CN116769695A - Culture media and methods for generating human cells and tissues from teratomas, organoids and embryoid bodies - Google Patents

Abstract

The present invention relates to media and methods for producing human cells and tissues from teratomas, organoids and embryoid bodies. The method of the invention for producing teratomas comprises the steps of transplanting primary, originating, 8clc or heavy primary PSCs into different organs or locations of an immunodeficient animal of interest and feeding the animal; wherein the method of making the primary PSCs/ICLCs comprises the step of culturing primate PSCs in a medium comprising an SAH/PRC/EZH2 inhibitor, an HDAC inhibitor and a WNT/beta-catenin signaling inhibitor; the preparation method of the 8CLCs comprises the steps of culturing primate PSCs or the original PSC/ICLC in a culture medium containing optimized doses of SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the heavy original state PSCs are obtained through induced differentiation of original state PSCs/ICLCs or 8 CLCs.

Description

Culture media and media for the generation of human cells and tissues from teratomas, organoids and embryoid bodies method

Technical field

The present invention relates to culture media and methods for generating human cells and tissues from teratomas, organoids and embryoid bodies.

Background technique

Human cells with potential therapeutic applications include fully differentiated cells such as T and B lymphocytes, somatic stem/progenitor cells such as hematopoietic stem cells (HSC) and hepatic progenitor cells (LPC). These cells can be obtained by isolation from the human body or differentiation from pluripotent/totipotent stem cells. Generally, cells isolated from the human body are functional and will not cause immune rejection whether used autologously or by individuals with appropriate HLA matching. However, not all types of cells can be isolated from the human body, and the cells that can be isolated are also rare in number. In addition, the isolated cells may function abnormally, and it also takes time to find patients with suitable HLA matches. Pluripotent/totipotent stem cells can be infinitely expanded and easily engineered, and both autologous and analog (HLA-generic, surface proteins removed to reduce immunogenicity) pluripotent/totipotent stem cells can be used to generate target derivatives. However, target derivatives produced from pluripotent/totipotent stem cells may not differentiate or function correctly.

In order for derivatives of pluripotent/totipotent stem cells to be suitable for clinical treatment, the cells need to achieve the correct cellular state and function during differentiation. In this regard, scientists have been working hard to develop various in vitro lineage-specific differentiation protocols for pluripotent stem cells (PSCs). Some solutions are more effective. For example, induction of differentiation in vitro to produce neural stem cells for transplantation has achieved partial success. Preclinical trials are underway in animal models including non-human primates, and several clinical trials are also underway. However, other scenarios such as HSC and LPC remain challenging due to limited efficiency and loss of functionality. The main reasons are as follows: 1) Starting PSCs have epigenetic abnormalities, which result from long-term culture or reprogramming, or limited flexibility of chromatin states; 2) In vitro differentiation protocols fail to fully simulate in vivo differentiation. way. Due to being at the beginning of epigenetic inheritance, naive stem cells or totipotent-like cells may have greater flexibility and greater differentiation potential than primary stem cells. In addition, the in vivo environment can effectively induce PSC differentiation, which is difficult to achieve in vitro. The combination of these two features may help generate functionally differentiated cells.

Contents of the invention

A first aspect of the present invention provides a method for producing teratomas, the method comprising converting original The steps of transplanting PSC/ICLC, primary PSC, 8CLC or reprimed PSC into different organs or locations of the immunodeficient animal of interest and raising the animal; wherein the preparation method of the original PSC/ICLC It includes the step of culturing primate PSC in a culture medium containing SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/β-catenin signaling inhibitor; the preparation method of 8CLC includes the step of culturing primate PSC in a medium containing an optimized dose of SAH/PRC The step of culturing primate PSC or the original PSC/ICLC in the medium of EZH2 inhibitor, HDAC inhibitor and WNT/β-catenin signaling inhibitor; the reestablishing the original PSC through the original PSC/ICLC or 8CLC were induced to differentiate.

The second aspect of the present invention provides a method for producing organoids, which method includes the step of suspending and culturing original PSC/ICLC, 8CLC or reconstituted original PSC, or in a culture medium that can differentiate into target organs in 3D. The step of cultivating the original PSC/ICLC, 8CLC or re-original PSC on the scaffold; wherein, the preparation method of the original PSC/ICLC includes a solution containing SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/β -The step of culturing primate PSCs in a medium containing an optimized dose of SAH/PRC/EZH2 inhibitors, HDAC inhibitors and WNT/β-catenin signaling inhibitors; the preparation method of the 8CLC includes: The step of culturing primate PSC or primitive PSC/ICLC in the culture medium; the re-primitive PSC is obtained by inducing differentiation of primitive PSC/ICLC or 8CLC.

A third aspect of the present invention provides a method for producing embryoid bodies, which method includes the step of suspending and culturing the original PSC/ICLC, 8CLC or reconstituted original PSC in a medium capable of differentiating into target organs; Wherein, the preparation method of original PSC/ICLC includes the step of culturing primate PSC in a culture medium containing SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/β-catenin signaling inhibitor; the 8CLC The preparation method includes the step of culturing primate PSCs or naive PSCs/ICLCs in a culture medium containing optimized doses of SAH/PRC/EZH2 inhibitors, HDAC inhibitors and WNT/β-catenin signaling inhibitors; the recombinant Naive PSCs are obtained through induced differentiation of naïve PSC/ICLC or 8CLC.

In one or more embodiments, the medium also adds one or more of L-ascorbic acid or a derivative thereof, a JAK/STAT3 signaling activator and a MAPK/ERK signaling inhibitor; optionally, the The medium is further supplemented with one or more of ACTIVIN/NODAL signaling activators, ROCK inhibitors, and extracellular matrix.

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

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

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

In one or more embodiments, the final concentration of the JAK/STAT3 signal activator is 10-50 ng/mL; the JAK/STAT3 signal activator is preferably LIF.

In one or more embodiments, the final concentration of the MAPK/ERK signaling inhibitor is 0.5-3 μM; the MAPK/ERK signaling inhibitor is preferably PD0325901.

In one or more embodiments, the final concentration of the ACTIVIN/NODAL signal activator is 10-25 ng/mL; preferably, the ACTIVIN/NODAL signal activator is selected from ACTIVIN A and NODAL.

In one or more embodiments, the final concentration of the ROCK inhibitor is 0.5-2 μM; preferably, the ROCK inhibitor is selected from Y27632, thiazovivin (CAS No.: 1226056-71-8; N-benzyl- 2-(pyrimidin-4-ylamino)thiazole-4-carboxamide) and hydroxyfasudil.

In one or more embodiments, the content of the extracellular matrix in the culture medium is 0.1-0.5% (v/v); preferably, the extracellular matrix is selected from Matrigel ^™ , Geltrex ^™ and ECM ^™ .

In one or more embodiments, the culture medium for preparing naive PSC/ICLC includes:

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

(B) L-ascorbic acid with a final concentration of 40-70 μg/mL;

(C) LIF with a final concentration of 10-30ng/mL;

(D) PD0325901 at a final concentration of 0.5-1.5 μM;

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

The culture medium also contains:

(1) ACTIVIN A or NODAL with a final concentration of 10-25ng/mL; Y27632, thiazovivin or hydroxyfasudil with a final concentration of 0.5-2μM; and extracellular extracellular fluid with a content of 0.1%-0.5% (v/v) substrate; or

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

(3) ACTIVIN A or NODAL with a final concentration of 10-25ng/mL; and extracellular matrix with a content of 0.1%-0.5% (v/v); or

(4) Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2 μM; and extracellular matrix at a content of 0.1%-0.5% (v/v); or

(5) ACTIVIN A or NODAL with a final concentration of 10-25ng/mL; or Y27632, thiazovivin or hydroxyfasudil with a final concentration of 0.5-2μM; or cells with a content of 0.1%-0.5% (v/v) Extramatrix.

In one or more embodiments, the culture medium for preparing naive PSC/ICLC includes 10 nMDZNep or 1 mM CPI-1205; 5 nM TSA, or 0.5mM VPA, or 0.5mM NaB; 50 μg/mL L-ascorbic acid; 20ng/mL LIF; 1μM PD0325901; and 5μM IWR1 or 5μM XAV939; and further add: (1) 20ng/mL of ACTIVINA or NODAL, 1μM of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (2) 20ng/mL ACTIVIN A or NODAL, and 1 μM Y27632, thiazovivin, or hydroxyfasudil; (3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) Extracellular matrix; or (4) 1 μM Y27632, thiazovivin, or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20 ng/mL ACTIVIN A or NODAL, or 1 μM Y27632 , thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix.

In one or more embodiments, the culture medium for preparing 8CLC includes DZNep at a final concentration of 40-70 nM or CPI-1205 at a final concentration of 2-4 mM; TSA at a final concentration of 10-30 nM, or TSA at a final concentration of 10-30 nM. VPA at a final concentration of 0.5-1.5mM or NaB at a final concentration of 0.5-1.5mM; L-ascorbic acid at a final concentration of 40-70μg/mL; LIF at a final concentration of 10-30ng/mL; final concentration 0.5-1.5μM PD0325901; and IWR1 or XAV939 at a final concentration of 3-6 μM respectively; and further add:

(1) ACTIVIN A or NODAL with a final concentration of 10-25ng/mL; Y27632, thiazovivin or hydroxyfasudil with a final concentration of 0.5-2μM; and extracellular extracellular fluid with a content of 0.1%-0.5% (v/v) substrate; or

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

(3) ACTIVIN A or NODAL with a final concentration of 10-25ng/mL; and extracellular matrix with a content of 0.1%-0.5% (v/v); or

(4) Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2 μM; and extracellular matrix at a content of 0.1%-0.5% (v/v); or

(5) ACTIVIN A or NODAL with a final concentration of 10-25ng/mL; or Y27632, thiazovivin or hydroxyfasudil with a final concentration of 0.5-2μM; or cells with a content of 0.1%-0.5% (v/v) Extramatrix.

In one or more embodiments, the medium for preparing 8CLC includes 50 nM DZNep or 3mMCPI-1205; 20nM TSA, or 1mM VPA, or 1mM NaB; 50μg/mL L-ascorbic acid; 20ng/mL LIF; 1μMPD0325901 ; and 5 μM IWR1 or 5 μM XAV939; and further add: (1) 20 ng/mL ACTIVIN A or NODAL, 1 μM MY27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (2) 20ng/mL ACTIVIN A or NODAL, and 1μM Y27632, thiazovivin or hydroxyfasudil; (3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (4) 1μM Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20ng/mL ACTIVIN A or NODAL, or 1 μM Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v /v) extracellular matrix.

In one or more embodiments, the basal medium used to prepare the original PSC/ICLC and 8CLC medium is selected from Dulbecco's modified Eagle medium (DMEM), minimal essential medium (MEM), basal medium Eagle (BME), RPMI1640, F10, F12, alpha minimum essential medium (αMEM), Glasgow minimum essential medium (GMEM), Iscove modified Dulbecco medium, neurobasal medium, advanced DMEM/F12, one or more; Among them, the basal medium is preferably a mixture of advanced DMEM/F12 and neural basal medium, with a ratio of 1:1 (v/v).

In one or more embodiments, one or more selected from the group consisting of serum substitutes, alternative carbon sources, non-essential amino acids, L-glutamine or its substitutes, and antibiotics are added to the culture medium.

In one or more embodiments, the serum substitute is selected from one or more of KOSR, N2 and B27; preferably, the serum substitute is a mixture of N2 and B27, with a ratio of 1:1 (w /w).

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

In one or more embodiments, the L-glutamine or substitute thereof is a Glutamax ^™ supplement containing L-alanyl-L-glutamine dipeptide in 0.85% NaCl.

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

In one or more embodiments, the method for preparing virgin PSC/ICLC includes the steps of:

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

(b) Culturing the genetically engineered cells obtained in step (a) in a medium containing: TSA with a final concentration of 3-30 nM, or VPA with a final concentration of 0.25-2mM, or a final concentration of 0.25- 2mM NaB; preferably, TSA at a final concentration of 3-10nM, or VPA at a final concentration of 0.25-1mM, or NaB at a final concentration of 0.25-1mM; and optionally, DZNep at a final concentration of 5-15nM or CPI-1205 at a final concentration of 0.5-2mM, or TSA at a final concentration of 3-10nM, or VPA at a final concentration of 0.25-0.5mM, or NaB at a final concentration of 0.25-0.5mM and optionally a final concentration of 5 -80nM, preferably 5-50nM DZNep, or CPI-1205 with a final concentration of 0.5-5mM; L-ascorbic acid with a final concentration of 40-70μg/mL; LIF with a final concentration of 10-30ng/mL; final concentration is PD0325901 at 0.5-1.5 μM; IWR1 or XAV939 at a final concentration of 3-6 μM; among which, the medium was further added with:

(1) ACTIVIN A or NODAL with a final concentration of 10-25ng/mL; Y27632, thiazovivin or hydroxyfasudil with a final concentration of 0.5-2μM; and extracellular extracellular fluid with a content of 0.1%-0.5% (v/v) substrate; or

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

(3) ACTIVIN A or NODAL with a final concentration of 10-25ng/mL; and extracellular matrix with a content of 0.1%-0.5% (v/v); or

(4) Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2 μM; and extracellular matrix at a content of 0.1%-0.5% (v/v); or

(5) ACTIVIN A or NODAL with a final concentration of 10-25ng/mL; or Y27632, thiazovivin or hydroxyfasudil with a final concentration of 0.5-2μM; or cells with a content of 0.1%-0.5% (v/v) extramatrix;

Preferably, the culture medium contains: 5nM TSA, or 0.5mM VPA, or 0.5mM NaB; 50μg/mL L-ascorbic acid; 20ng/mL LIF; 1μM PD0325901; 5μM IWR1 or 5μM XAV939; and optionally 10nM DZNep or 1mMCPI-1205; and wherein the medium is further supplemented with (1) 20ng/mL ACTIVIN A or NODAL, 1μMY27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (2) 20ng/mL ACTIVINA or NODAL, and 1μM Y27632, thiazovivin, or hydroxyfasudil; (3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (4) 1μM Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20ng/mL ACTIVIN A or NODAL, or 1μM Y27632, thiazovivin, or hydroxyfasudil, or 0.2% (v/v ) extracellular matrix.

In one or more embodiments, the steps of the method of preparing 8CLC include:

(a) Genetically engineer primate PSCs or naive PSCs/ICLCs to reduce SAH, PRC and/or EZH2 of PSCs by knocking down and/or knocking out one or more relevant genes in the cells or Activity of original PSC/ICLC;

(b) Culturing the genetically engineered cells obtained in step (a) in a culture medium containing: TSA with a final concentration of 10-30 nM, or MVPA with a final concentration of 0.5-1.5m or a final concentration of 0.5- NaB at 1.5mM; L-ascorbic acid at a final concentration of 40-70μg/mL; LIF at a final concentration of 10-30ng/mL; PD0325901 at a final concentration of 0.5-1.5μM; IWR1 or XAV939 at a final concentration of 3-6μM; and optionally, DZNep at a final concentration of 40-70 nM or CPI-1205 at a final concentration of 2-4 mM; and wherein the medium is further added:

(1) ACTIVIN A or NODAL with a final concentration of 10-25ng/mL; Y27632, thiazovivin or hydroxyfasudil with a final concentration of 0.5-2μM; and extracellular extracellular fluid with a content of 0.1%-0.5% (v/v) substrate; or

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

(3) ACTIVIN A or NODAL with a final concentration of 10-25ng/mL; and extracellular matrix with a content of 0.1%-0.5% (v/v); or

(4) Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2 μM; and extracellular matrix at a content of 0.1%-0.5% (v/v); or

(5) ACTIVIN A or NODAL with a final concentration of 10-25ng/mL; or Y27632, thiazovivin or hydroxyfasudil with a final concentration of 0.5-2μM; or cells with a content of 0.1%-0.5% (v/v) extramatrix;

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

In one or more embodiments, the primate PSC is selected from:

(i) Cells from the ESC line and/or ECC line;

(ii) cells of iPSC line;

(iii) ICM cells of preimplantation blastocysts cultured in vitro;

(iv) ICM cells of post-implantation blastocysts cultured in vitro;

(v) Cells of embryos cultured in vitro from the 8-cell stage to the morula stage.

