Culture medium for improving efficient transformation of embryonic stem cells or expanded pluripotent stem cells and application of culture medium

Abstract

The invention relates to an N2B27 culture medium capable of improving induction efficiency, a pharmaceutical composition is added into the N2B27 culture medium, the pharmaceutical composition is composed of 1-Azakenpaullone, B, FGF4 and 8Br-cAMP, the B is CD1530 or TTNPB, the 1-Azakenpaullone is 2.5 to 7.5 [mu] M, the B is 0.2 to 0.5 [mu] M, the FGF4 is 100 ng / ml, and the 8Br-cAMP is 1 mM. The invention provides application of a culture medium in improving efficient transformation of embryonic stem cells or expanding pluripotent stem cells and application of the culture medium in improving transformation and line establishment of PrE lineage cells. The method mainly realizes efficient conversion of mouse embryonic stem cells or expanded pluripotent stem cells into cells with early PrE pedigree characteristics, and realizes in-vitro long-term culture. According to the method, the convenience degree of transformation and line establishment of PrE lineage cells is remarkably improved, and the method is beneficial to research on second fate decision of embryos and screening of diseases.

Description

Technical Field

[0001] The present invention relates to the field of assisted reproductive technologies, and more particularly, to a culture medium for improving the efficient transformation of embryonic stem cells or expanding pluripotent stem cells and its application. Background Art

[0002] Before a mammalian embryo implants into the uterus, it needs to have three lineages of cells: epiblast (EPI), trophectoderm (TE), and primitive endoderm (PrE) to complete implantation and subsequent intrauterine development. Among them, the epiblast ultimately develops into fetal tissues, while the primitive endoderm and trophectoderm mainly form the yolk sac and placental tissues outside the embryo. 1-3 The yolk sac is responsible for the exchange of substances (such as antibodies, vitamins, lipids, and proteins) between the mother and the fetus in the early stage, is also the site of the initial hematopoietic movement, and maintains erythropoiesis throughout the murine embryonic period. 4,5 Therefore, the primitive endoderm (PrE) and its derived lineages play extremely important roles in embryo implantation and post-implantation development.

[0003] Some in vitro models have been established during the study of PrE lineage development, but their applications have great limitations. Early studies found that ESC would differentiate into PrE lineage cells during the formation of embryoid bodies (EBs). 6 Subsequently, Janet Rossant established a PrE-like cell line - XEN cells that can be cultured in vitro for a long time from embryos. 7 XEN cells can also be transformed from ESC, but the efficiency is low. Later, nEnd, pXEN and other PrE-like lineage cells were established. 8,9 In 2022, Yasuhide Ohinata established a cell line PrESC (Primitive endoderm stem cell) that simultaneously expresses OCT4 and GATA6 from blastocysts. 10 These cells are similar to the naive PrE cells in embryos in terms of expression profile and developmental potential, enabling people to study the maintenance and differentiation of naive PrE. In 2022, Nicolas C. Rivron developed a kind of embryoid body culture condition (CRFA) that can promote the generation of VE endoderm from embryoid bodies. 11

[0004] The differentiation of EBs is heterogeneous, and the resulting cell lineages are complex and disordered, leading to limited application of this system. Since XEN cells do not express the pluripotency gene Oct4 and tend to develop into the parietal endoderm in chimeric experiments, they can only represent a differentiated PrE cell state and cannot achieve the full developmental potential of PrE lineage cells. Although cells such as nEnd and pXEN in the PrE-like lineage have certain improvements at the chimeric and gene expression levels, there are still significant differences from embryonic PrE tissues. PrESCs need to be established from the already formed PrE tissues in the embryo, making it still difficult for us to study the formation mechanism of PrE in vitro, and its application potential is limited. Although the CRFA culture conditions can generate the VE, a derivative lineage of PrE, during the growth of embryoid bodies, a stable early PrE lineage cell line cannot be established in this process, and its induction efficiency also shows limitations.

[0005] In recent decades, although the primitive endoderm (PrE) lineage is very important in embryonic development, its research has lagged behind. On the one hand, because the formation and differentiation of PrE occur during the black box period of the peri-implantation stage, the number of cells is scarce and difficult to isolate, and it is extremely difficult to study its developmental process in vivo. On the other hand, compared with the epiblast (EPI) tissue, which has in vitro cell lines such as embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs), the PrE lineage has always lacked a good in vitro model. Therefore, in view of the obvious lag in PrE lineage research, it is very necessary to develop a perfect in vitro research system to explore the molecular mechanism. Summary of the Invention

[0006] The object of the present invention is to provide a culture medium for improving the transformation of embryonic stem cells or expanding pluripotent stem cells efficiently and its application in view of the deficiencies in the prior art.

