM markers KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL, and REX1 are significantly induced in

PSCs/ICLCs converted using 4CL medium 2, 4CL medium 3, and 4CL medium 4, respectively. These results demonstrate that 4CL medium devoid of either Geltrex ^TM, ROCK inhibitor, or ACTIVIN/NODAL activator is also effective.

Example 6: Generation of 8CLC using male and female cell lines

Materials and methods

4CL basal medium

Same as example 1.

e4CL supplements

e4CL medium, supplemented in the 4CL basal medium with:

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) , L-ascorbic acid (50 μg/mL) , JAK/STAT3 activator (20 ng/mL human LIF) , MAPK/ERK inhibitor (1 μM PD0325901) , tankyrase inhibitor (5 μM IWR1, or 5 μM XAV939) , ACTIVIN A/NODAL activator (20 ng/mL human ACTIVIN A, or 20 ng/mL human NODAL) , ROCK inhibitor (1 μM Y27632, or 1 μM thiazovivin , or 1 μM hydroxyfasudil) and extracellular matrix (0.2% (v/v) Geltrex ^TM or Matrigel ^TM) .

Cells

H9, H1, UH10 human PSC lines.

Procedures:

1) Conversion to 8CLCs from primed human PSCs

Primed human PSCs were cultured following the same procedures of example 1. One day before initiation of the conversion, primed human PSCs were dissociated into single cells and plated at 2,000 to 3,000 cells/cm ² on feeder using mTeSR1or E8 medium supplemented with 10 μM Y27632. Twenty-four hours later, culture medium was changed into e4CL medium, cells were cultured in incubator at 37 ℃, 5%CO ₂, hypoxic or normoxic condition. Medium was refreshed daily. Cells were passaged every 3 to 4 days. For passaging, cells were dissociated into single cells using TrypLE: 0.5 mM EDTA (1: 1) , plated at 2,000 to 3,000 cells/cm ² on feeder coated plates. Generally, cells were converted to 8CLCs in approximately one week.

2) Conversion to 8CLCs from

PSCs/ICLCs

One day before initiation of the conversion,

PSCs/ICLCs were dissociated into single cells and plated at 2,000 to 3,000 cells/cm ² on feeders using 4CL medium. Twenty-four hours later, culture medium is changed into e4CL medium. Medium was refreshed daily. Cells were converted to 8CLCs in 3 to 5 days without passaging.

Experimental results

RT-qPCR data in Figure 11 showed that human 8C specific markers ZSCAN4, TPRX1, ZIM3, ZSCAN5B, ZNF280A, and ARGFX are significantly induced in 8CLCs converted from primed human PSCs or

PSCs/ICLCs.

Example 7: Generation of 8CLC in suspension

Materials and methods

e4CL basal medium

Same as example 1.

e4CL supplements

Same as example 6.

Cells

H9 human ESC line.

Procedures:

Conversion to 8CLCs from

PSCs/ICLCs in suspension

PSCs/ICLCs were cultured following the same procedures of example 1. One day before initiation of the conversion,

PSCs/ICLCs were dissociated into single cells and resuspended in 4CL medium at a density of 300,000 cells/ml. The cell suspension was added into flasks for suspension culture (Greiner Bio-One, 658190) . Twenty-four hours later, cells formed small aggregates and medium changed to e4CL without adding Y27632. Medium was refreshed daily, and cells were converted to 8CLCs in 3 to 5 days without passaging.

Experimental results

RT-qPCR data in Figure 12 showed 8C markers ZSCAN4, ARGFX, TPRX1, ZNF280A, and ZSCAN5B are significantly induced in 8CLCs converted in suspension using e4CL medium. It indicates that e4CL medium is also effective for suspension culture.

Example 8: Generation of 8CLC using ESC and iPSC

Materials and methods

e4CL basal medium

Same as example 1.

e4CL supplements

Same as example 6.

Cells

Human PSC lines: HN10 and UH10

Procedures:

Same as example 6.

Experimental results

RT-qPCR data in Figure 13 showed 8C markers ZSCAN4, ARGFX, TPRX1, ZNF280A, ZSCAN5B, DUXA, DUXB, MBD3L2, STELLA, KLF17, and KHDC1L are significantly induced in 8CLCs converted from multiple hPSC lines. It indicates that e4CL medium is commonly applicable to human PSCs.