In one or more embodiments, the primate PSC or naive PSC/ICLC can be cultured under one or more conditions selected from: (i) on feeder cells; (ii) in the absence of feeder cells layer of extracellular matrix; (iii) in suspension without feeder cells; (iv) under hypoxic or normoxic conditions at about 37°C; (v) passaged as single cells every 3 to 4 days, with a division ratio 1:4 to 1:8; (vi) Change the culture medium daily.

In one or more embodiments, the method further includes culturing the somatic cells in the presence of a SAH/PRC/EZH2 inhibitor, an HDAC inhibitor, and a WNT/β-catenin signaling inhibitor to reprogram the somatic cells to produce primates. Animal-like primitive PSC/ICLC procedures.

A fourth aspect of the invention also provides a teratoma produced by a method as described in any embodiment herein, and cells isolated from the teratoma.

A fifth aspect of the invention also provides an organoid produced by the method described in any embodiment herein, and cells isolated from the organoid.

A sixth aspect of the invention also provides an embryoid body produced by the method described in any embodiment herein, and cells isolated from the embryoid body.

Description of drawings

Figure 1: (a) Schematic diagram of the process of inducing naive PSC/ICLC and 8CLC from primed PSC using 4CL, 4CL and e4CL medium combination (stepped e4CL) or e4CL alone (direct e4CL). D, days.

(b) Immunofluorescence staining images of KLF17 and TPRX1 in primary H9 ESCs that were not treated or transformed with 4CL (12 days) or e4CL (5 days). Scale bar, 20 μm.

(c) In NHSM, Heat map of enriched gene expression in human naive PSCs cultured in 5iLAF, 4CL and pre-implantation ICM (cell-like mass). Data were generated using H9 ESC.

(d) In NHSM, Heat map of totipotency gene expression in naive ESCs cultured with 5iLAF or step e4CL (day 5), EPSCs, and human 8C-embryonic cells. Data were generated using H9 ESC.

(e) RT-qPCR validation of totipotent genes in primary H9 ESC cultured in direct e4CL (day 7). Data are means ± SEM of fold changes compared to primary ESCs. n=3 biological replicates. P values were calculated using two-tailed unpaired Student's t test, ***P<0.001.

Figure 2: (a) UMAP plots of scRNA-seq time courses comparing stepwise or direct e4CL induction to return development from human E7 to E3 embryonic stages, respectively. Data were generated using H9 ESC.

(b) UMAP visualization of step-by-step e4CL-day 5 cell RNA-Seq data, showing 7 clusters, with cluster 5 (8CLC) accounting for 11.9% of the total cell number.

(c) Expression frequency and average expression of representative pluripotency and totipotency genes at early stages of human embryos and those not detected by 4CL (day 8 [passage 2] and day 12 [passage 3]) and Bubble plot of primary ESCs treated or transformed with e4CL (day 5 C5 [8CLC] and non-8CLC [all other clusters summed]). D, day.

(d) Log-normalized expression of representative early human embryonic enriched TEs at early stages of human embryos and human ESCs at passage 10 compared with naive ESCs, 4CL-day 12 naive ESCs, and 8CLCs. Violin diagram.

Figure 3: (a) Representative images of G-banded karyotypes of primary H9 and primary iPSC-4 cultured in 4CL for 15 generations. Twenty cells in metaphase were counted in each graph.

(b) Representative images of G-banded karyotypes of primary H9 and iPSC-4 cultured in step e4CL (day 5). Twenty cells in metaphase were counted in each graph.

Figure 4: (a) In the starting state, 4CL (day 12), 5iLAF, Violin plot of global CpG methylation levels detected by RRBS for human PSCs, human 8C embryos and ICM cultured in NHSM, stepwise e4CL (day 5) and direct e4CL (day 7). Generate data with H9 ESC

(b) In the starting state, 4CL (day 12), 5iLAF, Heat map of CpG methylation levels in imprinted control regions of human PSC and ICM and post-implantation embryos cultured with NHSM and step e4CL (day 5).

(c) Under starting conditions, 4CL (day 12), 5iLAF, CpG methylation of indicated naïve pluripotent (blue) and totipotent (red) sites in NHSM, stepwise e4CL (day 5) and direct e4CL (day 7), human 8C embryos and PSC cultured under ICM Horizontal genome browsing map trajectories. Each bar represents a single CpG, and the height indicates the percentage of methylation.

Figure 5: (a) Visualization of UMAP gene scores for all genes in scATAC-seq, primary ESC untreated (red), 4CL (day 12; blue), or step e4CL (day 5; green).

(b) and (c) UMAP visualization of gene scores based on panel a highlighting origin (ZIC2), shared origin pluripotency/8CLC (DPPA3) and totipotency (ZSCAN5B, ZNF280A, ARGFX) genes that project onto each cell in primary ESC-seq that was untreated or transformed by 4CL (day 12) and step e4CL (day 5).

(d) Genome view tracking the chromatin accessibility, H3K27ac levels and transcription factor DNA binding motif locations at the naive pluripotent KLF17 and totipotent ZSCAN4 loci.

Figure 6: (a) Top: Schematic representation of the insertion of EGFP into the TPRX1 site (for chimerism experiments), and the donor construct used to generate the TPRX1-EGFP reporter cell line. Bottom: TPRX1-EGFP knock-in cells were verified by e4CL step culture (day 5) and immunostaining with anti-TPRX1, and the results were consistent with the GFP ⁺ signal (left). Scale bar: 10μm. FACS analysis of GFP ⁺ cell percentage of TPRX1-EGFP cells in step e4CL (day 5) (right panel).

(b) Expression frequency and average expression of representative pluripotency and totipotency genes at human early embryonic stage and human ESC at passage 10 compared to primary ESC, day 12 4CL naive ESC and sorted 8CLC. Bubble chart.

Figure 7: Bar graph of expression levels of ICM and primer markers in H9, H1, HUES1 and WIBR3 human ESC lines that have been transformed into naive PSC/ICLC using 4CL Medium 1.

Figure 8: Pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4 ^, MAEL and REX1 were significantly induced in RT- qPCR data histogram.

Figure 9: Histogram of RT-qPCR data showing that pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL and REX1 were significantly induced in naive PSC/ICLC transformed using 4CL medium 1 suspension. . In the histogram, each gene in the left column represents cells cultured on feeder cells, and the right column represents cells cultured in suspension.

Figure 10: Original PSC/ICLC transformed using 4CL medium 2 (A), 4CL medium 3 (B), and 4CL medium 4 (C) respectively, pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, Histogram of RT-qPCR data where TFCP2L1, KLF4, MAEL and REX1 were significantly induced.

Figure 11: (A) Schematic diagram of two methods for generating 8CLC. Briefly, primary human PSC medium (such as mTeSR1) is replaced with e4CL medium or 4CL medium. Then, the cells were continuously cultured in e4CL, or were passaged twice in 4CL medium and then switched to e4CL. (B) Histogram of expression levels of selected naive multifunctional marker genes in H9 primary cells and H9-e4CL cells. (C) Histogram of expression levels of selected naive multifunctional marker genes in H9-e4CL cells and H9-4CL cells. (D) The expression levels of 8C-specific genes induced by the two methods are similar. (E) Immunofluorescence microscopy images of expression of ZSCAN4 (green) or DAPI nuclear counterstain (blue) in primary H9, H9-4CL, and H9-e4CL.

Figure 12: Histogram of RT-qPCR data showing that 8C marker genes ZSCAN4, ARGFX, TPRX1, ZNF280A and ZSCAN5B were significantly induced in 8CLC transformed using e4CL medium suspension. In the histogram, the left column for each gene represents cells cultured on feeder cells, and the right column represents cells cultured in suspension.

Figure 13: Histogram of RT-qPCR data showing that 8C marker genes ZSCAN4, ARGFX, TPRX1, ZNF280A, ZSCAN5B, DUXA, DUXB and MBD3L2 were significantly induced in 8CLC transformed from various hPSC lines. The figure shows that the expression levels of these genes in primary HN10 and UH10 are extremely low.

Figure 14: (a) Representative images of primary ESCs or teratomas derived from 4CL (passage 15) and sorted 8CLC. H9ESC was used in this experiment.

(b) Hematoxylin and eosin staining of teratomas generated from 4CL (passage 15) and sorted 8CLC. Representative images of tissues corresponding to the three germ layers are shown. Scale bar: 50 μm. H9 ESC was used in this experiment.

(c) UMAP visualization of identified cell types in scRNA-seq from sorted 8CLC, e4CL-day 5 cells, 4CL naive ESC, and primary ESC teratoma. H9 ESCs were used to generate these teratoma cell types.

Figure 15: UMAP visualization of identified cell types in scRNA-seq generated from sorted 8CLC, staged e4CL-day-5 cells, 4CL naive ESCs, and primed ESCs, based on Figure 14c highlighting respectively.

Figure 16: (a) Histogram of the proportion distribution of different teratomas against identified cell types.

(b) Histogram of the relative proportions of different teratomas for each germ layer (ectoderm, mesoderm, and endoderm) and extraembryonic (trophoblast) lineage.

Figure 17: (a) UMAP visualization of trophectoderm cell types identified from scRNA-seq of teratomas derived from sorted 8CLC, e4CL-day-5 cells, 4CL naive ESCs, and primary ESCs. All types of teratoma cells are cultured using H9 ESCs.

(b) Bubble plot of expression frequency and average expression levels of marker genes in extraembryonic trophoblast lineage cell subtypes.

(c) Histogram of the relative proportions of extraembryonic trophoblast lineage cell subtypes in teratomas produced by sorting 8CLC, e4CL-day-5 cells, 4CL naive ESCs and primary ESCs.

(d) UMAP visualization showing the distribution and expression of relevant markers in the indicated teratoma trophoblast cells according to Figure 14c.

Figure 18: (a) UMAP visualization of annotated cell subpopulations of primed PSCs, 4CL naive ESCs, sorted e4CL-day-5 cells, and sorted 8CLC-derived teratoma immune cells.

(b) Bubble chart of expression frequency and average expression level of immune marker genes in different immune cell subtypes.

(c) Distribution histogram of different immune cell subtypes in sorted 8CLC, step e4CL-day-5, 4CL-naive and primary PSC-derived teratomas.

Figure 19: (a) UMAP visualization of the distribution of teratoma cells obtained from primary PSCs, 4CL naive PSCs, e4CL cells and sorted 8CLC.

(b) UMAP visualization of annotated subtypes of neuronal cells from primary PSCs, 4CL naive PSCs, e4CL cells, and sorted 8CLC-derived teratomas.

(c) Histogram of the relative contribution of different neuronal cell types to the indicated teratomas.

(d) Histogram of the contribution of different teratomas to identified cell types.

Figure 20: (a) UMAP visualization of cell distribution in brain organoids derived from primary and 4CL naive PSCs.

(b) UMAP visualization of annotated subtypes of neural cells from naive and 4CL naïve PSC-derived brain organoids.

(c) Histogram of the proportion of each cell type in two brain organoids.

(d) Histogram of the proportion of two brain organoids in each cell type.

Figure 21: (a) UMAP visualization of the distribution of EBs (embryoid bodies) derived from primary and 4CL naive PSCs.

(b) UMAP visualization of annotated cell types in EBs derived from primary and 4CL naive PSCs.

(c) Histogram of the proportion of different cell types in designated EBs.

(d) Histogram of the proportion of different EBs in designated cell types.

Detailed ways

Current methods to generate and maintain naive human PSCs (Chan, Goke et al., 2013; Takashima, Guo et al., 2014; Theunissen, Powell et al., 2014) and display similar properties to human pre-implantation ICMs. There are still some problems that need to be solved in the use of existing methods to generate naive human PSCs, such as long induction time, different expression levels of naive-specific genes, transgene dependence, genome instability and loss of imprinting, low multi-lineage differentiation ability, and lack of xenogeneic Chimeric ability. None of these studies reported the generation of cells approaching the 8C stage. Furthermore, there are no reports describing the generation of extraembryonic lineages in teratomas generated using human naive or primary PSCs, nor has the in vivo differentiation potential of naive PSCs been expanded upon compared with untransformed primary PSCs.

In order to overcome the above problems, the inventors first screened a group of inhibitors that target epigenetic regulators and multiple signaling pathways related to the development of the human preimplantation inner cell mass, and found three basic Modulators (JAK/STAT3 activators, MAPK/ERK inhibitors, and tankyrase inhibitors) activate the molecular network that controls the preimplantation intracellular mass-like state of human PSCs. The inventors also found that SAH/PRC/EZH2 inhibitors and HDAC inhibitors make the epigenetic state and transcriptomic state of cultured cells closer to the human pre-implantation inner cell mass, and they convert primary PSCs into their original state. PSC, which possess all the main characteristics of the human preimplantation inner cell mass as described in the background section. It is worth noting that the inventors also found that activation of the WNT/β-catenin signaling pathway inhibited the transition from primary PSC to naive PSC/ICLC. Therefore, regulators such as the GSK inhibitor CHIR99021 (a widely used activator of the WNT/β-catenin signaling pathway in published original and expanded PSC culture conditions) that activates WNT/β-catenin signaling should be removed from the culture medium, and Regulators that inhibit the WNT/β-catenin signaling pathway, such as IWR1 and XAV939, are necessary. Therefore, in preferred embodiments of various aspects of the present application, including the media, kits, compositions, and methods described herein, CHIR99021, GSK inhibitors, and any agents that activate the WNT/β-catenin signaling pathway are not included in In culture media, kits or compositions, not for use in methods of culturing cells.

Therefore, the present application provides a variety of methods and chemically defined media to promote the stable generation of primate PSC/ICLC. The methods described herein are applicable to a number of human and non-human primate PSC lines that are in the primed state as confirmed by surface markers such as SSEA-3, SSEA-4, TRA-1-81 and TRA-1-60 and other pluripotent surface marker genes, or in a pre-implantation ICM-like state, as verified by the expression of genes such as DNMT3L, STELLA, DPPA5 and KLF17. Primate PSC lines that can be used in this application include, but are not limited to, traditional primate PSC and ICM-like PSC. The method described here can also be used to isolate naive PSC/ICLC from the primate preimplantation inner cell mass. The method described does not require transgenes, and primate PSCs can be converted into naive PSCs/ICLCs under one culture condition in approximately 2 weeks.