[0007] In the first aspect, the present invention provides a pharmaceutical composition for improving induction efficiency, which is composed of 1-Azakenpaullone, B, FGF4 and 8Br-cAMP, wherein B is CD1530 or TTNPB, 1-Azakenpaullone is 2.5 - 7.5 μM, B is 0.2 - 0.5 μM, FGF4 is 100 ng / ml, and 8Br-cAMP is 1 mM.

[0008] As a preferred example, the pharmaceutical composition is composed of 2.5 μM 1-Azakenpaullone, 0.2 μM TTNPB, 100 ng / ml FGF4 and 1 mM 8Br-cAMP.

[0009] As a preferred example, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

[0010] In a second aspect, the present invention provides the use of a pharmaceutical composition in the preparation of a culture medium product for enhancing the efficient transformation of embryonic stem cells or extended pluripotent stem cells.

[0011] In a third aspect, the present invention provides the use of a pharmaceutical composition in the preparation of a culture medium product for enhancing the transformation and cell line establishment of PrE lineage cells.

[0012] In a fourth aspect, the present invention provides an N2B27 culture medium for enhancing induction efficiency, wherein the N2B27 culture medium is added with a pharmaceutical composition, and the pharmaceutical composition is composed of 1-Azakenpaullone, B, FGF4, and 8Br-cAMP. B is CD1530 or TTNPB, 1-Azakenpaullone is 2.5 - 7.5 μM, B is 0.2 - 0.5 μM, FGF4 is 100 ng / ml, and 8Br-cAMP is 1 mM.

[0013] As a preferred example, the pharmaceutical composition is composed of 2.5 μM 1-Azakenpaullone, 0.2 μM TTNPB, 100 ng / ml FGF4, and 1 mM 8Br-cAMP.

[0014] In a fifth aspect, the present invention provides the use of a culture medium in enhancing the efficient transformation of embryonic stem cells or extended pluripotent stem cells.

[0015] In a sixth aspect, the present invention provides the use of a culture medium in enhancing the transformation and cell line establishment of PrE lineage cells.

[0016] The advantages of the present invention are as follows: The present invention mainly realizes the efficient transformation of mouse embryonic stem cells or extended pluripotent stem cells into cells with early PrE lineage characteristics and realizes long-term in vitro culture. This method significantly improves the convenience of the transformation and cell line establishment of PrE lineage cells, which is beneficial to the research on the second embryonic fate determination and disease screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Improvement of the PrELC induction system. (A) Flow chart of the generation, isolation, and amplification of PrE lineage cells induced from EPSC / ESC in vitro. (B) Proportion of OCT4+ / PDGFRα+ cells generated from EPSC and ESC under different induction conditions detected by flow cytometry.

[0018] Figure 2Establishment of the PrELC culture system. (A) Bright-field and immunofluorescence images of PrELC derived from EPSC and ESC. Scale bar = 100 μm. (B) Flow cytometry analysis of the proportion of OCT4+PDGFRα+ cells in PrELC derived from EPSC and ESC after 15 passages of culture. (C) Immunofluorescence images of PrELC. Scale bar = 10 μm. (D) Karyotype banding pattern of the GBL-1 PrELC cell line at passage 15. (E) Statistical chart of the cell cycle proportions of the three cell lines as shown. (F) Cell proliferation curve of different cell lines as shown.

[0019] Figure 3 PrELC is transcriptionally similar to naive PrE cells. (A) Scatter plot of differentially expressed genes between PrELC and ESC analyzed based on a large amount of mRNA-seq data. (B) qPCR detection of the expression of marker genes in ESC and PrELC. ***p < 0.001. (C) Heat map showing the GO analysis of differentially expressed genes between PrELC and ESC. (D) Principal component analysis of PrELC, PrESC10, ESC, and embryonic tissues. (E) Gene set enrichment analysis (GSEA) of PrELC, ES, and XEN on PrE, EPI, and co-expressed specific genes. (F) Heat map showing the expression of pluripotency and PrE-specific expressed genes in different samples.