Example 9: Teratoma generation

Materials and methods

DMEM/F12,

Cells:

Human 8CLC,

PSC/ICLC from Example 1, or reprimed PSC obtained by culturing human ESCs in mTeSR1.

Procedures

1) Animal preparation

Male NOD-scid-IL2Rg ^-/-mice were maintained in SPF facility until 6-8 weeks old.

2) Primed PSC,

PSC/ICLC and 8CLC suspension preparation

One million primed PSC,

PSC/ICLC or 8CLC were dissociated from culture, collected and resuspended with 200 μL prechilled 1: 1 mixture of DMEM/F12 and

The suspension was placed on ice until transplantation. Transplantation should be done immediately.

3) Transplantation

Primed,

PSC/ICLC or 8CLC suspension was collected to a 1 mL syringe and injected subcutaneously to 6-8 weeks old male NOD-scid-IL2Rg ^-/-mice.

To avoid stress response, mice need to be prepared in advanced at least one week before. For teratoma formation, one million cells were counted in suspension with a 200 μl prechilled 1:1 mixture of DMEM/F12 and Matrigel in 1 ml sterile injection syringe. Hold the mouse from back, exposing the abdomen to inject easily. The needle is penetrated into the skin inclined and manipulating needle horizontally to determine the subcutaneous location. The cell suspension was injected subcutaneously into 6 to 8-week-old male NOD-scid IL2Rg ^-/-mice and pushed out the needle slowly to prevent cell suspension outflow of the injection position.

4) Animal monitoring during teratoma formation

After injection, mice were maintained in SPF facility up to 8 weeks. Size of teratoma grew in injection site was observed by eyes. Usually, the teratoma is ready to be explanted upon 6-8 weeks post injection.

5) Collection of human cells from teratoma

Upon 8 weeks post injection, mice were sacrificed, and teratoma were explanted. The explanted teratoma was dissected for cryosection, cell isolation, single-cell transcriptomic analysis or other utility.

Results

Teratoma can be formed with primed cells,

PSCs/ICLCs (4CL) and 8CLC, as demonstrated by Fig. 3 (a) to 3 (c) , which show representative images of teratomas derived from primed ESC converted by 4CL (passage 15) and sorted 8CLC, hematoxylin and eosin staining of teratomas derived from primed ESC converted by 4CL (passage 15) and sorted 8CLC, and UMAP visualization of the identified cell types in scRNA-seq of teratomas derived from sorted 8CLC, e4CL-day 5 cells, 4CL

ESC and primed ESC.

Fig. 15 shows teratoma cell annotation for different starting cell type. UMAP visualization based on Figure 14 (c) highlights the identified cell types in the scRNA-seq datasets generated from primed ESC, 4CL

PSC, stepwise e4CL-day 5 cells and sorted 8CLC.

As shown in Fig. 16, embryonic (3 germ layers cell types) and extraembryonic trophoblast can be derived from

PSC/ICLC teratoma or 8CLC teratoma, wherein Fig. 16 (a) shows the contribution of the different teratomas to the identified cell types, and Fig. 16 (b) shows the relative contribution of the different teratomas to each embryonic (ectoderm, mesoderm, and endoderm) and extraembryonic (trophoblast) lineage.

Teratoma trophoblast cell subtypes in teratoma can further generate cytotrophoblast, villous cytotrophoblast and placental endothelial cell, as demonstrated in Fig. 17 (a) to 17 (d) , which respectively show UMAP visualization of the identified cell types in scRNA-seq of teratomas derived from sorted 8CLC, e4CL-day 5 cells, 4CL

ESC and primed ESC, the frequency of expression and average expression of marker genes in cell subtypes of the extraembryonic trophoblast lineage, the relative contribution of sorted 8CLC, e4CL-day 5 cells, 4CL

ESC and primed ESC-derived teratomas to cell subtypes of the extraembryonic trophoblast lineage, and the distribution and the expression of related markers in trophoblast cells from the indicated teratomas.

Annotation and quantification of teratoma immune cell subtypes, including granulocyte-macrophage progenitor, hemogenic endothelial cell, mast cell, lymphocyte and eruthrocyte, are shown in Fig. 18, and annotation and quantification of teratoma neural cell subtypes, including cycling EOMES ⁺ IPCs, DA progenitors, dopaminergic neurons, GABAergic neurons, glutamatergic neurons, immature neurons, neuroblasts, radial glial, serotonergic neurons, are shown in Fig. 19.