To the best of our knowledge, there is currently no method for inducing primate 8CLC in vitro. In order to achieve this, the inventors further optimized the formula for inducing ICLC and found that only increasing the dosage of SAH/PRC/EZH2 inhibitors and HDAC inhibitors in the culture medium can transform primary human PSCs and/or ICLCs. for 8CLC. Accordingly, the present application provides a chemically defined culture medium that promotes the generation of primate 8-cell embryonic-like cells (8CLC). The methods described herein can be applied to a number of human and non-human primate PSC lines in the primed state, such as SSEA-3, SSEA-4, TRA-1-81 and TRA-1-60 Verified by the expression of pluripotent surface marker genes, or in a pre-implantation ICM-like state, as verified by the expression of genes such as DNMT3L, STELLA, DPPA5 and KLF17. Primate PSC lines that can be used in this application include, but are not limited to, primary primate PSC and ICM-like PSC. The methods described herein can also be used to isolate 8CLC from primate 8-cell embryos. The method described does not require transgenes and converts to 8CLC under one culture condition in approximately 1 week. In fact, activation of the WNT/β-catenin signaling pathway also inhibited the formation of 8CLC. Therefore, regulators such as the GSK inhibitor CHIR99021 (a WNT/β-catenin signaling pathway activator widely used in published original and expanded PSC culture conditions) that activates the WNT/β-catenin signaling pathway should be excluded from the culture conditions. Regulators that inhibit the WNT/β-catenin signaling pathway, such as IWR1 or XAV939, are required.

ICLC/original PSC and 8CLC can capture the characteristics of their corresponding in vivo embryonic development stages and have fewer epigenetic abnormalities. However, differentiation protocols for ICLC/naïve PSC and 8CLC are still lacking. Therefore, we used these cells for various types of in vivo and in vitro differentiation to form teratomas, brain organoids, and EBs.

Compared with teratomas generated from naive PSCs, teratomas generated from ICLC/naïve PSCs and/or 8CLC contained a high percentage of HSCs, LPCs, and a significant percentage of extraembryonic cells. By reconstructing the differentiation trajectories of HSCs and LPCs, we demonstrate that HSCs and LPCs can differentiate into functional cell types of the hematopoietic lineage, hepatic lineage, and extraembryonic lineage, respectively. Brain organoids generated from ICLC/naïve PSCs include a higher percentage of neuroepithelial cells and retinal progenitor cells, which can give rise to different types of neuronal cells. A higher percentage of progenitor cells can lead to higher complexity and more functional brain organoids. In addition, EBs generated from ICLC/naïve PSCs contain more neuroepithelial cells, trophoblast cells, and endodermal epithelial cells, which can serve as a good source of derived human cells or tissues. In summary, we developed a method to produce primate tissues, cells, and biomolecules with potential for therapeutic applications.

Details of the present invention will be described below. It should be understood that the features described in the various embodiments can be combined with each other to form preferred technical solutions, which are also within the scope of the present application.

I. Terminology

Unless otherwise specified, all terms used herein have the meaning commonly understood by those skilled in the art. To facilitate understanding of the present invention, some terms used herein are defined below.

As used in the specification and claims, the singular "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "(a) cell" includes a plurality of cells, including mixtures thereof.

All numerical specifications such as pH, temperature, time, concentration and molecular weight (including ranges) are approximate. It is important to understand that, although this is not always stated explicitly, all numerical indicators are preceded by the word “approximately”. It is also to be understood that, although not always explicitly described, the agents described herein are examples only and equivalents are known in the art.

The term "basal medium" as used herein refers to any medium capable of supporting cell growth. Basal media provides standard inorganic salts such as zinc, iron, magnesium, calcium and potassium, as well as vitamins, glucose, buffer systems and key amino acids. Basic media that can be used in this application include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI1640, F10, F12, α Minimum Essential Medium ( αMEM), Glasgow 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 medium suitable for the cells being cultured. In a preferred embodiment, the basal medium used in this application is a 1:1 (w/w) mixture of DMEM/F12 and neural basal medium.

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

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

Those skilled in the art can conveniently configure serum substitutes based on existing technology, cell types 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 1:1 (w/w) mixture of N2 and B27.

The term "primate" or "primate" as used herein refers to animals belonging to the order Primate. Primates include humans and non-human primates. Non-human primates include animals of the suborder Prosimian and the suborder Simiae. Specific non-human primates include, but are not limited to, macaques, lemurs, gibbons, orangutans, and baboons.

As used herein, "pluripotent stem cells" (PSCs) refer to pluripotent cells obtained from embryos at any time before gastrulation and iPSCs generated by somatic cell reprogramming. Depending on their origin and culture method, PSCs may be in different states, including primary PSCs, naive PSCs, expanded PSCs, and extended PSCs (Gafni et al., 2013; Gao et al., 2019; Takashima et al., 2014; Theunissen et al., 2014; Yang et al., 2017). PSCs are characterized by their ability to produce progeny of different cell types under appropriate conditions, which are derivatives of the three germ layers (endoderm, mesoderm and ectoderm). These can be determined according to technical tests standard in the art, such as the ability of 6 to 12 week old SCID mice to form teratomas, and also to generate the different cell types of the placenta under appropriate conditions. A PSC culture is described as "undifferentiated" when a significant proportion of the stem cells and their derivatives in the population display morphological characteristics of undifferentiated cells, distinguishing them from differentiated cells of embryonic or adult origin. It is understood that colonies of undifferentiated cells within the population may be surrounded by adjacent differentiated cells.

Various types of stem cells can be used in this application. Particularly suitable for this application are primate pluripotent stem cells. Non-limiting examples are primary cultures or established lines of ESCs and iPSCs. Pluripotent stem cells from any non-primate mammal may also be used in this application.

In one or more embodiments, primate PSCs useful in the present application may be selected from:

(i) Cells from the ESC line and/or ECC line;

(ii) cells of iPSC line;

(iii) ICM cells of preimplantation blastocysts cultured in vitro;

(iv) cells of the ICM of post-implantation blastocysts cultured in vitro; and

(v) Cells of embryos cultured in vitro from the 8-cell stage to the morula stage.

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

As used herein, the term "teratoma" is a multilineage developmental model for studying human and other animal development that encompasses various cell types from the three germ layers. Teratomas can be used to simultaneously analyze the effects of genetic perturbations in multiple cell types and can be molecularly sculpted to enrich specific tissues through microRNA (miRNA)-regulated suicide gene expression. Teratomas can be used as platforms for modeling multilineage development, pan-tissue functional genetic screens, and tissue engineering.

As used herein, the term "organoid" refers to 3D "mini-organs" derived from naive PSCs, naive PSC/ICLCs or 8CLCs, in which cells spontaneously assemble into differentiated cells that structurally and functionally mimic their in vivo counterparts. Functional cell types. Organoids can be used to model specific developmental stages of interest in humans or animals in a 3D environment, which can be particularly beneficial for studying genetic diseases or cancers where models are scarce.

As used herein, the term "embryoid body (EB)" refers to an aggregate of pluripotent cells induced to differentiate by a combination of changing the culture medium (removing factors that support pluripotency) and allowing cells to interact in a 3D structure. EBs form 3D structures under differentiation conditions and are often used as the initiating process for directed differentiation. EBs contain different germ layer cell types and can be used as a model for rapid generation of target cell types in vitro.

II. Culture medium

The culture medium disclosed herein is a chemically defined culture medium that can effectively convert primate PSCs from the primary state to the pre-implantation inner cell mass-like state, thereby generating the pre-implantation inner cell mass within 2 weeks. Such pristine PSC/ICLC without the need for colony selection. The culture medium of the present application can also transform primate PSCs from the primary state and/or the pre-implantation inner cell mass-like state to the 8-cell embryonic-like state within about 1 week, producing 8-cell embryonic-like cells (8CLC). Therefore, this medium may also be referred to as "transformation medium" in this application. In some embodiments, the media of the present application can also support the generation of cells in a pre-implantation intracellular clump-like state, survival, self-renewal and proliferation after passage and/or recovery. In some other embodiments, the culture medium of the present application can also support the survival, self-renewal and proliferation of cells in a pre-implantation intracellular mass-like state after passage and/or recovery on the extracellular matrix without the need for feeder cells or Conditioned media. In some embodiments, the culture medium of the present application can also support survival, self-renewal and proliferation after passage and/or recovery of suspension cells in a pre-implantation intracellular clump-like state without the need for feeder cells or conditioned media. . In some other embodiments, the culture medium of the present application can also support the survival, self-renewal and proliferation of cells in a pre-implantation intracellular clump-like state after passage and/or recovery on feeder cells. Preferably, the chemically defined medium of the present application is a serum-free medium.

The medium of the present application contains basal medium supplemented with PRC and/or EZH2 inhibitors, HDAC inhibitors and WNT/β-catenin signaling pathway inhibitors, and optionally selected from L-ascorbic acid, JAK/STAT3 signaling One or more components of activators and inhibitors of MAPK/ERK signaling. The basal culture medium can support cell growth, particularly the growth of human and non-human primate PSCs. Preferably, the basal medium used in this application is a 1:1 (v/v) mixture of advanced DMEM/F12 and neural basal medium. It should be understood that inhibitors of SAH can also achieve the effect of inhibiting PRC and EZH2. Thus, in some embodiments, the PRC and/or EZH2 inhibitor is a SAH inhibitor. In this application, the term "SAH/PRC/EZH2 inhibitor" refers to inhibitors of SAH, PRC and/or EZH2.

Under the culture conditions described, the presence of SAH/PRC/EZH2 inhibitors is critical for the induction of multiple regulators, including STELLA, DNMT3L and MAEL, that control naive molecular networks in humans. STELLA is a DNA methylation modulator. Its ectopic overexpression in somatic cells induces global demethylation of DNA by interfering with the function of the DNA methylation regulator UHRF1. UHRF1 dysfunction caused by STELLA deletion leads to the accumulation of abnormal DNA methylation during oogenesis (Li et al., 2018). Induction of STELLA is dose-dependent. The inventors further revealed the functional role of STELLA and found that STELLA knockout hindered the induction of naive PSC/ICLC and 8CLC. During the transformation process from primary PSC to naive PSC/ICLC, STELLA deletion caused pre-implantation ICM markers including KLF17, DPPA5, DNMT3L, TFCP2L1 and MAEL to fail to be induced. During the transformation of primary PSC and ICLC into 8CLC, 8C markers including TPRX1, TRIM60, KHDC1L, YPEL2, ALPG, ZNF280F, FAM151A and CCNA1 failed to be induced in the absence of STELLA. As demonstrated herein, during transformation using 4CL or e4CL medium, overall DNA methylation levels were significantly increased in STELLA knockout cells compared to wild type. Therefore, STELLA is required for controlled demethylation of DNA during conversion to ICLC and 8CLC. In conclusion, the present application found that SAH/PRC/EZH2 inhibitors can promote the induction of ICLC and 8CLC by resetting histone modification and DNA methylation status.

Any substance that can act as a SAH/PRC/EZH2 inhibitor can be used in the culture medium of this application, including but not limited to DZNep (CAS number: 102052-95-9, a SAH inhibitor) and CPI-1205 (CAS number: 1621862-70-1, a PRC/EZH2 inhibitor). In the culture medium of the present application, SAH/PRC/EZH2 inhibitors can be used alone or in combination, usually in their respective conventional amounts, and the amount used will not cause cell death. For example, the final concentration of DZNep in the culture medium can be 5 to 80 nM, preferably 5 to 50 nM, and the final concentration of CPI-1205 can be 0.5 to 5mM, preferably 1 to 3mM. In one or more embodiments, the SAH/PRC/EZH2 inhibitor is a PRC inhibitor.

Any substance that can act as an HDAC inhibitor can be used in the culture medium of the present application, including but not limited to trichostatin A (TSA), valproic acid (VPA), and sodium butyrate (NaB). In the culture medium of the present application, HDAC inhibitors can be used alone or in combination, and are usually used in their respective conventional amounts, and the amount used will not cause cell death. For example, the final concentration of TSA in the culture medium can be from 3 to 30 nM, preferably from 3 to 25 nM, the final concentration of VPA can be from 0.25 to 2mM, preferably from 0.5 to 1.5mM, and the final concentration of NaB can be from 0.25 to 2mM, preferably 0.5 to 1.5mM.

The inventors also found that when both SAH/PRC/EZH2 inhibitors and HDAC inhibitors are used at relatively high concentrations, primary PSCs and/or ICLCs can be converted into 8CLCs by the culture medium of the present application. Specifically, in some embodiments, to produce 8CLC, when each is used alone, DZNep can be 40 nM or higher, such as 40-80 nM, preferably about 50 nM; CPI-1205 can be 2 mM or higher, such as 2- 5mM, preferably about 3mM; TSA can be 10nM or higher, such as 10-30nM, preferably about 20nM; VPA can be 1mM or higher, such as 1-2mM, preferably about 1.5mM; and NaB can be 1mM or higher, Such as 1-2mM, preferably about 1.5mM. It should be understood that when two or more SAH/PRC/EZH2 inhibitors or two or more 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 8CLC by a combination of these SAH/PRC/EZH2 inhibitors or HDAC inhibitors. These amounts can be readily determined by those skilled in the art based on the disclosure of this application and common knowledge in the art.

In addition, it should be understood that excessive amounts of SAH/PRC/EZH2 inhibitors and HDAC inhibitors may lead to cell death. Therefore, to induce naive PSCs/ICLC while minimizing cell death, one or both SAH/PRC/EZH2 inhibitors and HDAC inhibitors can be used at lower concentrations. Specifically, when each is used alone, the final concentration of DZNep can be 5 to 15nM, preferably about 10nM, the final concentration of CPI-1205 can be 0.5 to 3mM, preferably about 1mM, and the final concentration of TSA can be 3 to 10nM, Preferably 4 to 6 nM, more preferably about 5 nM, the final concentration of VPA may be 0.25 to 1 mM, preferably 0.5mM, and the final concentration of NaB may be 0.25 to 1mM, preferably 0.5mM. In some embodiments, the SAH/PRC/EZH2 inhibitor is used in a relatively high concentration range. For example, the final concentration of DZNep can be 5 to 80 nM, preferably 5 to 50 nM, and the final concentration of CPI-1205 can be 0.5 to 5mM. , preferably 1 to 3mM, while HDAC inhibitors are used in a relatively low concentration range. For example, the final concentration of TSA can be 3 to 10nM, preferably 4 to 6nM, the final concentration of VPA can be 0.25 to 0.5mM, and the final concentration of NaB The final concentration may be 0.25 to 0.5mM. In some embodiments, SAH/PRC/EZH2 inhibitors are used in relatively low concentration ranges, such as DZNep at a final concentration of 5 to 15 nM, CPI-1205 at a final concentration of 0.5 to 2 mM, and HDAC inhibitors. Then it is used in a relatively high concentration range. For example, the final concentration of TSA can be 3 to 30 nM, preferably 3 to 25 nM, the final concentration of VPA can be 0.25 to 2mM, and the final concentration of NaB can be 0.25 to 2mM. This type of culture medium can convert primate PSCs into naive PSCs/ICLCs.