[0020] Figure 4 PrELC has good in vivo chimeric ability. (A) Schematic diagram of the PrELC chimeric experiment. (B) Immunofluorescence detection of WT and E4.5 blastocysts after PD03 treatment for 48 hours. CDX2 and GATA6 were detected. (C and D) Bright-field and fluorescence images of E7.5 and E12.5 chimeric embryos. (E) Bright-field and fluorescence images of E7.5 chimeric embryos with in vitro fertilized embryos as recipients. (F) Statistical chart of the chimeric efficiency of E7.5 chimeric embryos with in vitro fertilized embryos as recipients. Detailed implementation manners

[0021] The present invention will be further described below in conjunction with the detailed implementation manners. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0022] Example 1

[0023] I. Method

[0024] Animals

[0025] All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals approved by Tongji University. All animals were housed and bred in the specific pathogen-free animal research facility at Tongji University in Shanghai, China. C57BL / 6, ICR, and 129 mice at 6 - 8 weeks of age were purchased from Vital River Laboratories (China) in Beijing. B6.Cg-Tg(CAG-GFP)Smoc mice, in which the fluorescent gene was stably expressed in all organs, were obtained from Shanghai Model Organisms (China). TgOG2 mice carrying the △PE-Oct4-GFP fluorescent reporter gene (referred to as Oct4-GFP in this article) were a gift from Dr. Jeff R. Mann. CDX2-mCherry fluorescent reporter mice were constructed by Professor Jiayu Chen at Tongji University. TgOG2 mice (female, 6 - 8 weeks old) were crossed with CDX2-mCherry fluorescent reporter mice (male) to obtain Oct4-GFP and CDX2-mCherry double reporter system mice. The chimeric cell line was derived from the F1 embryos generated by crossing B6.Cg-Tg(CAG-GFP)Smoc (male, 8 - 10 weeks old) and ICR (female, 6 - 8 weeks old). Chimeric donor embryos were obtained from C57BL / 6 females at 6 - 8 weeks of age. Pseudopregnant recipients were ICR females at 8 - 10 weeks of age.

[0026] Cell line

[0027] Oct4-GFP and CDX2-mCherry double fluorescent reporter mouse ESC / EPSC were derived from E3.5 blastocysts (Oct4-GFP and CDX2-mCherry double fluorescent reporter mice); WT mouse ESC / EPSC were derived from E3.5 blastocysts (129 female mice crossed with male OG2 mice); ESC / EPSC with persistent CAG-GFP fluorescence were derived from E3.5 blastocysts (ICR female mice crossed with male B6.Cg-Tg(CAG-GFP)Smoc).

[0028] Mouse stem cell culture

[0029] Mouse fibroblasts were treated with mitomycin (MMC; Sigma-Aldrich, M0503) and used as feeder layers to culture mouse stem cells. All mouse stem cells were cultured under 5% CO2 at 37°C. The mouse stem cell medium was changed daily or every other day, and subculture was performed using 0.05% trypsin-EDTA when the cell density was high (80% confluence). ESC medium (ESM) is the basal medium used to culture ESCs, mainly composed of DMEM (Sigma-Aldrich, D5671), which contains 1 mM L-glutamine (Millipore, 25030-081), 0.1 mM β-mercaptoethanol (GIBCO, 2285-023), 1× non-essential amino acids (Millipore, e, TSM-001-C), 1× penicillin-streptomycin (Thermo Fisher, 15140122) and 1× nucleosides (Millipore, ES-008-D) and 15% (v / v) fetal bovine serum (FBS) (HyClone, SH30070.03). In this study, ESCs were cultured in ESM supplemented with 2i / LIF (1 μM MEK inhibitor PD0325901 Selleck 1036S; 3 μM GSK-3 inhibitor CHIR99021, Selleck S2924 and 10 ng / ml LIF, Millipore, ESG1107).

[0030] EPSCs were cultured in the EPSC medium described in the published research. N2B27 basal medium: DMEM / F-12 (GIBCO, 11330-032) and Neurobasal (GIBCO, 21103-049) were mixed at a ratio of 1:1, and then 1× non-essential amino acids, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 5% serum replacement (GIBCO 10828-028), 0.5× N2 supplement (Invitrogen, 17502-048), and 0.5× B27 supplement (Invitrogen, 17504-044) were added. The EPSC medium was prepared by adding 3 μM CHIR99021, 2 μM (S)-(+)-dimethylene maleate (Tocris, 1425), 2 μM minocycline hydrochloride (Selleck, s4226), and 10 ng / mL recombinant human LIF (Millipore, LIF1050) to the N2B27 basal medium. BPSCs were cultured in N2B27 medium supplemented with 10 nM LY2090314 (MedChem1Express SHY-16294) and 0.6 μM of AS1842856 (MedChemExpress HY-100596).