Example 10. Brain organoid generation

Materials and methods

DMEM-F12 (Invitrogen, cat. no. 11330-032 or 31330-038, depending on location)

Neurobasal medium

GlutaMAX (Invitrogen, cat. no. 35050-038)

MEM-NEAA (Sigma, cat. no. M7145) Penicillin-Streptomycin

N2 supplement (Invitrogen)

B27 without vitamin A (Invitrogen, cat. no. 12587010)

B27 with vitamin A (Invitrogen, cat. no. 17504044)

Y27632 (Axon Medchem 1681) ,

2-mercaptoethanol (Merck, cat. no. 8057400005)

Heparin (Sigma, cat. no. H3149)

Insulin (Sigma, cat. no. I9278-5ML)

BDNF (R&D 248-BDB-250)

Reduced growth factor Matrigel (BD Biosciences 356230)

U-bottom ultralow attachment plates, 96 well (Corning, cat. no. CLS7007)

Ultralow attachment plates, 24 well (Corning, cat. no. CLS3473)

60×15mm style dish, Ultra-Low Attachment Surface (Corning, cat. No. 3261)

Medium:

Cells

Human 8CLC,

Procedures

Day0

Grow cells until they are 70–80%confluent, then the colonies be dissociated to single cells with Accutase, and cells were plated in 150 uL of EBM in each well of ultralow attachment 96-well plates (9000 cells/well) , culture plates to form uniform EBs.

Day1

Observe the plate under the tissue culture microscope 24 h later. Small EBs with clear borders could be visible. Continue to culture EBs in the tissue culture incubator at 37 ℃ and 5%CO ₂.

Day2

Feed the EBs with 150ul EBM by gently aspirating approximately half of the medium without disturbing the EB at the bottom of the well.

Day4

Feed the EBs with EBM (without Y27632) .

Day6

Transfer each EB with a cut 200-μl pipette tip to one well of a low-attachment 24-well plate containing 500 μl of NIM.

Day8

Feed the EBs by adding another 500 μl of NIM.

Day10: transferring neuroepithelial tissues to Matrigel droplets

1. Thaw Matrigel on ice at 4 ℃ for 30min.

2. Prepare dimpled Parafilm substrate for the generation of Matrigel droplets by layering a square of Parafilm (4×4) over an empty tip tray for size 200-μl tips. Press your gloved finger into the Parafilm over each hole in the tip tray to create small dimples in the Parafilm.

3. Use a cut 200-μl tip to transfer the neuroepithelial tissues one by one to each dimple in the Parafilm.

4. Remove excess medium from each tissue by carefully sucking off the fluid with an uncut 200-μl tip.

5. Immediately add droplets of Reduced growth factor Matrigel to each aggregate by dripping ～30 μl onto each tissue so that the droplet fills the Parafilm dimple.

6. Position each aggregate in the center of the droplet using a 10-μl pipette tip to move the tissue within the droplet

7. Place the 60-mm dish containing droplets on Parafilm back into the 37 ℃ incubator, and incubate it for 20-30 min to allow the Matrigel to polymerize.

8. Add 5 ml of cerebral organoid differentiation medium without vitamin A to the 60-mm dish.

9. Remove the Matrigel droplets from Parafilm by first using sterile forceps to turn the Parafilm sheet over and by agitating the dish until the droplets fall off the sheet. Any remaining droplets can be removed by using forceps to shake the Parafilm sheet in the medium more vigorously. Continue culturing the tissue droplets in a CO ₂ incubator.

Day12

Feed the EBs with 5 ml of CDM without vitamin A.

Day14

Feed the EBs with 5 ml of CDM with vitamin A, then transfer the embedded organoids to orbital shaker installed in the incubator, shaking at 70 r. p. m. Change the medium completely, every 3–4 d.

Day30

Feed the EBs with 5 ml of CDM with 14 ng/mL BDNF, Change the medium completely, every 3–4 d for long term culture.

Experimental results

Annotation and quantification of brain organoid cell types, including cycling neural progenitor, granulocyte-macrophage progenitor, oligodendrocyte precursor cell, radial glia and schwann cell precursor, are shown in Fig. 20.