The WNT/β-catenin signaling pathway inhibitors in the culture medium of the present application include tankyrase inhibitors that inhibit classical WNT signaling. Any known tankyrase inhibitor can be used, especially those commonly used in stem cell culture, including but not limited to IWR1 (CAS No.: 1127442-82-3) and XAV939 (CAS No.: 284028-89- 3). Tankyrase inhibitors can be used in amounts commonly used in culturing stem cells. Exemplary final concentrations of tankyrase inhibitors may range from 2 to 8 μM, preferably 3 to 6 μM. For example, for IWR1 and XAV939, their respective final concentrations in the culture medium of the present application may be in the range of 2 to 8 μM, preferably 3 to 6 μM, and more preferably about 5 μM. Two or more tankyrase inhibitors can be used in combination while reducing the amount of each inhibitor used.

L-ascorbic acid was found to improve the generation and maintenance of mouse iPSCs (similar to mouse ESCs) from somatic cells by enhancing Jumonji domain-containing histone demethylases, as described in Application No. CN 200910041331.9, content of the application Incorporated herein by reference. Therefore, the inventors hypothesized that L-ascorbic acid also has a similar effect on the formation of a cell mass-like state in primates before implantation. Through appropriate testing, the inventors found that L-ascorbic acid can potentially increase the expression levels of inner cell mass 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.

L-ascorbic acid derivatives may also be used in this application, which refer to similar compounds with similar structures and antioxidant activity to L-ascorbic acid. These derivatives are more stable or more easily absorbed by cells while maintaining the biological activity of L-ascorbic acid. L-ascorbic acid derivatives include, but are not limited to, L-ascorbic acid phosphate and L-ascorbic acid organic esters, such as L-ascorbic acid palmitate. The amount of L-ascorbic acid derivative in the culture medium is not limited, but should generally be sufficient to produce a sufficient amount of L-ascorbic acid as described above.

The culture medium of the present application may contain one or more activators of Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) (i.e., JAK/STAT3) signaling, which can synergistically induce the early embryonic specificity of the present application. Gene subsets. Any known JAK/STAT3 activator may be used, in particular, those commonly used in stem cell culture are preferred. One such JAK/STAT3 activator is LIF. As used herein, LIF refers to leukemia inhibitory factor, a growth factor commonly added to culture stem cells. The preferred LIF is human LIF. The amount of JAK/STAT3 activator is a common amount that can be used in stem cell culture, and an exemplary final concentration can generally be 10 to 50 ng/mL. For example, for LIF, especially human LIF, the final concentration in the culture medium of the present application may be 10 to 50 ng/mL, preferably 10 to 30 ng/mL, and more preferably about 20 ng/mL.

The culture medium of the present application may contain one or more inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) (i.e., MAPK/ERK) signaling, which helps to interact with the culture medium of the present application. Other components in synergistically reduce DNA methylation. Any known MAPK/ERK inhibitor may be used, in particular, those commonly used in stem cell culture are preferred. One of the MAPK/ERK inhibitors is PD0325901 (CAS number: 391210-10-9). The amount of MAPK/ERK inhibitor is a common amount that can be used in stem cell culture, and an exemplary final concentration range can be 0.5 to 3 μM, preferably 0.5 to 1.5 μM. For example, for PD0325901, its final concentration in the medium of the present application can be 0.5 to 3 μM, preferably 0.5 to 1.5 μM, and more preferably about 1 μM.

In one or more embodiments, the culture medium contains DZNep at a final concentration of 5 to 15 nM or CPI-1205 at a final concentration of 0.5 to 2 mM; TSA at a final concentration of 3 to 30 nM, or 0.25 to 2 mM VPA or NaB with a final concentration of 0.25 to 2mM; preferably TSA with a final concentration of 3~10nM, or VPA with a final concentration of 0.25~1mM or NaB with a final concentration of 0.25~1mM; L-ascorbic acid of 40~70ug/mL ; LIF with a final concentration of 10-30ng/mL; PD0325901, with a final concentration of 0.5-1.5μM; the final concentration of IWR1 or XAV939 is 2-8μM respectively, preferably 3-6μM; in one or more embodiments, The culture medium for this application includes DZNep with a final concentration of 5-80nM (preferably 5-50nM) or CPI-1205 with a final concentration of 0.5-5mM (preferably 0.5-3mM); the final concentration of TSA is 3-10M, and the final concentration of VPA is 0.25～0.5mM, the final concentration of NaB is 0.25～0.5mM; the final concentration of IWR1 or XAV939 is 2～8μM respectively, preferably 3～6μM; the final concentration of L-ascorbic acid is 40～70μg/ml; the final concentration is 10～ LIF at 30ng/mL; PD0325901, final concentration is 0.5~1.5μM, MIWR1 or XAV939 is 3~6μM. More preferably, the culture medium for this application includes 10nMDZNep or 1mM CPI-1205; 5nM TSA, or 0.5mM VPA, or 0.5mM NaB; 50μg/ml L-ascorbic acid; 20ng/mL LIF; 1μM PD0325901; 5μM IWR1 or 5 μM XAV939. These media can be used to convert primate PSCs into naive PSCs/ICLCs.

In one or more preferred embodiments, the culture medium for this application includes DZNep at a final concentration of 40 to 70 nM or CPI-1205 at a final concentration of 2 to 4 mM; the final concentration of TSA is 10~30 nM, and the final concentration of VPA is 0.5~ 1.5mM, final concentration of NaB is 0.5~1.5mM; final concentration of L-ascorbic acid is 40~70g/ml; final concentration of LIF is 10~30ng/mL; PD0325901, final concentration is 0.5~1.5μM; final concentration of IWR1 or XAV939 The concentrations were 3-6μM respectively. The culture medium for this application more preferably includes 50nM DZNep or 3mM CPI-1205; 20nM TSA, or 1mM VPA, or 1mM NaB; 50ug/ml L-ascorbic acid; 20ng/mL LIF; 1μM PD0325901; 5μM IWR1 or 5μM XAV939. These media are preferably used to convert primate PSCs or naive PSCs/ICLCs into 8CLCs.

The culture medium used in the present invention may further contain at least one or more additives, which are composed of extracellular matrix, ACTIVIN/NODAL signal activator and ROCK inhibitor.

Compared with human primary PSCs, the expression level of NODAL (an activator of ACTIVIN/NODAL signaling) was increased in naive PSCs/ICLCs and 8CLCs. This observation suggests that ACTIVIN/NODAL signaling is endogenously/spontaneously activated during switching processes and self-renewal. Therefore, in some embodiments of the present application, the culture medium further includes an activator of ACTIVIN/NODAL signaling to accelerate the conversion process. Any known ACTIVIN/NODAL signaling activator can be added to the culture medium for this application, including but not limited to human ACTIVIN A and human NODAL, whose amino acid sequences are well known. In the culture medium for this application, the final concentration is 10-25ng/mL, preferably around 20ng/mL human ACTIVIN/NODAL. A combination of ACTIVIN/NODAL can also be used. Generally, the total concentration of human ACTIVINA and human NODAL in the culture medium is between 10 and 25ng/mL, preferably around 20ng/mL.

After conversion to naive PSCs/ICLCs and/or 8CLCs, cell survival at single cell passage no longer requires inhibition of ROCK signaling. However, providing low concentrations of ROCK inhibitors can increase the yield of naive PSCs/ICLCs and 8CLCs, which will facilitate the expansion of culture scale. Therefore, in some embodiments of the invention, the culture medium further includes a ROCK inhibitor. Any known ROCK inhibitor can be used in currently used media, including but not limited to Y27632 (CAS No. 146986-50-7), thiazovivin (CAS No. 1226056-71-8), hydroxyfasudil (CAS No. 105628-72-6). ROCK inhibitors can be used at final concentrations between 0.5 and 2uM, preferably around 1μM. Two or more ROCK inhibitors can be used in combination, with a total concentration of 0.5 to 2 μM in the culture medium, preferably about 1 μM.

The present invention found that when PSCs are cultured in the culture medium of the present invention, they can be transformed and maintained in suspension culture without feeder cells, and the transformed cells can self-renew and proliferate in the form of spherical clones. Accordingly, in some embodiments of the present application, the methods, culture conditions and media are feeder-free.

In some other embodiments, the inventors have discovered that providing additional cell matrix will promote spheroidal formation of cell colonies during transformation and maintenance. Under these conditions, more than 90% of PSCs can transform into hemispherical colonies during the transformation process, expressing inner cell mass markers such as DNMT3L and KLF17. Therefore, in some embodiments, naive PSCs/ICLCs and 8CLCs are cultured using extracellular matrix in the culture medium. The extracellular matrix is a dissolvable basement membrane preparation extracted from Engelbreth-Holm-Swarm mouse sarcoma (Matrigel ^™ or Geltrex ^™ or ECM ^™ ), or is a preparation containing the human matrix protein collagen IV and at least one selected from fibronectin , laminin and vitamin C components. Extracellular matrix is typically present in the culture medium of the present application in an amount of 0.1% to 0.5% (v/v). If necessary, a combination of different types of extracellular matrices can be used, and their total amount in the culture medium should also be in the range of 0.1% to 0.5% (v/v). Preferably, extracellular matrix is typically present in the culture medium of the present application in an amount of 0.2% (v/v).

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

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

(B) L-ascorbic acid at a final concentration of 40 to 70 μg/mL;

(C) Human 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 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 of 0.5 to 2 μM; and extracellular matrix at 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 0.1% to 0.5% (v/v) of extracellular matrix; or

(4) Y27632, Thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM; and 0.1% to 0.5% (v/v) extracellular matrix; 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 extracellular matrix at 0.1% to 0.5% (v/v) . These media are preferably used to convert primate PSCs into naive PSCs/ICLCs.

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

In one or more preferred embodiments, the culture medium of the present application contains 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 0.5 to 30 nM. VPA at 1.5mM, or NaB at a final concentration of 0.5 to 1.5mM; L-ascorbic acid at a final concentration of 40 to 70μg/mL; LIF at a final concentration of 10 to 30ng/mL; PD0325901 at a final concentration of 0.5 to 1.5μM; and IWR1 or XAV939 at a final concentration of 3 to 6 μM; and further supplemented by:

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

(4) Y27632, Thiazovivin, or hydroxyfasudil at a final concentration of 0.5 to 2 μM; and 0.1% to 0.5% (v/v) extracellular matrix; 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 extracellular matrix at 0.1% to 0.5% (v/v) . These media are preferably used to convert primate PSCs or naive PSCs/ICLCs into 8CLCs.

More preferably, the medium of the present application contains 50 nM DZNep or 3 mM CPI-1205; 20 nM TSA, or 1 mM VPA, or 1 mM NaB; 50 μg/mL L-ascorbic acid; 20 ng/mL LIF; 1 μM PD0325901 ; and 5 μM IWR1 or 5 μM XAV939; and further supplemented with (1) 20 ng/mL ACTIVIN A or NODAL, 1 μM Y27632, Thiazovivin, or hydroxyfasudil, and 0.2% (v/v) extracellular matrix ; or (2) 20ng/mL of ACTIVIN A or NODAL, and 1 μM of Y27632, Thiazovivin, or hydroxyfasudil; (3) 20ng/mL of ACTIVIN A or NODAL, and 0.2% (v/v) extracellular Matrix; or (4) 1 μM Y27632, Thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20 ng/mL ACTIVIN A or NODAL, or 1 μM Y27632, Thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix. These media are preferably used to convert primate PSCs or naive PSCs/ICLCs into 8CLCs.

In addition to the above components, the medium of the present application may also contain other additives commonly used in culture medium for culturing stem cells, including but not limited to serum substitutes, such as N2 and/or B27; alternative carbon sources, such as pyruvate, such as Sodium pyruvate; non-essential amino acids; L-glutamine or its substitutes, such as Glutamax ^TM supplement containing L-alanyl-L-glutamine dipeptide in 0.85% sodium chloride; and antibiotics, such as Penicillin, streptomycin, or a mixture of penicillin and streptomycin. The amounts of these additives may be those commonly used for cell culture, especially stem cell culture.

Ⅲ.Method

The culture medium of the present invention can be used to reprogram primate somatic cells into primitive PSCs/ICLCs, convert primate PSCs into primitive PSCs/ICLCs, and convert primate PSCs or primitive PSCs/ICLCs. Convert to 8CLCs.

Therefore, one aspect of the present invention discloses a method for reprogramming primate somatic cells into naive PSCs/ICLCs, including culturing the somatic cells in a solution containing SAH/PRC/EZH2 inhibitors, HDAC inhibitors, L-ascorbic acid, JAK/STAT3 signaling activator, MAPK/ERK signaling inhibitor and tankyrase inhibitor, and optionally ACTIVIN/NODAL signaling activator and/or ROCK inhibitor, in transformation medium with or without extracellular matrix. The resulting pristine PSCs/ICLCs can be used in methods to convert pristine PSCs/ICLCs into 8CLCs. Preferably, the transformation medium is the medium described in any embodiment of the present invention.

Another aspect of the invention discloses a method for converting primate PSCs into naive PSCs/ICLCs, or a method for converting primate PSCs or naive PSCs/ICLCs into 8CLCs, including culturing primate PSCs. Contains SAH/PRC/EZH2 inhibitors, HDAC inhibitors, L-ascorbic acid, JAK/STAT3 signaling activators, MAPK/ERK signaling inhibitors and tankyrase inhibitors, and optional ACTIVIN/NODAL signaling activators and/or ROCK inhibitors in transformation medium with or without extracellular matrix. In a preferred embodiment, the transformation medium is the medium described in any embodiment of the present invention.

In one or more preferred embodiments, the method is a method of converting primate PSCs into naive PSCs/ICLCs, and the transformation medium is SAH/PRC/EZH2 with a relatively low concentration as defined in any of the above embodiments. Inhibitor and HDAC inhibitor media.

In other preferred embodiments, the method is to convert primate PSCs or naïve PSCs/ICLCs into 8CLCs with a relatively high concentration of SAH/PRC/EZH2 inhibitor defined in the culture medium of any of the above embodiments. and HDAC inhibitors.

Traditional stem cell culture conditions can be used to convert PSCs into naive PSCs/ICLCs or 8CLCs. For example, single-cell primary PSCs are inoculated in traditional culture media such as mTeSR1 or E8, and can be supplemented with 5 to 15 μM ROCK inhibitors, such as Y27632. After culturing for a period of time, such as 24 hours, the medium is replaced with the medium of the present invention, and the cells continue to be cultured in the medium until original PSCs/ICLCs or 8CLCs are produced. During the culture period, it is best to replace the medium with fresh medium every day. During passage, the small clones are digested into single cells using traditional methods, and after inoculation, continue to be cultured with the culture medium of the present invention until original PSCs/ICLCs or 8CLCs are formed. It is preferable to conduct single cell passage in 3-4 days according to the ratio of 1:4-1:8. Usually, cells can be converted from primary PSCs to original PSCs/ICLCs in about 2 weeks, and cells can be converted from primary PSCs to 8CLCs in about one week. , cells can be transformed from original PSCs/ICLCs into 8CLCs in 3 to 5 days when cultured with high concentrations of SAH/PRC/EZH2 inhibitors and HDAC inhibitors. The naïve PSCs/ICLCs used for conversion to 8CLC may be naïve PSCs/ICLCs obtained by culturing primate PSCs by any of the methods described herein, or may be known naïve PSCs/ICLCs or derived from any known Original PSCs/ICLCs prepared by the method.