[0031] TSCs were cultured in TSM. Preparation of TSM basal medium: RPMI 1640 medium was supplemented with 20% (v / v) FBS, 1× glutamine (Thermo Fisher, 35050-061), 0.1 mM β-mercaptoethanol, 1× sodium pyruvate (sigma-aldrich, S8636), and 1× penicillin-streptomycin. TSM was based on the TSM basal medium and then supplemented with 25 ng / mL FGF4 (Sino Biological 16043-HNAE-100) and 1 μg / mL heparin (sigma-aldrich H3149-500KU).

[0032] The PrE induction medium consisted of N2B27 basal medium supplemented with 2.5 μM of 1-Azakenpaullone (SelleckChemicals, S7193), 100 ng / mL recombinant human FGF4 (PeproTech, 100-31), 0.2 μM TTNPB (Selleck, S4627), and 1 mM 8Br-cAMP (Selleck, S7857).

[0033] Chemically induced BPSCs

[0034] Generation of BPSC from murine ESC / EPS by chemical induction

[0035] Starting from mouse ESCs / EPSCs carrying the dual-fluorescent reporters Oct4-GFP and CDX2-mCherry, they were cultured in N2B27 basal medium containing 10 nM LY2090314 (MedChem Express HY-16294) and 0.6 μM AS1842856 (MedChem Express HY-100596) for 2 - 3 days. BPSCs with dual positivity for Oct4-GFP and CDX2-mCherry were detected by flow cytometry (FACS) and fluorescence microscopy.

[0036] Derivation of BPSC from mouse morulae BPSCs were derived from mouse morulae (16 - to 32 - cell stage) from mice carrying the dual-fluorescent reporters Oct4-GFP and CDX2-mCherry. After culturing in BPSC medium, their karyotypes could be identified after several passages.

[0037] Differentiation of BPSC

[0038] For the spontaneous differentiation of BPSC: BPSCs were cultured in cytokine-free TSM basal medium for 2 days.

[0039] Induced differentiation of BPSC:

[0040] To induce the differentiation of BPSC, we cultured BPSCs in TSM medium containing 25 μg / mL FGF4 and 1 μg / mL heparin for 3 days. Then, we screened and collected cells positive for CDX2-mCherry and CDCP1 (R&D Systems AF4515-SP) by flow cytometry (FACS). These markers helped us identify and isolate cells with trophectoderm characteristics. BPSCs induced to differentiate for 2 days with TSM were used to construct chimeric embryos to evaluate their potential for in vivo development.

[0041] Chimera construction and microinjection of differentiated cells (BPSC differentiated using TSM) and ESCs

[0042] Chimeras were constructed by microinjecting differentiated cells (BPSC differentiated in TSM) and ESCs into normal 8-cell donor embryos. The constructed chimeras were cultured in G-1PLUS (Vitrolife 10128) medium and transferred into the uteri of pseudopregnant recipient mice at the E3.5 blastocyst stage, which were usually at 2.5 days after pseudopregnancy.

[0043] Tetraploid complementation assay

[0044] Collect 2-cell stage recipient embryos from the oviducts of mated ICR female mice and culture them under mineral oil using the microdrop method in G-1 PLUS medium at 37 °C and 5% CO2. Generate tetraploid embryos at the late 2-cell stage by electrofusion technology. Treat BPSC clones with 0.05% trypsin-EDTA in a 37 °C cell culture incubator for approximately 4 minutes to obtain a single-cell suspension. Transfer 10 - 15 single cells into the depressions of an aggregation plate. Digest the zona pellucida of tetraploid embryos with 20 mg / mL PE (Pronase E; Sigma, #P8811) at the "4-cell stage", and then place every two embryos as a group into one depression. Culture the aggregated embryos in G-1 PLUS medium until the blastocyst stage, and then transfer them into the uteri of pseudopregnant recipient mice, which are usually at 2.5 days post coitum (2.5 dpc).

[0045] Construct embryo-like structures using the IVC and EUCM culture systems

[0046] Construct embryo-like cells by sequential induction.