Example 11: Embryoid body generation

Materials and methods

Accutase

AggreWell ^TM plate

Low attachment 10cm plate

EB differentiation medium

Cells:

Human 8CLC and

Procedures

Primed,

PSCs/ICLCs or 8CLCs grow until they are 70–80%confluent, then the colonies are dissociated to single cells with Accutase, 6 x 10 ⁵ cells/well of 24 well AggreWell ^TM plate were resuspended in 2mL EB differentiation medium + Y-27632. Centrifuge the AggreWell ^TM plate at 100 x g immediately for 3 minutes to capture cells in the microwells. Observe plate under a microscope to verify cells that are evenly distributed among the microwells. Incubate the plate at 37℃ with 5%CO ₂ and 95%humidity for 24 hours. Observe the plate under the tissue culture microscope 24 hour later. Slowly remove 1 -1.5 mL of medium from each well. Replace with 1 -1.5 mL of fresh complete EB formation medium by slowly pipetting down the wall of the well. Slowly dispense the medium to prevent displacement of EBs/spheroids from the microwells. 2-4 days after culture in AggreWell ^TM plate, the EBs are uplifted from the plate and then transferred to low attachment plate and cultured in suspension for 20 days before harvesting (method 1) or cultured in suspension for 20 days following 15 days differentiation attached on Gelatin coated plate before harvesting (method 2) for consequent cell sorting, sequencing or other utility.

EB harvest:

Transfer EBs into 15ml or 50ml centrifuge tubes with pasteur pipette. Dispense 1 mL warm medium across the entire surface of the well to dislodge any remaining EBs/spheroids. Repeat this wash step 3 times and transfer the EBs into the same centrifuge tube. Keep the tube steady for 5 mins to let the EBs sediment. Gently aspirate the upper layer medium. Add 2ml DPBS into the centrifuge tube and mix the EBs with DPBS carefully, keep the tube steady for 5 mins to let the EBs sediment and gently aspirate the upper layer DPBS. Repeat the washing step once more and digest the EBs for consequent study.

Experimental results

Annotation and quantification of EB cell types, including endoderm epithelial, endothelial cell, arterial endothelial cell, midgut epithelial cell, hindgut epithelial cell, neural stem cell, neuroblast, neuroepithelial, retinal progenitor, schwann cell precursor, smooth muscle cell and trophoblast, are shown in Fig. 21.

Claims

A method for producing a teratoma, comprising the step of transplanting
PSCs/ICLCs, 8CLCs, or reprimed PSCs into different organ or position of recipient immunocompromised animals of interest and culturing the animals; wherein the
PSCs/ICLCs are obtained by a method comprising a step of culturing primate PSCs in a culture medium containing a SAH/PRC/EZH2 inhibitor, a HDAC inhibitor and a WNT/β-catenin signaling inhibitor, the 8CLCs are obtained by a method comprising a step of culturing the primate PSCs or the
PSCs/ICLCs in a culture medium containing dose optimized SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/β-catenin signaling inhibitor, the reprimed PSC are obtained through guided differentiation of
PSCs/ICLCs or 8CLCs.
A method for producing an organoid, comprising the step of culturing
PSCs/ICLCs, 8CLCs or reprimed PSCs as an aggregate or in a 3D scaffold in the presence of a culture medium allowing targeted organ differentiation; wherein the
PSCs/ICLCs are obtained by a method comprising a step of culturing primate PSCs in a culture medium containing a SAH/PRC/EZH2 inhibitor, a HDAC inhibitor and a WNT/β-catenin signaling inhibitor, the 8CLCs are obtained by a method comprising a step of culturing the primate PSCs or the
PSCs/ICLCs in a culture medium containing dose optimized SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/β-catenin signaling inhibitor, the reprimed PSC are obtained through guided differentiation of
PSCs/ICLCs or 8CLCs.
A method for producing an embryoid body, comprising the step of culturing
PSCs/ICLCs, 8CLCs or reprimed PSCs in suspension in the presence of a culture medium allowing automatic differentiation; wherein the
PSCs/ICLCs are obtained by a method comprising a step of culturing primate PSCs in a culture medium containing a SAH/PRC/EZH2 inhibitor, a HDAC inhibitor and a WNT/β-catenin signaling inhibitor, the 8CLCs are obtained by a method comprising a step of culturing the primate PSCs or
PSCs/ICLCs in a culture medium containing dose optimized SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/β-catenin signaling inhibitor, the reprimed PSC are obtained through guided differentiation of
PSCs/ICLCs or 8CLCs.
The method according to any one of claims 1-3, wherein the culture medium is further supplemented with one or more components selected from a group consisting of L-ascorbic acid or a derivative thereof, an activator of JAK/STAT3 signaling, and an inhibitor of MAPK/ERK signaling; optionally, the culture medium is further supplemented with one or more components selected from a group consisting of an activator of ACTIVIN/NODAL signaling, a ROCK inhibitor, and an extracellular matrix.
The method according to any one of claims 1-4, wherein:

the PRC/EZH2 inhibitor or the SAH inhibitor is selected from a group consisting of DZNep and CPI-1205; preferably, the final concentration of DZNep in the culture medium is from 5 to 80 nM, preferably 5 to 50 nM; preferably, the final concentration of CPI-1205 in the culture medium is from 0.5 to 5 mM, preferably 1 to 3 mM; and/or

the HDAC inhibitor is selected from a group consisting of TSA, VPA and NaB; preferably, the final concentration of TSA in the culture medium is from 3 to 30 nM, preferably 3 to 25 nM; preferably, the final concentration of VPA in the culture medium is from 0.25 to 2 mM, preferably 0.5 to 1.5 mM; preferably, the final concentration of NaB in the culture medium is from 0.25 to 2 mM, preferably 0.5 to 1.5 mM; and/or the final concentration of the WNT/β-catenin signaling inhibitor in the culture medium is from 2 to 8 μM; preferably, the WNT/β-catenin signaling inhibitor is selected from a group consisting of IWR1 and XAV939.
The method according to claim 4, wherein:

the final concentration of L-ascorbic acid in the culture medium is 40 to 70 μg/mL; and/or

the final concentration of the activator of JAK/STAT3 signaling in the culture medium is 10 to 50 ng/mL; preferably, the activator of JAK/STAT3 signaling is LIF; and/or

the final concentration of the inhibitor of MAPK/ERK signaling in the culture medium is 0.5 μM to 3 μM; preferably, the inhibitor of MAPK/ERK signaling is PD0325901; and/or

the final concentration of the activator of ACTIVIN/NODAL signaling is from 10 to 25 ng/mL; preferably, the activator of ACTIVIN/NODAL signaling is selected from a group consisting of ACTIVIN A and NODAL; and/or

the final concentration of the ROCK inhibitor in the culture medium is from 0.5 to 2 μM; preferably, the ROCK inhibitor is selected from a group consisting of Y27632, thiazovivin and hydroxyfasudil; and/or

the amount of the extracellular matrix in the culture medium is 0.1 to 0.5% (v/v) ; preferably, the extracellular matrix is selected from a group consisting of Matrigel ^TM, Geltrex ^TM and ECM ^TM.
The method according to any of claims 1-3, wherein the culture medium for producing the
PSCs/ICLCs comprises:

(A) DZNep at a final concentration of 5 to 15 nM or CPI-1205 at a final concentration of 0.5 to 2 mM, and TSA at a final concentration of 3 to 30 nM, or VPA at a final concentration of 0.25 to 2 mM, or NaB at a final concentration of 0.25 to 2 mM, preferably TSA at a final concentration of 3 to 10 nM, or VPA at a final concentration of 0.25 to 1 mM, or NaB at a final concentration of 0.25 to 1 mM; or DZNep at a final concentration of 5 to 80 nM, preferably 5 to 50 nM or CPI-1205 at a final concentration of 0.5 to 5 mM, preferably 0.5 to 3 mM, and TSA at a final concentration of 3 to 10 nM, or VPA at a final concentration of 0.25 to 0.5 mM, or NaB at a final concentration of 0.25 to 0.5 mM;

(B) L-ascorbic acid at a final concentration of 40 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;

(E) IWR1 or XAV939 at a final concentration of 3 to 6 μM;

and the culture medium is further supplemented with:

(1) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; and an 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; and Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; or