Generally, cells can be cultured under normoxic conditions (5% CO ₂ ) or hypoxic conditions (5% CO ₂ and 5% O ₂ ) at 37°C. There is no specific limit on the culture time, which can be easily determined by those skilled in the art based on the present disclosure and conventional techniques in the art. The addition/plating concentration can be determined by those skilled in the art based on common knowledge in the art and actual production conditions.

In some embodiments of the present application, cells can be cultured under one or more conditions selected from: (i) on feeder cells; (ii) on extracellular matrix without a feeder layer; (iii) without In a suspension of feeder cells; (iv) under hypoxic or normoxic conditions at about 37°C; (v) passaged as single cells every 3 to 4 days, with a division ratio of 1:4 to 1:8; (vi) Change culture medium daily.

In some embodiments, to convert primate PSCs to naive PSCs/ICLCs, single naive primate PSCs are added to mTeSR1 or E8 cultures supplemented with 5 to 15 μM of a ROCK inhibitor (e.g., Y27632) Culture on the base feeder layer for a period of time, such as 24 hours, and then replace the mTeSR1 or E8 medium with the transformation medium containing relatively low concentrations of SAH/PRC/EZH2 inhibitors and HDAC inhibitors as described herein, and incubate Cells were cultured under hypoxic or normoxic conditions at approximately 37°C, and the culture medium was updated every day. During the culture process, cells were passaged every 3 to 4 days with a division ratio of 1:4 to 1:8 until original PSCs/ICLCs were obtained. In some embodiments, single primary primate PSCs are cultured as described above, but the cells are added to an extracellular matrix, such as Geltrex ^™ , on a dish coated in DMEM-F12 instead of on feeder cells.

In some embodiments, to transform primate PSCs into naive PSCs/ICLCs, single naive primate PSCs are cultured using mTeSR1 or E8 medium supplemented with 5 to 15 μM of a ROCK inhibitor (e.g., Y27632). Add to the plate and culture for a period of time, such as 24 hours, then replace the mTeSR1 or E8 medium with transformation medium containing relatively low concentrations of SAH/PRC/EZH2 inhibitors and HDAC inhibitors, and culture under hypoxic conditions cells; after forming pellets, move the pellets into a flask for suspension culture, and update the culture medium every day; single cells are passaged every 4 to 5 days, with a division ratio of 1:4 to 1:8, until the original PSCs/ICLCs are obtained.

In some embodiments, to convert primate PSCs into 8CLCs, single primary primate PSCs are added to a feeder layer of mTeSR1 or E8 medium supplemented with 5 to 15 μM of a ROCK inhibitor (e.g., Y27632) Culture for a period of time, such as 24 hours, and then replace the medium with a transformation medium containing relatively high concentrations of SAH/PRC/EZH2 inhibitors and HDAC inhibitors as described herein, and incubate under normoxic or hypoxic conditions. Culture cells under conditions; passage single cells every 3 to 4 days with a division ratio of 1:4 to 1:8.

In some embodiments, to convert naive PSCs/ICLCs into 8CLCs, single cells are isolated from the naive PSCs/ICLCs and treated with relatively low concentrations of SAH/PRC/EZH2 inhibitors and HDAC inhibitors on the feeder layer. The transformation medium of the present application is cultured for a period of time, such as 24 hours, and then the medium is replaced with the transformation medium of the present application with a higher concentration of SAH/PRC/EZH2 inhibitors and HDAC inhibitors described herein, without passage. The case is cultured for 3 to 5 days, and the culture medium is updated daily.

In some embodiments, to convert naive PSCs/ICLCs into 8CLCs, single cells are isolated from the naive PSCs/ICLCs and suspended in the present application with relatively low concentrations of SAH/PRC/EZH2 inhibitors and HDAC inhibitors. For a period of time in the transformation medium, the transformation medium is additionally supplemented with 5 to 15 μM ROCK inhibitor (such as Y27632); after the formation of small aggregates, the medium is replaced with a higher concentration of SAH/PRC/EZH2 inhibitor and HDAC inhibition Transformation was carried out for several days in the application's transformation medium without additional ROCK inhibitor (such as Y27632) without passage, and the medium was updated every day.

In some embodiments, teratomas are generated by subcutaneously injecting naive PSCs/ICLCs, 8CLCs or re-naive cells as described in any embodiment herein into different organs or locations in immunodeficient mice or other immunodeficient animals. produced. Organs or locations that may be injected include, but are not limited to, the back, neck, legs, and testicles. The immunodeficient mice or other immunodeficient animals include but are not limited to immunodeficient 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-3NPG mice, hSCF1 NPG mice, hSCF2 NPG small mice, NPG-Fah mice and F344RG rats. Taking NOD-scid IL2Rg/immune deficient mice as an example, in order to avoid stress reactions, the mice need to be prepared one week in advance. Count 1 million cells and resuspend in 200ul of pre-chilled DMEM/F12 and Matrigel 1:1 mixture in a 1ml sterile injection syringe. Secure the mouse from its back and expose the abdomen for easy injection. Insert the needle diagonally into the skin and move the needle horizontally to determine the subcutaneous location. Inject the cell suspension subcutaneously into 6- to 8-week-old male NOD-scid IL2Rg/mouse, and withdraw the needle slowly to prevent the cell suspension from flowing out of the injection site. Teratomas can be seen within 3-4 weeks and collected for subsequent experiments at 7-8 weeks.

In some embodiments, the naive PSCs/ICLCs, 8CLCs or de novo PSCs described in any embodiment herein are allowed to form EBs in the form of spheroids in suspension culture in a medium that differentiates by itself. In some embodiments, these cells can be seeded onto the walls to form spheroids. The plate used to seed the cells can be a plate well known in the art for generating EBs, including but not limited to ultralow attachment plates and AggreWell ^™ plates. Any known medium that allows spontaneous differentiation can be used in this application. Culture conditions are well known in the art and are usually carried out at 37°C and 5% _CO2 .

In some embodiments, naive PSC/ICLC, 8CLC, or re-naive PSC as described in any embodiment herein are first cultured to form uniform EBs, and then the EBs are cultured in a medium that allows differentiation into the target organ, thereby Formation of organoids. The EB can be prepared using the method for preparing EB described in any embodiment herein. The organ can be any organ of interest, including but not limited to brain, liver, kidney, heart, lung, spleen, and intestine. Typically, naive PSC/ICLC, 8CLC or de novo PSC are cultured in the medium used to form EBs, the medium can be changed every day or every two days, and the resulting EBs are transferred to a medium on approximately day 6 to allow differentiation into The organ is placed in the culture medium and continues to be cultured for several days, such as continuing to culture for 2-6 days. In some embodiments, the cells are seeded into the wells of an ultra-low adsorption plate and cultured in suspension in a medium that allows for spontaneous differentiation (such as mTesR supplemented with Y-27632) to form EBs; after about 3 or 4 days, the cells are The medium is replaced with the same medium without Y-27632; after 1-3 days of culture, transfer the EBs to ultra-low adsorption plates at approximately day 6 and continue culturing for several days in a medium that allows differentiation into target organs. days, such as 2-6 days. The tissue thus formed can be cultured to form aggregates or 3D structures in a differentiation medium suitable for preparing the desired organ, for example on an orbiting shaker installed in an incubator that can rotate at 50-100 RPM. By proceeding, organoids of the organ of interest have been generated. The 3D structure (3D scaffold) used herein includes but is not limited to Matrigel, Geltrex, etc. The medium that allows differentiation into the organ of interest can be any medium known in the art for differentiating EBs into the organ of interest. In some embodiments, for cerebral organoids, neural induction medium and brain differentiation medium may be used. Change EB medium every day or every two days, then transfer to neural induction medium on day 6 and continue culturing for 4 days. EB will form neuroepithelial tissue. The tissue can be embedded within a 3D structure such as a Matrigel drop and cultured in brain differentiation medium mounted on an orbital shaker in an incubator that can rotate at 50-100 RPM. Brain organoids can be collected for later use.

In some embodiments, brain organoids are generated by first seeding 9000 primary PSC, naive PSC/ICLC or 8CLC cells in an ultra-low-attachment 96-well plate containing EB medium to form uniform EB spheres. Replace the EB medium every two days, update the neural induction culture medium on the low-adsorption plate on the 6th day, and continue to culture in the neural induction medium for 4 days. EBs will form neuroepithelial tissue, and then the tissue will be embedded into Matrigel droplets, cultured in brain differentiation medium, and placed on an orbital shaker installed in an incubator with a rotation speed of 70 rpm. Brain organoids can be harvested on day 30 for further use.

The re-primitive PSCs described herein can be obtained by directional differentiation of naive PSCs/ICLCs or 8CLCs using methods commonly used in this field. For example, human ESCs or iPSCs can be cultured in commercially available media such as TeSR or Essential 8 to obtain reconstituted PSCs.

Reprogramming somatic primate cells into naive PSCs/ICLCs, converting primate PSCs into naive PSCs/ICLCs, or converting primates into naive PSCs/ICLCs using any of the transformation media described in any embodiment of the invention. Animal PSCs or original PSCs/ICLCs are converted into 8CLCs, primate somatic cells are reprogrammed into original PSCs/ICLCs using a culture medium or a kit, or primate PSCs are converted into original PSCs /ICLCs, or converting primate PSCs or naive PSCs/ICLCs into 8CLCs using a culture medium or a kit to generate teratomas, organoids or EBs are also included in this application.

In some embodiments, the present application also includes SAH/PRC/EZH2 inhibitors and HDAC inhibitors for use in reprogramming primate somatic cells into iPSCs, or transforming primate PSCs into naive PSCs/ICLCs, or Use in culture media or kits for transforming primate PSCs or original PSCs/ICLCs into 8CLC. Preferably, the culture medium or kit may further comprise one or more components selected from the group consisting of L-ascorbic acid, JAK/STAT3 signaling activator, MAPK/ERK signaling inhibitor and tankyrase inhibitor, and optionally ACTIVIN/NODAL signaling activator and optional ROCK inhibitor (such as Y27632), and optional extracellular matrix.

In some embodiments, methods of converting primate PSCs into naive PSCs/ICLCs and converting primate PSCs or naive PSCs/ICLCs into 8CLCs may include a genetic engineering step through knockdown and/or Or knock out one or more related genes in the cells to reduce the activity of SAH, PRC and/or EZH2, and/or HDAC of PSCs, and then use the medium of the present application to culture the modified PSCs. Preferably, in order to reduce the activity of SAH, PRC and/or EZH2 in PSCs, the expression of any SAH, PRC and EZH2 regulators can be reduced by knockdown (such as siRNA technology), or knockout (such as CRISPR/Cas9 technology) . Likewise, HDAC regulon expression can be reduced in the same manner as described above. After PSCs are transformed, they can be cultured using the culture medium of the present application according to the above method. In some embodiments, when the activities of SAH, PRC and/or EZH2 of PSCs are reduced due to modification, the culture medium used to culture the above-mentioned modified PSCs may or may not contain SAH/PRC/EZH2 inhibitors. Likewise, if the HDAC activity of PSCs is reduced due to engineering, the culture medium may or may not contain HDAC inhibitors. In cases where SAH, PRC, and/or EZH2 activity and HDAC activity are both reduced due to engineering, the culture medium may contain neither SAH/PRC/EZH2 inhibitors nor HDAC inhibitors, or it may contain SAH/PRC/ EZH2 inhibitors or HDAC inhibitors.

Therefore, in some embodiments, the application further provides neither a SAH/PRC/EZH2 inhibitor nor a SAH/PRC/EZH2 inhibitor or an HDAC inhibitor, or a SAH/PRC/EZH2 inhibitor or an HDAC inhibitor. The remaining components of the culture medium are the same as the components and dosage of the culture medium described in the second part of this application. In some embodiments, the culture medium may contain reagents for transfection using liposomes. For example, in the above method, primate PSCs are cultured in a culture medium containing liposomes encapsulating a vector of shRNA targeting SAH, PRC and/or EZH2 regulators, and in addition to the vector and liposome, the The culture medium also contains other components described in Section II for the culture medium of the present invention, and the culture medium may or may not contain a SAH/PRC/EZH2 inhibitor.

Ⅳ. Cells

This application provides human cells/tissues from teratomas, organoids and EBs. Human cells/tissues have transcriptomes similar to those of cell/tissue types in the human body, including but not limited to airway epithelial cells, epithelial cells, epithelial progenitor cells, radial glia, periodic radial glia, neurons, melanocytes , mesenchymal stem cells, pericyclic mesenchymal stem cells, adipogenic mesenchymal stem cells, vascular endothelial cells, smooth muscle cells, pericytes, fibroblasts, fibroblast progenitor cells, hematopoietic endothelial cells, immune cells, red blood cells, primitive intestine Endodermal cells, trophoblasts, cytotrophoblasts, villous cytotrophoblasts, placental endothelial cells, granulocyte-macrophage progenitor cells, hematopoietic endothelial cells, mast cells, lymphocytes, neuroblasts, serotonergic neurons, Cycling neural progenitor cells, granulocyte-macrophage progenitor cells, oligodendrocyte precursor cells, Schwann cell precursor cells, endothelial cells, arterial endothelial cells, midgut epithelial cells, hindgut epithelial cells, neural stem cells, Neuroblasts, neuroepithelial cells, retinal precursor cells.

This application also provides isolation of primary PSCs/ICLCs. The original PSCs/ICLCs of the present application have a transcriptome close to the human pre-implantation inner cell mass, a transposable element profile close to the human pre-implantation inner cell mass, and a DNA methylation group close to the human pre-implantation inner cell mass. It is close to the chromatin open state of the human pre-implantation inner cell mass and close to the metabolic state of the human pre-implantation inner cell mass.

In this article, the term "close to" refers to being essentially the same, or without substantial differences. Those skilled in the art can confirm based on known techniques in the art that even though there may be some minor differences, the cells of the present application, including those from the present application, The cells of ICLC and 8CLC described in the application are essentially the same as natural ICM cells or 8-cell embryonic cells.

Preferably, the pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, MAEL and REX1 are significantly induced in the original PSCs/ICLCs of the present application. More preferably, among the pre-implantation ICM marker genes in the original PSCs/ICLCs of the present application, the expression level of at least one marker gene is 10 times the expression level of the corresponding pre-implantation ICM marker gene in the primary human PSCs. Above; preferably, the expression levels of all the above marker genes in original PSCs/ICLCs are more than 10 times higher than the corresponding pre-implantation ICM marker gene expression levels in primary human PSCs.

Preferably, the original PSCs/ICLCs of the present application also have one or more of the following characteristics:

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

2) Maintain the stability of the genome in culture according to the karyotype;

3) Cells capable of producing 3 germ layers;

4) Able to produce primordial germ cell-like cells;

5) Able to be chimeric into mouse embryos and differentiate into embryonic and extraembryonic tissues;

6) Capable of conversion to an extraembryonic cell fate in vitro; and

7) Ability to form blastocyst-like structures in vitro.

Such naive PSCs/ICLCs can be obtained by culturing primate PSCs using any method described in any embodiment of the present application. Therefore, in some embodiments, the present application also includes cells obtained by any method described herein, especially naive PSCs/ICLCs.

The present application also provides isolated 8CLCs that express 8-cell (8C) state-specific marker genes at a much higher level than cells in the pre-implantation intracellular mass-like state or primary state. In some embodiments, the 8-cell state specific marker genes include ZSCAN4, TPRX1, ZIM3, ZSCAN5B, ZNF280A, and ARGFX. Preferably, among the specific marker genes, the expression level of at least one specific marker gene is more than 5 times the expression level of the corresponding 8-cell specific marker gene in primary PSCs or naive PSCs/ICLCs. Preferably, the expression level of all the above-mentioned specific marker genes is more than 5 times the expression level of the corresponding 8-cell-specific marker genes in primary PSCs or naive PSCs/ICLCs.