[0047] We constructed embryo-like structures using the IVC and EUCM culture systems according to the method reported previously. Induce BPSC in TSM for one day, and then further induce PrE for one day. Subsequently, these cells are digested into single cells and seeded in Aggrewell at a density of 31,200 cells per well. On the first day, use FM plus 10 μM Y-27632. On the second day, rinse twice with fresh FM and then continue to culture with fresh FM. On the third day, replace 1.5 mL of IVC2 per well, and on the fourth day, replace 2 mL of IVC2 per well. On the fifth day, transfer the embryoid bodies to an ultra-low attachment 6-well plate and continue to culture with IVC2 for one day, and then transfer the well-shaped embryoid bodies to EUCM for two days. In the next two days, culture under roller culture conditions (rotation speed of 30 revolutions per minute), replace fresh EUCM every day, and add an additional 3 mg / L glucose on the last day.

[0048] Construct embryo-like structures by inducing TS and PrE separately

[0049] First, the starting cells, EPSCs or ESCs, are divided into two parts. One part is first converted into BPSCs and then transferred to the TSM for culturing for two days. The other part is cultured in the PrE induction medium for one day (EPSC) or two days (ESC). Then, these cells are digested into single cells and seeded in Aggrewell at a density of 43,200 cells per well (35,200 TS-induced cells plus 8,000 PrE-induced cells). On the first day, a mixed medium (1:1) is used, including FM and N2B27 basal medium plus 10 μM Y27. On the second day, continue to culture with fresh mixed medium without Y27. On the third day, replace 1.5 mL of IVC2 per well. On the fourth day, all aggregates are transferred to an ultra-low attachment 6-well plate under shaking conditions (rotation speed of 70 rpm) and continue to be cultured with IVC2 (3 mL per well) for one day. On the fifth day, well-shaped embryoid bodies are transferred to EUCM for culturing for one day under static culture conditions. In the next three days, culture is carried out under roller bottle culture conditions (rotation speed of 30 rpm, Eastmo Biotechnology Co., Ltd., WEC001-GT4), with fresh EUCM replaced daily, and an additional 3 mg / L glucose is added on the last day.

[0050] FM consists of DMEM containing 15% (v / v) fetal bovine serum (FBS), 1 mM L-glutamine, 1× non-essential amino acids, 1× penicillin-streptomycin, and 1× sodium pyruvate.

[0051] IVC2 consists of CMRL 1066 (Thermo Fisher Scientific 11530037), containing 20% (v / v) FBS, 1 mM sodium pyruvate, 1× penicillin-streptomycin, 2 mM L-glutamine, 1× N2 supplement, and 0.25× B27 supplement.

[0052] EUCM consists of 25% (v / v) DMEM (Sigma-Aldrich, D5671) or DMEM / F12 (GIBCO, 11330–032), 25% (v / v) adult serum (Sigma-Aldrich, H4522-100ML), 50% rat serum (Charles River), 1 mM sodium pyruvate, 1× non-essential amino acids, 1× penicillin-streptomycin, and 2 mM L-glutamine.

[0053] Generation of BPSC-like blastocysts

[0054] Place 6000 - 12000 cells in one well of a 24 - well Aggrewell - 400 (STEMCELL Technologies, 34415) plate and culture them using blastocyst - like medium. The blastocyst - like medium consists of the following components: 25% TSM basal medium, 25% (v / v) N2B27 basal medium, and 50% (v / v) KSOM (AibeiBiotechnology, M1430) or G - 1PLUS, and supplemented with 2 μM ROCK inhibitor Y - 27632 (Selleck, S049), 12.5 ng / mL recombinant human FGF4, 0.5 μg / mL heparin (Sigma–Aldrich, H3149), 3 μM CHIR99021, 5 ng / mL recombinant human BMP4 (PeproTech, 12–05ET), and 0.5 μM A83–01 (Axon Medchem, 1421). On the next day, replace the old medium with fresh medium without Y - 27632.

[0055] Immunofluorescence

[0056] Before performing the immunofluorescence experiment, all cell clones and embryos need to be fixed with 4% paraformaldehyde (Servicebio, China) at 4 °C for 24 hours. After fixation, morulae, blastocysts, E6.5 embryos, cell clones, and embryo bodies similar to E5.5 need to be incubated with a permeabilization reagent at room temperature for 30 minutes. The permeabilization reagent is prepared by adding 0.3% Triton X - 100 (Sigma–Aldrich, 93443) to DPBS (Gibco). Then, the cell clones are blocked in DPBS containing 3% bovine serum albumin (BSA, MP Biomedicals) for 1 hour. Different from cell clones, embryos and E5.5 - like structures need to be permeabilized and blocked in DPBS with 0.5% Triton X - 100 and 3% BSA for 2 hours. After dilution in the blocking reagent, the primary antibody is incubated with the samples at 4 °C overnight. After incubation, the samples are washed three times with DPBS containing 0.01% Triton X - 100. The secondary antibody is diluted in 3% BSA - DPBS and incubated with the samples at room temperature for 2 hours. The cell nuclei are stained with DAPI (Invitrogen D3571) for 15 - 20 minutes. After staining, the samples are washed with DPBS. The samples that have completed immunofluorescence staining are photographed and processed using a ZEISS LSM 880 confocal microscope.