(3) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; and an 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 an 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; or Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 to 2 μM; or an extracellular matrix in an amount of 0.1%to 0.5% (v/v) .
The method according to claim 7, wherein the culture medium comprises 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 is further supplemented with (1) 20 ng/mL of ACTIVIN A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an extracellular matrix; or (2) 20 ng/mL of ACTIVIN A or NODAL, and 1 μM of Y27632, thiazovivin or hydroxyfasudil; (3) 20 ng/mL of ACTIVIN A or NODAL, and 0.2%(v/v) of an extracellular matrix; or (4) 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an 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 an extracellular matrix.
The method according to any of claims 1-3, wherein the culture medium for producing the 8CLCs comprises DZNep at a final concentration of 40 to 70 nM or CPI-1205 at a final concentration of 2 to 4 mM; TSA at a final concentration of 10 to 30 nM, or VPA at a final concentration of 0.5 to 1.5 mM or NaB at a final concentration of 0.5 to 1.5 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 each at a final concentration of 3 to 6 μM; and is further supplemented with:

(1) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; and an 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; and 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 an 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 an 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; or Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; or an extracellular matrix in an amount of 0.1%to 0.5% (v/v) .
The method according to claim 9, wherein the culture medium comprises 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 is further supplemented with (1) 20 ng/mL of ACTIVIN A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an extracellular matrix; or (2) 20 ng/mL of ACTIVIN A or NODAL, and 1 μM of Y27632, thiazovivin or hydroxyfasudil; (3) 20 ng/mL of ACTIVIN A or NODAL, and 0.2%(v/v) of an extracellular matrix; or (4) 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2%(v/v) of an 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 an extracellular matrix.
The method according to any of claims 1 to 10, wherein the basal medium of the culture medium for producing the
PSCs/ICLCs and the 8CLCs is selected from a group consisting of Dulbecco's modified eagle's medium (DMEM) , minimal essential medium (MEM) , basal medium Eagle (BME) , RPMI1640, F10, F12, α minimal essential medium (α MEM) , Glasgow's minimal essential medium (GMEM) , Iscove's modified Dulbecco's medium, Neurobasal Medium, DMEM/F12 and Advanced DMEM/F12 and a combination thereof; preferably, the basal medium is a mixture of Advanced DMEM/F12 and Neurobasal Medium in a ratio of 1: 1 (v/v) .
The method according to any of claims 1 to 11, wherein the culture medium is further supplemented with one or more components selected from a group consisting of serum replacement, alternative carbon source, non-essential amino acid, L-glutamine or its alternative and antibiotic.
The method according to claim 12, wherein:

the serum replacement is selected from a group consisting of KOSR, N2 and B27, and combinations thereof; preferably, the serum replacement is a mixture of N2 and B27 in a ratio of 1: 1 (w/w) ;

the alternative carbon source is pyruvate, such as sodium pyruvate;

the L-glutamine or its alternative is Glutamax ^TM supplement comprising L-alanyl-L-glutamine dipeptide in 0.85%NaCl; and/or

the antibiotic is selected from a group consisting of penicillin, streptomycin, or a mixture of penicillin and streptomycin.
The method according to any of claims 1-3, wherein the
PSCs/ICLCs are obtained by a method comprising:

(a) genetically engineering the primate PSCs to reduce the activity of SAH, PRC and/or EZH2 of the PSCs by knockdown and/or knockout of one or more relevant genes in the cells; and

(b) culturing the genetically engineered cells obtained in step (a) in a culture medium comprising: TSA at a final concentration of 3 to 30 nM, or VPA at a final concentration of 0.25 to 2 mM, or NaB at a final concentration of 0.25 to 2 mM, preferably TSA at a final concentration of 3 to 10 nM, or VPA at a final concentration of 0.25 to 1 mM, or NaB at a final concentration of 0.25 to 1 mM, and optionally DZNep at a final concentration of 5 to 15 nM or CPI-1205 at a final concentration of 0.5 to 2 mM, or TSA at a final concentration of 3 to 10 nM, or VPA at a final concentration of 0.25 to 0.5 mM, or NaB at a final concentration of 0.25 to 0.5 mM and optionally DZNep at a final concentration of 5 to 80 nM, preferably 5 to 50 nM or CPI-1205 at a final concentration of 0.5 to 5 mM; 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; IWR1 or XAV939 at a final concentration of 3 to 6 μM; wherein the culture medium is further supplemented with:

(1) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; and an 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; and Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; or

(3) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; and an 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 an 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; or Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5 to 2 μM; or an extracellular matrix in an amount of 0.1%to 0.5% (v/v) ;

preferably the culture medium comprises: 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; 5 μM IWR1 or 5 μM XAV939; and optionally 10 nM DZNep or 1 mM CPI-1205; and wherein the culture medium is further supplemented with (1) 20 ng/mL of ACTIVIN A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an extracellular matrix; or (2) 20 ng/mL of ACTIVIN A or NODAL, and 1 μM of Y27632, thiazovivin or hydroxyfasudil; (3) 20 ng/mL of ACTIVIN A or NODAL, and 0.2% (v/v) of an extracellular matrix; or (4) 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an 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 an extracellular matrix.
The method according to any of claims 1-3, wherein the 8CLCs are obtained by a method comprising:

(a) genetically engineering the primate PSCs or
PSCs/ICLCs to reduce the activity of SAH, PRC and/or EZH2 of the PSCs or
PSCs/ICLCs by knockdown and/or knockout of one or more relevant genes in the cells;

(b) culturing the genetically engineered cells obtained in step (a) in a culture medium comprising: TSA at a final concentration of 10 to 30 nM, or VPA at a final concentration of 0.5 to 1.5 mM or NaB at a final concentration of 0.5 to 1.5 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; IWR1 or XAV939 each at a final concentration of 3 to 6 μM;and optionally DZNep at a final concentration of 40 to 70 nM or CPI-1205 at a final concentration of 2 to 4 mM; and wherein the culture medium is further supplemented with:

(1) ACTIVIN A or NODAL at a final concentration of 10 to 25 ng/mL; Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; and an 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; and 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 an 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 an 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; or Y27632, thiazovivin or hydroxyfasudil at a final concentration in a range 0.5 to 2 μM; or an extracellular matrix in an amount of 0.1%to 0.5% (v/v) ;

preferably, the culture medium comprises: 20 nM TSA, or 1 mM VPA, or 1 mM NaB; 50 μg/mL L-ascorbic acid; 20 ng/mL LIF; 1 μM PD0325901; 5 μM IWR1 or 5 μM XAV939; and optionally 50 nM DZNep or 3 mM CPI-1205; and wherein the culture medium is further supplemented with (1) 20 ng/mL of ACTIVIN A or NODAL, 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an extracellular matrix; or (2) 20 ng/mL of ACTIVIN A or NODAL, and 1 μM of Y27632, thiazovivin or hydroxyfasudil; (3) 20 ng/mL of ACTIVIN A or NODAL, and 0.2% (v/v) of an extracellular matrix; or (4) 1 μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of an 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 an extracellular matrix.
The method according to any of claims 1-15, wherein the primate PSCs are selected from a group consisting of:

(i) cells from an ESC line and/or an ECC line;

(ii) cells from an iPSC line;

(iii) cells from ICM of a preimplantation blastocyst cultured in vitro;

(iv) cells from ICM of a post-implantation blastocyst cultured in vitro;

(v) cells from an embryo of 8C stage to morula stage cultured in vitro.
The method according to any of claims 1-16, wherein the primate PSCs or the
PSCs/ICLCs are cultured under one or more conditions selected from a group consisting of: (i) on feeder cells; (ii) on an extracellular matrix devoid of feeders; (iii) in suspension devoid of feeder cells; (iv) in hypoxic or normoxic condition at about 37℃ temperature; (v) passaging as single cells every 3 to 4 days with a split ratio of 1: 4 to 1: 8; (vi) changing medium daily.
The method according to any of claims 1-3, wherein the method further comprises a step of culturing somatic cells in the presence of the SAH/PRC/EZH2 inhibitor, the HDAC inhibitor and the WNT/β-catenin signaling inhibitor to reprogram the somatic cells to produce the primate
PSCs/ICLCs.
Teratoma produced by the method described in any one of claims 1 and 4-18, and cells dissociated from the teratoma.
Organoid produced by the method described in any one of claims 2 and 4-18, and cells dissociated from the organoid.
Embryoid body produced by the method described in any one of claims 3 and 4-18, and cells dissociated from the embryoid body.