Preferably, the isolated 8CLC of the present application has transcriptome, transposable element characteristics, and chromatin open state close to those of human 8-cell embryos. More preferably, the 8CLC of the present application also has one or more of the following characteristics:

1) Maintain the stability of the genome in culture according to the karyotype;

2) Able to produce cells of 3 germ layers;

3) Able to produce primordial germ cell-like cells;

4) Able to be chimeric into mouse embryos and differentiate into embryonic and extraembryonic tissues;

5) Capable of conversion to an extraembryonic cell fate in vitro; and

6) Ability to form blastocyst-like structures in vitro.

Naive PSCs/ICLCs obtained by somatic cell reprogramming of somatic cells using the transformation medium of the present application are also within the scope of the present application.

The present application also provides a cell culture medium containing the cells of the present application, especially the original PSCs/ICLCs and/or 8CLCs of the present application. The cell culture medium may also include the culture medium described in any embodiment of the application.

The invention is described in the following non-limiting examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way. Various changes and modifications may be made within the spirit of this application. Unless otherwise stated, the technologies involved are conventional technologies in various fields such as molecular biology, cell biology, and biochemistry that are well known to those skilled in the art.

Example 1: Generation of naive PSCs/ICLCs and 8CLCs

1. Generation of original PSCs/ICLCs

Materials and methods

4CL basic medium

1:1 mixture of Neurobasal Medium (Gibco) and Advanced DMEM/F12 (Gibco) supplemented with N2 Supplement (1X, Gibco), B27 Supplement (1X, Gibco) (homemade N2 and B27), sodium pyruvate (1X, Hyclone), non-essential amino acids (NEAA) (Gibco), glutaminase ^™ (1X, Gibco), and penicillin-streptomycin (1X, Gibco).

4CL supplement

4CL Medium 1 is supplemented with 4CL Basic Medium:

SAH/PRC/EZH2 inhibitor (10nM DZNep), HDAC inhibitor (5nM TSA), L-ascorbic acid (50μg/mL), JAK/STAT3 activator (20ng/mL human LIF), MAPK/ERK inhibitor (1μM PD0325901 ), tankyrase inhibitor (5μM IWR1), ACTIVIN A/NODAL activator (20ng/mL human ACTIVIN A), extracellular matrix (0.2% (v/v) Geltrex ^TM ), ROCK inhibitor (1μM Y27632) . Table 1 lists the trademarks and catalog numbers of all 4CL supplements.

Table 1

cell

H9 human ESC line

method

1) Maintenance of primary human PSCs

All human PSCs provided were routinely maintained in mTeSR1 or E8 medium on Matrigel ^™ or Geltrex ^™ coated plates. Cells are generally passaged with 0.5mM EDTA every 4 to 5 days. During passage, cells were washed once with PBS and treated with 0.5mM EDTA for 5 minutes. Then, remove the EDTA and detach the cells into small pieces using a Pasteur pipette with mTeSR1 or E8 medium. Human PSCs were cultured in an incubator under normoxic conditions (37°C, 5% CO ₂ ).

2) Convert to original PSCs/ICLCs on the feeder layer

One day before the start of transformation, primary human PSCs were washed once with PBS, isolated into single cells, and added to a feeder layer of mTeSR1 or E8 medium supplemented with 10 μM Y27632 at a density of 1000 to 1500 cells/ ^cm superior. After 24 hours, the medium was changed to 4CL medium 1. Replace the medium with the same medium every 24 hours. Colonies become round and hemispherical within 24 to 48 hours. Cells were passaged every 3 to 4 days. When passaging, use TrypLE: 0.5mM EDTA (1:1) to separate the cells into single cells and add them to the feeder layer at a density of 1000 to 1500 cells/cm ² (the feeder layer is inoculated after coating with Geltrex ^TM /Matrigel ^TM on the petri dish). The induction and maintenance of original PSCs/ICLC can be carried out under hypoxic (37°C, 5% CO ₂ , 5% O ₂ ) or normoxic (37°C, 5% CO ₂ , 21% O ₂ ) conditions, preferably low oxygen conditions.

2.8 Generation of CLC

Materials and methods

4CL basic medium

Same as Example 1.

e4CL supplement

e4CL medium is supplemented with 4CL basic medium:

SAH/PRC/EZH2 inhibitor (50nM DZNep or 3mM CPI-1205), HDAC inhibitor (20nM TSA or 1mMVPA or 1mM NaB), tankyrase inhibitor (5μM IWR1 or 5μM XAV939), L-ascorbic acid (50μg/ mL), JAK/STAT3 activator (20ng/mL human LIF), MAPK/ERK inhibitor (1μM PD0325901), ACTIVIN A/NODAL activator (20ng/mL human ACTIVIN A or 20ng/mL human NODAL), ROCK inhibitor (1 μM Y27632 or 1 μM Thiazovivin or 1 μM hydroxyfasudil) and extracellular matrix (0.2% (v/v) Geltrex ^™ or Matrigel ^™ ).

cell

H9, H1, and UH10 human ESC lines.

method

1) Conversion of primary human PSCs into 8CLCs

Primary human PSCs were cultured according to the same method as Example 1. One day before the start of transformation, primary human PSCs were isolated into single cells and seeded onto the feeder layer at 2,000 to 3,000 cells/ ^cm with mTeSR1 or E8 medium supplemented with 10 μM Y27632. After 24 hours, the medium was replaced with e4CL medium, and the cells were cultured at 37°C, 5% CO ₂ , hypoxia or normoxia. Renew the culture medium daily. Cells were passaged every 3 to 4 days. For passage, dissociate into single cells using TrypLE:0.5mM EDTA (1:1) and add to feeder-coated plates at a density of 2000 to 3000 cells/ ^cm . Typically, cells convert to 8CLCs in about a week.

2) Convert from original PSCs/ICLCs to 8CLCs

One day before the start of transformation, naive PSCs/ICLCs were isolated into single cells and seeded on feeder cells using 4CL medium 1 at 2000-3000 cells/ ^cm2 . After 24 hours, the medium was changed to e4CL medium. Renew the culture medium daily. The cells transform into 8CLCs within 3 to 5 days without passage.

Experimental results

The results are shown in Figure 1-6. Figure 1. Induction of naive PSCs/ICLCs and 8CLCs expressing naive or 8C-specific genes by untreated or transformed primary H9ESCs with 4CL (day 12) or step e4CL (day 5). The immunostaining images of KLF17 and TPRX1 are demonstrated, as shown in Figure 1(b); the heat map shown in Figure 1(c) shows that in NHSM, Expression of preimplantation ICM-enriched genes in human naïve PSCs cultured in 5iLAF, 4CL, and human ICM cells; the heat map shown in Figure 1(d) shows that in NHSM, /> Expression of totipotency genes in naive ESCs cultured in 5iLAF or step e4CL (day 5), EPSCs and human 8C embryonic cells; validation by RT-qPCR in naive H9 ESCs cultured in direct e4CL (day 7) The totipotent gene in , as shown in Figure 1(e).

The results shown in Figure 2 demonstrate that the transcriptomes of naive PSCs/ICLCs and 8CLCs match the transcriptomes of human embryos. UMAP was used to compare the results of return development from human E7 to E3 embryonic stages during the scRNA-seq time course of stepwise or direct e4CL induction, as shown in Figure 2(a); the analysis shown in Figure 2(b) UMAP visualization of step e4CL-day5 cells, which shows 7 clusters, with cluster 5 (8CLC) accounting for 11.9% of the total cell number; through the bubble chart shown in Figure 2(c), representative cells in the early stages of human embryos are shown Expression frequency and average expression of pluripotency and totipotency genes, and those not expressed by 4CL (day 8 [passage 2] and day 12 [passage 3]) and e4CL (day 5 C5 [8CLC] and non-8CLC [all other clusters added]) treated or transformed primary ESC; the violin plot shown in Figure 2(d) shows that compared with primary ESC, 4CL-day 12 original ESC and 8CLC, Log-normalized expression of representative early human embryonic enriched TEs at early stages of human embryos and at passage 10 in human ESCs.

The results shown in Figure 3 show that the karyotype of cells induced by original PSC medium (4CL) and 8CLC medium (e4CL) is normal after long-term culture. Among them, as shown in Figure 3(a), representative images of G-banded karyotypes of primary H9 and primary iPSC-4 cultured in 4CL for 15 generations. Each panel counts 20 cells in metaphase and the G-banded karyotype of primary H9 and iPSC-4 cultured in step e4CL (day 5) as shown in Figure 3(b) Representative image.

The results shown in Figure 4 indicate that cells cultured in naive PSC medium (4CL) and 8CLC medium (e4CL) have lower DNA methylation levels compared with primary PSCs. Among them, the violin plot in Figure 4(a) shows that under the initial conditions, 4CL (day 12), 5iLAF, Global CpG methylation levels measured by RRBS for NHSM, stepwise e4CL (day 5) and direct e4CL (day 7) cultured human PSCs as well as human 8C embryos and ICM pass; Figure 4(b), Shown in the starting state, 4CL (day 12), 5iLAF, /> CpG methylation levels of human PSCs cultured under NHSM and step e4CL (day 5) conditions, as well as imprinting control regions in ICM and post-implantation embryos; Genome Browse traces show that in primary conditions, 4CL (day 12 )、5iLAF、/> CpG methylation of indicated naïve pluripotent (blue) and totipotent (red) sites in NHSM, stepwise e4CL (day 5) and direct e4CL (day 7), human 8C embryos and PSC cultured under ICM horizontal, as shown in Figure 4(c).

The results shown in Figure 5(a)-(d) demonstrate that naive PSC/ICLC and 8CLC have matching chromatin accessibility to human embryos. Among them, the UMAP gene score of all genes in scATAC-seq is visualized, with primary ESC untreated (red), 4CL (day 12; blue) or step e4CL (day 5; green), as shown in Figure 5(a) ); the UMAP visualization is based on panel a and highlights gene scores for the originating state (ZIC2), shared naive state pluripotency/8CLC (DPPA3) and totipotent genes that were either untreated or induced by 4CL (day 12 ) and stepwise e4CL (day 5) expressed in every cell in primary ESCs transformed as shown in Figure 5(b); genome reader tracking reveals chromatin accessibility, H3K27ac levels and transcription factors The location of the DNA binding motif is at the original pluripotent KLF17 and totipotent ZSCAN4 sites.

The results shown in Figure 6 demonstrate the promotion of 8CLC by sorting TPRX1-GFP reporter cells. As shown in the upper panel of Figure 6(a), EGFP was inserted into the TPRX1 site of H9 ESCs or HN10-DsRed ESCs (for chimerism experiments), and the donor construct used to generate the TPRX1-EGFP reporter cell line. The lower figure shows that TPRX1-EGFP was knocked into cells and verified by e4CL step culture (day 5) and anti-TPRX1 immunostaining, and the results were consistent with the GFP ⁺ signal (left figure). FACS analysis of TPRX1-EGFP cells in step e4CL (day 5) shows the percentage of GFP ⁺ cells (right panel). As shown in the bubble chart in Figure 6(b), it represents the representative pluripotency of 4CL original ESC and sorted 8CLC at the human early embryonic stage and the 10th generation of human ESC compared with the primary ESC. and expression frequency and average expression of totipotency genes.

Example 2: Generation of naive PSCs/ICLCs via ESCs or iPSCs

Materials and methods

4CL basic medium

Same as Example 1.

4CL supplement

Same as Example 1.

cell

Human ESC lines: H1 (male), HN10 (female), HUES1 (male) and WIBR3 (female); human iPSC lines: CBC14 (made by the inventor, female), C11 (made by the inventor, female), Phoenix ( Gift from Ulrich Martin Laboratory, female), DiPS 1016SevA (purchased from Harvard Stem Cell Institute, male), STiPS O-XX1 (purchased from Harvard Stem Cell Institute, female), UH10 (male).

method

The same method as in Example 1 was used.

Experimental results

The RT-qPCR data in Figure 7 shows that in the original PSC/ICLC transformed from various primary human PSC lines, the pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL and REX1 were Significantly induced. This proves that 4CL medium 1 has broad applicability for inducing human PSCs.

Example 3: Generation of naive PSCs/ICLCs in extracellular matrix

Materials and methods

4CL basic medium

Same as Example 1.

4CL supplement

Same as Example 1.

cell

H9 human ESC line.

method

The same method as in Example 1 was used, except that cells were added to culture dishes coated with DMEM-F12 (cat#) containing 1% (v/v) Geltrex ^™ instead of feeder cells.

Experimental results

The RT-qPCR data in Figure 8 shows that in the original PSCs/ICLCs transformed on Geltrex ^TM- coated culture dishes containing 4CL medium 1, the pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, and KLF4 , MAEL and REX1 were significantly induced, which was similar to the original PSCs/ICLCs on the feeder layer. This shows that 4CL medium 1 is also effective without feeder cells.

Example 4: Generation of pristine PSCs/ICLCs in suspension

Materials and methods

4CL basic medium

Same as Example 1.

4CL supplement

Same as Example 1.

cell

H9 human ESC line.

method

Primary human PSCs were cultured according to the same method as Example 1. One day before the start of transformation, primary human PSCs were isolated into single cells and added to Aggrewell ^™ 800 plates at 60,000 cells/well using mTeSR1 or E8 medium supplemented with 10 μM Y27632. After 24 hours, the medium was changed to 4CL medium 1, and then converted to hypoxic culture. Cells formed pellets within 3 days. The pellets were then resuspended and transferred to low-adsorption culture bottles (Greiner Bio One, 658190) for suspension culture. Renew the culture medium daily. Cells were passaged every 4 to 5 days. For passage, cells were dissociated into single cells using TrypLE:0.5mM EDTA (1:1) and resuspended in 4CL Medium 1 at a density of 150,000 cells/mL. The resuspended cells were then added to a low-adsorption culture bottle (Greiner Bio One, 658190) for suspension culture. Cells formed small aggregates within 24 hours. Typically, cells convert to naive PSCs/ICLCs within approximately 3 weeks of initiation.

Experimental results

Figure 9 is RT-qPCR data showing that in the original PSCs/ICLCs transformed using 4CL medium 1 suspension, the pre-implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL and REX1 were significantly induced. It shows that 4CL medium 1 is equally effective for suspension culture.

Example 5: Generation of naive PSCs/ICLCs in 4CL without ECM/ROCK inhibitor/ACTIVIN/NODAL activator

Materials and methods

4CL basic medium

Same as Example 1.

4CL supplement

4CL medium 2 (minus extracellular matrix) is supplemented with 4CL basal medium:

SAH/PRC/EZH2 inhibitor (10nM DZNep), HDAC inhibitor (5nM TSA), L-ascorbic acid (50μg/mL), JAK/STAT3 activator (20ng/mL human LIF), MAPK/ERK inhibitor (1μM PD0325901 ), tankyrase inhibitor (5 μM IWR1), ACTIVIN A/NODAL activator (20 ng/mL human ACTIVIN A), and ROCK inhibitor (1 μM MY27632).

4CL Medium 3 (minus ROCK inhibitor) is supplemented with 4CL Base Medium:

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

4CL Medium 4 (minus ACTIVIN/NODAL activator) is in 4CL Base Medium supplemented with:

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

cell

H9 human ESC line.

method

The same method as in Example 1 was used.

Experimental results

Figure 10 is RT-qPCR data showing that in the original PSCs/ICLCs transformed using 4CL medium 2 (Picture A), 4CL medium 3 (Picture B), and 4CL medium 4 (Picture C) respectively, before implantation ICM marker genes KLF17, DNMT3L, DPPA5, STELLA, TFCP2L1, KLF4, MAEL and REX1 were significantly induced. These results indicate that 4CL medium without Geltrex ^™ , ROCK inhibitors or ACTIVIN/NODAL activators is also effective.

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

Materials and methods

4CL basic medium

Same as Example 1.

e4CL supplement

e4CL medium is supplemented with 4CL basic medium:

SAH/PRC/EZH2 inhibitor (50nM DZNep or 3mM CPI-1205), HDAC inhibitor (20nM TSA or 1mMVPA or 1mM NaB), L-ascorbic acid (50μg/mL), JAK/STAT3 activator (20ng/mL human LIF ), MAPK/ERK inhibitor (1μM PD0325901), tankyrase inhibitor (5μM IWR1 or 5μM XAV939), ACTIVIN A/NODAL activator (20ng/mL human ACTIVIN A or 20ng/mL human NODAL), ROCK inhibitor (1 μM Y27632 or 1 μM Thiazovivin or 1 μM hydroxyfasudil) and extracellular matrix (0.2% (v/v) Geltrex ^™ or Matrigel ^™ ).

cell

H9, H1, and UH10 human ESC lines.

method

1) Conversion from primary human PSCs to 8CLCs

Primary human PSCs were cultured according to the same method as Example 1. One day before the start of transformation, primary human PSCs cells were isolated into single cells and added to the feeder layer with mTeSR1 or E8 medium supplemented with 10 μM Y27632 at 2,000 to 3,000 cells/ ^cm . After 24 hours, the medium was replaced with e4CL medium, and the cells were cultured at 37°C, 5% CO ₂ , hypoxia or normoxia. Renew the culture medium daily. Cells were passaged every 3 to 4 days. For passage, cells were dissociated into single cells using TrypLE:0.5mM EDTA (1:1) and added to feeder-coated plates at a density of 2000 to 3000 cells/ ^cm . Typically, cells convert to 8CLCs in about a week.

2) Convert from original PSCs/ICLCs to 8CLCs

One day before the start of transformation, ICLC were dissociated into single cells and loaded on the feeder layer with 4CL medium ¹ at 2000-3000 cells/cm. After 24 hours, the medium was changed to e4CL medium. Renew the culture medium daily. The cells transform into 8CLC within 3 to 5 days without passage.

Experimental results

Figure 11 RT-qPCR data shows that human 8C-specific marker genes ZSCAN4, TPRX1, ZIM3, ZSCAN5B, ZNF280A and ARGFX are significantly induced in 8CLCs transformed from primary human PSCs or naive PSCs/ICLCs.

Example 7: Generation of 8CLC in suspension

Materials and methods

4CL basic medium

Same as Example 1.

e4CL supplement

Same as Example 6.

cell

H9 human ESC line.

method

Conversion from suspension cultured original PSCs/ICLCs to suspension cultured 8CLCs

The original PSCs/ICLCs were cultured according to the same method as Example 1. One day before the start of transformation, naive PSCs/ICLCs were isolated into single cells and resuspended in 4CL medium 1 at a density of 300,000 cells/ml. The cell suspension was then added to a low-adsorption culture bottle (Greiner Bio One, 658190) for suspension culture. After 24 hours, cells formed small aggregates and the medium was switched to e4CL medium. The culture medium is updated daily, and cells transform into 8CLC within 3 to 5 days without passage.

Experimental results

The RT-qPCR data in Figure 12 shows that in 8CLC transformed using e4CL medium suspension, the 8C marker genes ZSCAN4, ARGFX, TPRX1, ZNF280A and ZSCAN5B were significantly induced. This shows that e4CL medium is equally effective for suspension culture.

Example 8: Generation of 8CLC by ESCs and iPSCs

Materials and methods

4CL basic medium

Same as Example 1.

e4CL supplement

Same as Example 6.

cell

Human ESC lines: HN10 and UH10

method

Same as Example 6.

Experimental results

The RT-qPCR data in Figure 13 shows that the 8C marker genes ZSCAN4, ARGFX, TPRX1, ZNF280A, ZSCAN5B, DUXA, DUXB, MBD3L2, STELLA, KLF17 and KHDC1L were significantly induced in 8CLCs transformed from various human PSCs lines. This indicates that e4CL medium has broad applicability to human PSCs.

Example 9: Generation of teratoma

Materials and methods

DMEM/F12,

cell:

Human 8CLC, naive PSC/ICLC were obtained from Examples 1-8 or de novo PSC were obtained from human ESCs cultured in mTeSR1.

method

1) Animal preparation

Male NOD-scid-IL2Rg-/- mice were maintained in an SPF facility environment until 6-8 weeks of age.

2) Preparation of primary PSC, primary PSC/ICLC and 8CLC suspensions

Isolate 1 million primary PSC, original PSC/ICLC or 8CLC from the culture medium. After collection, use 200 μL of 1:1 DMEM/F12 and Resuspend in pre-cooled solution. The resuspension was placed on ice until transplantation. Transplantation should be performed immediately.

3) Transplantation

Collect the primary, naive PSC/ICLC or 8CLC suspension into a 1ml syringe and inject it subcutaneously into 6-8 week old male NOD-scid-IL2Rg-/- mice.

To avoid stress reactions, mice need to be prepared at least a week in advance. When teratoma is formed, count 1 million cells in a 1 mL sterile syringe using 200 μl of pre-chilled 1:1 DMEM/F12 and Matrigel mixture. Secure the mouse from its back and expose the abdomen for easy injection. Insert the needle diagonally into the skin and work the 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 the needle was slowly pushed out to prevent the cell suspension from flowing out of the injection site.

4) Animal monitoring during teratoma formation

Mice were maintained in SPF grade facilities for 8 weeks after injection. Visually observe the size of the teratoma growing at the injection site. Typically, teratomas are isolated 6-8 weeks after injection.

5) Collect human cells from teratomas

Mice were sacrificed 8 weeks after injection, and teratomas were isolated. Teratomas are collected for cryosectioning, cell isolation, single-cell transcriptome analysis, or other uses.

Experimental results

The results shown in Figure 14 indicate that primary cells, naive PSCs/ICLCs (4CL) and 8CLC can form teratomas. As shown in Figure 14(a), representative images of teratomas are derived from primary ESCs transformed by 4CL (passage 15) and 8CLC; as shown in Figure 14(b), hematoxylin and The eosin staining picture shows the primary ESC teratoma transformed from 4CL (passage 15) and sorted 8CLC. Representative images of tissues corresponding to the three germ layers are shown; as shown in Figure 14(c), UMAP visualization showing scRNA from sorted 8CLC, e4CL-day5 cells, 4CL naive ESC and primary ESC teratoma -The cell type identified in the seq. H9ESCs were used to generate these teratoma cell types.

The results shown in Figure 15 are annotations of teratoma cells with different starting cell types. The UMAP visualization is based on Figure 14c and shows the cell types identified in scRNA-seq datasets generated from primed ESCs, 4CL naive PSCs, step e4CL-day 5 cells, and sorted 8CLCs.

The results shown in Figure 16 represent teratoma cell type quantification. Primitive PSC/ICLC teratomas or 8CLC teratomas can derive embryonic (3 germ layer cell types) and extraembryonic trophoblast. The histogram shown in Figure 16(a) shows the pairs of identified cells in different teratomas. Distribution of types, the histogram shown in Figure 16(b) shows the relative distribution of different teratomas for each embryonic (ectodermal, mesodermal and endodermal) and extraembryonic (trophoblast) lineage.

The results shown in Figures 17(a) to 17(d) represent the annotation and quantification of teratoma trophoblast subtypes, and the trophoblasts of teratomas can further generate cytotrophoblasts, villous cytotrophoblasts, and placental endothelial cells; Figure 17 Represents UMAP visualization of cell types identified from scRNA-seq of teratomas generated from sorted 8CLC, e4CL-day 5 cells, 4CL naive ESCs, and primed ESCs, respectively, among extraembryonic trophoblast lineage cell subtypes Expression frequency and average expression level results of marker genes, and the relative distribution of extraembryonic trophoblast lineage cell subtypes in teratomas produced by sorted 8CLC, e4CL-day-5 cells, 4CL naive ESCs, and primary ESCs. Figure 14c shows UMAP visualization of the distribution and expression of relevant markers in the indicated teratoma trophoblast cells.

Figure 18 represents annotation and quantification of teratoma immune cell subtypes, including granulocyte-macrophage progenitor cells, hematopoietic endothelial cells, mast cells, lymphocytes and erythrocytes. For example, a. UMAP visualization shows annotated immune cell subpopulations (left picture) and primary PSCs, 4CL naive ESCs, step-by-step e4CL-day-5 cells, and sorted 8CLC-derived teratoma cells (right picture) ).

b. Bubble chart shows the expression frequency and average expression level of marker genes in different immune cell subtypes.

c. Bar graph showing the distribution of sorted 8CLC, staged e4CL-day-5, 4CL-naïve and primary PSC-derived teratomas against different immune cell subtypes.

Figure 19 shows annotation and quantification of teratoma neural cell subtypes, including circulating EOMES+ intermediate progenitor cells, dopaminergic neuron progenitor cells, dopaminergic neurons, gama-GABAergic neurons, glutamatergic neurons, and Mature neurons, neuroblasts, radial glia, serotonergic neurons.

a.UMAP visualization shows the distribution of primary PSCs, 4CL original PSCs, e4CL cells and teratoma cells obtained from sorted 8CLC;

b. UMAP visualization shows annotated neuronal cell subclusters derived from primary PSCs, 4CL naive PSCs, e4CL cells and sorted 8CLC-derived teratomas;

c. Bar graph showing the relative contribution of different neuronal cell types to the indicated teratomas;

d. Bar graph showing the contribution of different teratomas to the identified cell types.

Example 10: Generation of cerebral organoids

Materials and methods

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

Neurobasal medium (Gibco)

GlutaminaseTM (Invitrogen cat.no.35050-038)

Non-essential amino acids (Sigma cat.no.M7145)

Penicillin-Streptomycin (Sigma)

N2 supplement (Invitrogen)

B27 supplement does not contain vitamin A (Invitrogen cat.no.12587010)

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

Y27632 (Axon Medchem 1681)

Mercaptoethanol (Merck cat.no.8057400005)

Heparin (Sigma cat.no.H3149)

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

Brain-derived neurotrophic factor (R&D Company 248-BDB-250)

Matrigel Growth Reduction Factor (BD Biosciences 356230)

U-shaped bottom low adsorption 96-well plate (Corning company cat.no.CLS7007)

Low adsorption 24-well plate (Corning cat.no.CLS3473)

60×15mm low-adsorption tissue culture dish (Corning company cat. No. 3261)

medium

cell

The human 8CLC, naive PSC/ICLC or de novo PSC in Example 1 were obtained from human ESCs cultured in mTeSR1.

method

Day 0

When the cell growth ratio accounts for 70-80% of the culture plate area, use accutase to digest the clones into single cells, resuspend 9000 cells/well in 150 μL EBM and seed them in a 96-well low-adsorption culture plate.

Day 1

Observe under a tissue culture microscope after 24 hours, and small embryoid bodies with clear boundaries can be observed. Continue to culture the embryoid bodies in a cell culture incubator at 37°C and 5% _CO2 .

Day 2

Remove the old culture medium to avoid touching the embryoid bodies at the bottom of the culture plate, and add 150 μL of fresh EBM to the culture plate.

Day 4

Add Y-free fresh EBM to the culture plate.

Day 6

Use a 200 μL expanded pipette tip to transfer the embryoid bodies to a 24-well low-adsorption plate and add 500 μL of NIM to it.

Day 8

Add 500ul of fresh NIM to the culture plate.

Day 10: Transfer neuroepithelialoid tissue pieces into Matrigel drops

1. Dissolve Matrigel at 4℃ for 30 minutes.

2. Place the sealing film on the 200 μL pipette tip with an area of (4x4) 16-well size, and press the sealing film with your fingers to form a small depression. UV sterilization for 30 minutes.

3. Use a 200 μL expanded pipette tip to transfer the neuroepithelial-like tissue pieces into each small groove prepared in advance.

4. Use a 200 μL pipette tip to carefully remove excess culture medium from each groove.

5. Immediately drop a volume of approximately 30 μL of growth factor-reduced Matrigel onto the tissue block in each groove to fill the grooves.

6. Use a 10 μL pipette tip to move the neuroepithelial-like tissue block to the center of the droplet.

7. Place the grooved device in a 60mm ² culture dish and incubate it at 37°C for 20-30 minutes. The purpose is to allow the Matrigel droplets to wrap around the tissue block.

8. Add 5 mL of vitamin A-free CDM to a 60 mm ² culture dish.

9. Use sterile forceps to turn the sealing film and shake the Petri dish to make the Matrigel droplets fall off the sealing film. If there are still droplets remaining on the sealing film, you can use tweezers to fix one end of the sealing film and shake the sealing film in the culture medium to make the remaining droplets fall off. Place the fallen Matrigel droplets in a cell culture incubator to continue culturing.

Day 12

Add 5 mL of vitamin A-free CDM to the culture plate.

Day 14

Add 5 mL of CDM containing vitamin A to the culture plate, transfer the culture plate to an incubator with a 70 rpm shaker, and replace the culture medium every 3-4 days.

Day 30

Add 5 mL of CDM containing 14 ng/mL BDNF to the culture plate, and replace the medium every 3-4 days.

Experimental results

Figure 20 represents annotation and quantification of brain organoid cell types, including cycling neural progenitors, granulocyte-macrophage progenitors, oligodendrocyte precursors, radial glia, and Schwann cell precursors.

a.UMAP visualization shows the proportion of brain organoids derived from PSCs in the primary state and 4CL naive state;

b.UMAP visualization shows the annotated neural cell subpopulations in brain organoids obtained from primary and 4CL naive PSCs;

c. Histogram showing the proportion of each cell type in the two brain organoids;

d. Histogram shows the proportion of two brain organoids in each cell type.

Example 11: Generation of embryoid bodies

Materials and methods

Accutase

AggreWell ^TM plate

Low adsorption ^10cm2 culture plate

EB differentiation medium

cell

Human 8CLC and naive PSC/ICLC in Example 1 or de novo PSCs were obtained by culturing human ESCs in mTeSR1.

method

When the growth ratio of primary, original PSCs/ICLCs and 8CLCs accounts for 70-80% of the culture plate area, use accutase to digest the clones into single cells. 6×10 ⁵ cells were resuspended in 2 ml of EB differentiation medium containing Y-27632 and seeded in a 24-well AggreWell ^TM plate. The AggreWell ^TM plate was immediately centrifuged at 100 g for 3 minutes to capture the cells in the microwells. , and observe under a microscope to ensure that the cells are evenly distributed in the microwells. Observe under the microscope after culturing for 24 hours at 37°C, 5% _CO2 and 95% humidity. Slowly aspirate 1-1.5mL of culture medium and add 1.5mL of fresh EB differentiation medium along the wall to avoid loss of EB balls. After continuing to culture for 2-4 days, transfer the floating EB balls to a low-adsorption culture plate. Before harvesting the sample, continue the suspension culture for 20 days (method 1) or continue the suspension culture for 20 days and then adhere to the gelatin-coated culture plate for 15 days. Samples were harvested days later. The samples are used for subsequent cell sorting, sequencing or other purposes.

Acquisition of EB

Use a Pasteur dropper to transfer EB beads to a 15 mL or 50 mL centrifuge tube. Use 1 mL of warm culture medium to pipette the bottom of the plate to fully obtain the EB balls. Transfer the EB ball to the same centrifuge tube by pipetting three times. Keep the centrifuge tube stable to allow the EB balls to fully precipitate. Gently remove the upper culture medium. Add 2ml DPBS to the centrifuge tube, carefully mix the EB balls, keep the centrifuge tube stable for 5 minutes to allow the EB balls to sink to the bottom, and gently remove the upper layer of DPBS. Wash and digest EBs once more for subsequent studies.

Experimental results

Figure 21 represents annotation and quantification of EB cell types, including endodermal epithelial cells, endothelial cells, arterial endothelial cells, midgut epithelial cells, hindgut epithelial cells, neural stem cells, neuroblasts, neuroepithelial cells, retinal precursor cells, Schwann cell precursor cells, smooth muscle cells, and trophoblast cells. Among them, the UMAP visualization shown in Figure 21(a) shows the distribution of cells in different germ layers of EB obtained from PSCs in the primary state and 4CL original state; the UMAP visualization shown in Figure 21(b) shows the distribution of cells in the different germ layers of EB obtained from PSCs in the primary state and 4CL original state. Cell types annotated in EBs obtained from 4CL original PSCs; the histogram shown in Figure 21(c) shows the proportion of different cell types in the specified EBs; the histogram shown in Figure 21(d) shows the different The proportion of EBs in the specified cell type.

Claims (21)

1. A method of producing teratomas, said method comprising the steps of transplanting primary, originating, 8clc or heavy primary PSCs into different organs or locations of an immunodeficient animal of interest and feeding said animal;

wherein the method of making the primary PSCs/ICLCs comprises the step of culturing primate PSCs in a medium comprising an SAH/PRC/EZH2 inhibitor, an HDAC inhibitor and a WNT/beta-catenin signaling inhibitor; the preparation method of the 8CLCs comprises the steps of culturing primate PSCs or the original PSC/ICLC in a culture medium containing optimized doses of SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the heavy original state PSCs are obtained through induced differentiation of original state PSCs/ICLCs or 8 CLCs.

2. A method of producing organoids, said method comprising the step of suspension culturing primary, 8clc or heavy primary PSCs, or culturing said primary, 8clc or heavy primary PSCs on a 3D scaffold in a medium that allows differentiation into a target organ; wherein the method of preparing the primary PSCs/ICLCs comprises the step of culturing primate PSCs in a medium comprising an SAH/PRC/EZH2 inhibitor, an HDAC inhibitor and a WNT/beta-catenin signal inhibitor; the preparation method of the 8CLCs comprises the steps of culturing primate PSCs or original PSCs/ICLCs in a culture medium containing optimized doses of SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the heavy original state PSCs are obtained by inducing differentiation of original state PSCs/ICLCs or 8 CLCs.

3. A method of producing embryoid bodies, comprising the step of suspension culturing said primary, 8clc or heavy primary PSCs in a medium that allows differentiation into a target organ; wherein the method of preparing the primary PSCs/ICLCs comprises the step of culturing primate PSCs in a medium comprising an SAH/PRC/EZH2 inhibitor, an HDAC inhibitor and a WNT/beta-catenin signal inhibitor; the preparation method of the 8CLCs comprises the steps of culturing primate PSC or primary PSC/ICLC in a culture medium containing optimized doses of SAH/PRC/EZH2 inhibitor, HDAC inhibitor and WNT/beta-catenin signal inhibitor; the heavy original PSCs are obtained by inducing differentiation of original PSCs/ICLCs or 8 CLCs.

4. The method of any one of claims 1-3, wherein the medium is further supplemented with one or more of L-ascorbic acid or a derivative thereof, a JAK/STAT3 signaling activator, and a MAPK/ERK signaling inhibitor;

optionally, the medium is further supplemented with one or more of an ACTIVIN/NODAL signal activator, a ROCK inhibitor, and an extracellular matrix.

5. The method according to any one of claims 1 to 4, wherein,

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

The HDAC inhibitor is selected from TSA, VPA and NaB; preferably, the final concentration of TSA in the medium is 3-30nM, more preferably 3-25nM; preferably, the final concentration of VPA in the medium is 0.25-2mM, more preferably 0.5-1.5mM; preferably, the NaB is present in the medium at a medium concentration of 0.25-2mM, more preferably 0.5-1.5mM; and/or, preferably, the final concentration of the WNT/beta-catenin signal inhibitor in the medium is 2 to 8. Mu.M; preferably, the WNT/beta-catenin signal inhibitor is selected from IWR1 and XAV939.

6. The method according to claim 4, wherein:

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

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

The final concentration of the MAPK/ERK signal inhibitor in the culture medium is 0.5-3 mu M; preferably, the MAPK/ERK signaling inhibitor is PD0325901; and/or

The final concentration of the ACTIVIN/NODAL signal activator is 10-25ng/mL; preferably, the activator of the ACTIVIN/NODAL signal is selected from ACTIVIN a and NODAL; and/or

The final concentration of the ROCK inhibitor is 0.5-2 mu M; preferably, the ROCK inhibitor is selected from Y27632, thiazovivin, and hydroxyfasudil; and/or

The extracellular matrix is contained in the culture medium in an amount of 0.1-05% (v/v); preferably, the extracellular matrix is selected from Matrigel ^TM 、Geltrex ^TM And ECM ^TM 。

7. A method according to any one of claims 1-3, wherein the medium used to prepare the pristine PSCs/ICLCs comprises:

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

(B) L-ascorbic acid with a final concentration of 40-70 mug/mL;

(C) LIF with a final concentration of 10-30 ng/mL;

(D) PD0325901 with a final concentration of 0.5-1.5 mu M;

(E) IWR1 or XAV939 at a final concentration of 3-6. Mu.M;

the medium further comprises:

(1) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2. Mu.M; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

(2) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; and a final concentration of 0.5-2 μm Y27632, thiazovivin or hydroxyfasudil; or (b)

(3) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

(4) Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2. Mu.M; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

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

8. The method of claim 7, wherein the step of determining the position of the probe is performed,

the medium included 10nM DZNep or 1mM CPI-1205;5nM TSA, or 0.5mM VPA, or 0.5mM NaB;50 μg/mL L-ascorbic acid; 20ng/mL LIF;1 μM PD0325901; and 5 μM IWR1 or 5 μM XAV939;

And further supplement:

(1) 20ng/mL ACTIVIN A or NODAL, 1. Mu.M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (b)

(2) 20ng/mL ACTIVIN A or NODAL, and 1. Mu.M Y27632, thiazovivin or hydroxyfasudil;

(3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (b)

(4) 1. Mu.M of Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) of extracellular matrix; or (b)

(5) 20ng/mL ACTIVIN A or NODAL, or 1. Mu.M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix.

9. A method according to any one of claims 1-3, wherein the medium used to prepare 8CLCs contains a final concentration of 40-70nM of DZNep or a final concentration of 2-4mM of CPI-1205; TSA at a final concentration of 10-30nM, or VPA at a final concentration of 0.5-1.5mM or NaB at a final concentration of 0.5-1.5 mM; l-ascorbic acid with a final concentration of 40-70 mug/mL; LIF with a final concentration of 10-30 ng/mL; PD0325901 with a final concentration of 0.5-1.5 mu M; and IWR1 or XAV939 at a final concentration of 3-6. Mu.M, respectively; and further supplement:

(1) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2. Mu.M; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

(2) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; and a final concentration of 0.5-2 μm Y27632, thiazovivin or hydroxyfasudil; or (b)

(3) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

(4) Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2. Mu.M; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

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

10. The method of claim 9, wherein the medium comprises 50nM DZNep or 3mM CPI-1205;20nM TSA, or 1mM VPA, or 1mM NaB;50g/mL L-ascorbic acid; LIF at 20 ng/mL; 1 μM PD0325901; and 5 μM IWR1 or 5 μM XAV939;

and further supplement:

(1) 20ng/mL ACTIVIN A or NODAL,1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (b)

(2) 20ng/mL ACTIVIN A or NODAL, and 1 μ M Y27632, thiazovivin or hydroxyfasudil;

(3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (b)

(4) 1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (b)

(5) 20ng/mL ACTIVIN A or NODAL, or 1 μ M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix.

11. The method according to any one of claims 1-10, wherein the basal medium of the medium used to prepare the original PSCs/ICLCs and 8CLCs is selected from Dulbecco's Modified Eagle Medium (DMEM), minimal Essential Medium (MEM), basal Medium Eagle (BME), RPMI1640, F10, F12, alpha minimal essential medium (alpha MEM), glass Minimal Essential Medium (GMEM), iscove's modified Dulbecco's medium, neural basal medium, DMEM/F12 and advanced DMEM/F12 and combinations thereof; preferably, the basal medium is a mixture of higher DMEM/F12 and neural basal medium in a ratio of 1:1 (v/v).

12. The method according to any one of claims 1 to 11, wherein one or more selected from the group consisting of serum substitutes, substituted carbon sources, non-essential amino acids, L-glutamine or substitutes thereof, and antibiotics are further added to the medium.

13. The method of claim 12, wherein,

The serum replacement is selected from the 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 pyruvic acid, such as sodium pyruvate;

the L-glutamine or the substitute thereof is Glutamax containing L-alanyl-L-glutamine dipeptide ^TM A supplement; and/or

The antibiotic is selected from penicillin, streptomycin or a mixture of penicillin and streptomycin.

14. A method according to any one of claims 1 to 3, wherein the method of preparing PSCs/ics in raw form comprises:

(a) Gene editing primate PSCs by knocking down and/or knocking out one or more associated genes in the cell to reduce the activity of SAH, PRC and/or EZH2 of the PSCs; and

(b) Culturing the genetically engineered cell obtained in step (a) in a medium comprising: TSA at a final concentration of 3-30nM or VPA at a final concentration of 0.25-2mM or NaB at a final concentration of 0.25-2mM, preferably TSA at a final concentration of 3-10nM or VPA at a final concentration of 0.25-1mM or NaB at a final concentration of 0.25-1mM and optionally DZNep at a final concentration of 5-15nM or CPI-1205 at a final concentration of 0.5-2mM, or TSA at a final concentration of 3-10nM or VPA at a final concentration of 0.25-0.5mM or NaB at a final concentration of 0.25-0.5mM and optionally DZNep at a final concentration of 5-80nM, preferably 5-50nM or CPI-1205 at a final concentration of 0.5-5 mM; l-ascorbic acid with a final concentration of 40-70 mug/mL; LIF with a final concentration of 10-30 ng/mL; PD0325901 with a final concentration of 0.5-1.5 mu M; IWR1 or XAV939 at a final concentration of 3-6. Mu.M; wherein the culture medium further comprises:

(1) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2. Mu.M; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

(2) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; and a final concentration of 0.5-2 μm Y27632, thiazovivin or hydroxyfasudil; or (b)

(3) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

(4) Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2. Mu.M; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

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

preferably, the medium contains:

5nM TSA, or 0.5mM VPA, or 0.5mM NaB;50 μg/mL L-ascorbic acid; 20ng/mL LIF;1 μM PD0325901;5 μM IWR1 or 5 μM XAV939; and optionally 10nM DZNep or 1mM CPI-1205; wherein the medium further comprises: (1) 20ng/mL ACTIVIN A or NODAL,1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (2) 20ng/mL ACTIVIN A or NODAL, and 1 μ M Y27632, thiazovivin or hydroxyfasudil; (3) 20ng/mL ACTIVIN A or NODAL, and 0.2% (v/v) extracellular matrix; or (4) 1 μ M Y27632, thiazovivin or hydroxyfasudil, and 0.2% (v/v) extracellular matrix; or (5) 20ng/mL ACTIVIN A or NODAL, or 1 μ M Y27632, thiazovivin or hydroxyfasudil, or 0.2% (v/v) extracellular matrix.

15. The method of any one of claims 1-3, wherein the method of preparing 8CLCs comprises:

(a) Gene editing primate PSCs or primary PSCs/ICLCs by knocking down and/or knocking out one or more associated genes in the cell to reduce the SAH, PRC and/or EZH2 activity of the PSCs;

(b) Culturing the genetically engineered cell obtained in step (a) in a medium comprising: TSA at a final concentration of 10-30nM, or VPA at a final concentration of 0.5-1.5mM or NaB at a final concentration of 0.5-1.5 mM; l-ascorbic acid with a final concentration of 40-70 mug/mL; LIF with a final concentration of 10-30 ng/mL; PD0325901 with a final concentration of 0.5-1.5 mu M; IWR1 or XAV939 at a final concentration of 3-6. Mu.M; and optionally, DZNep at a final concentration of 40-70nM or CPI-1205 at a final concentration of 2-4 mM; and wherein the medium further comprises:

(1) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2. Mu.M; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

(2) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; and a final concentration of 0.5-2 μm Y27632, thiazovivin or hydroxyfasudil; or (b)

(3) ACTIVIN a or NODAL at a final concentration of 10-25 ng/mL; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

(4) Y27632, thiazovivin or hydroxyfasudil at a final concentration of 0.5-2. Mu.M; and an extracellular matrix in an amount of 0.1% to 0.5% (v/v); or (b)

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

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

16. The method of any one of claims 1-15, wherein the primate PSC is selected from the group consisting of:

(i) Cells of the ESC and/or ECC series;

(ii) Cells of the iPSC line;

(iii) Cells of the Inner Cell Mass (ICM) of the pre-implantation blastocyst cultured in vitro;

(iv) Cells of the Inner Cell Mass (ICM) of the blastocyst after implantation cultured in vitro;

(v) Cells of embryos from 8 cells (8C) stage to morula stage cultured in vitro.

17. The method of any one of claims 1-16, wherein the primate PSC or primary PSC/ICLC is cultured under one or more conditions selected from the group consisting of: (i) on feeder cells; (ii) on an extracellular matrix without feeder cells; (iii) in suspension without feeder cells; (iv) under hypoxic or normoxic conditions at about 37 ℃; (v) Passaging every 3 to 4 days with single cells at a split ratio of 1:4 to 1:8; (vi) daily medium changes.

18. The method of any one of claims 1-3, further comprising the step of culturing somatic cells in the presence of a SAH/PRC/EZH2 inhibitor, an HDAC inhibitor, and a WNT/β -catenin signaling inhibitor to reprogram the somatic cells to produce primate primary PSC/ICLC.

19. A teratoma produced according to the method of any one of claims 1 and 4-18, and cells isolated from the teratoma.

20. Organoids produced according to the method of any one of claims 2 and 4-18, and cells isolated from organoids.

21. An embryoid body produced by the method of any one of claims 3 and 4-18, and cells isolated from the embryoid body.