[0057] Whole immunostaining of D8 embryoid bodies. For whole immunostaining of D8 embryoid bodies, we first cut them out from the amnion and placed them in 4% paraformaldehyde, fixed overnight at 4°C. Before incubating with the primary antibody, we used an animal tissue optical clearing kit (Beyotime, P0112L) to clear the embryoid bodies. Then, immunofluorescence staining was performed according to the above method, and the incubation times of the secondary antibody and DAPI were doubled. The samples after immunofluorescence treatment were photographed and processed by an Olympus FVMPE-RS microscope.

[0058] The primary antibodies used and their dilution ratios are as follows: mouse anti-CDX2 (1:400; Bio-Genex, MU392A-UC); anti-OCT4 (1:200; Santa Cruz, sc-5279); anti-GATA6 (1:200; R&D Systems, AF1700); anti-SOX2 (1:200; R&D Systems, AF2018-SP); anti-Brachyury (1:200; R&D Systems, AF2085-SP); anti-TFAP2C (1:200; Santa Cruz, sc-12762); anti-SOX17 (1:200; R&D Systems, AF1924-SP); anti-SOX1 (1:200; Cell Signaling Technology, 4194S); anti-MHCII (1:200; R&D Systems, MAB4470-SP)

[0059] anti-HOXB4 (1:100; Abcam, ab133521); anti-TPBPA (1:500; Abcam, ab104401); The secondary antibodies were purchased from the Alexa series of Thermo Fisher and diluted at a ratio of 1:200. Series, and diluted at a ratio of 1:200.

[0060] Fluorescent positive cell sorting

[0061] Cells were digested with 0.05% trypsin-EDTA and washed with DPBS containing 2% FBS. Subsequently, the cells were incubated with anti-CDCP1 (1:50; R&D Systems, AF4515-SP) or anti-PDGFRα (1:200; Abcam, ab203491) antibodies, and after staining, they were washed again with DPBS containing 2% FBS and resuspended. Cells with Oct4-GFP and CDX2-mCherry dual fluorescence reporter genes were directly analyzed by FACS without staining. All cells were analyzed and collected by a MoFlo XDP cell sorter (Beckman Coulter).

[0062] Compound library screening

[0063] High-throughput screening was performed using extended pluripotent stem cells (EPS) with dual labels of Oct4-GFP and CDX2-mCherry. These EPSCs were seeded into 384-well plates 24 hours after the feeder cells were inoculated. After a 12-hour cell attachment time, a total of 1859 compounds (final concentration 1 μM) were added to the cells. Each compound was tested in three replicate wells. After 72 hours of culture, the cells were fixed with 4% paraformaldehyde (PFA) for 20 minutes. Subsequently, the cell nuclei were stained with DAPI. High-content imaging was performed using an Operetta CLS system (PerkinElmer, USA), and image analysis was performed using Harmony 4.0 software (PerkinElmer, USA).

[0064] Bulk mRNA-seq library preparation

[0065] In a 35-mm culture dish, when the cells reached 80% confluence, they were digested with 0.05% trypsin-EDTA. After washing with DPBS, the cells were collected into a 1.5-ml centrifuge tube containing 1 ml of TRIzol (Takara Bio, 9109). After vortexing for 5 minutes, the cells could be transferred to -80 °C for storage until RNA extraction. Total RNA was extracted using the phenol-chloroform extraction method. The KAPA Stranded mRNA-Seq Kit (KAPA, KK8421) was used to construct bulk mRNA sequencing libraries. The constructed mRNA sequencing libraries were sequenced on an Illumina Novaseq 6000 with paired-end 150-bp reads. The Nanjing Jiangbei New Area Biomedical Public Service Platform performed the sequencing and quality control of the libraries.

[0066] Preparation of single-cell RNA sequencing (scRNA-seq)

[0067] Digest the cells with 0.05% trypsin-EDTA and wash them three times with DPBS containing 0.04% BSA. Digest the embryoid bodies with a 1:1 mixture of Accumax (Sigma-Aldrich A7089-100ML) and collagenase (Thermo Fisher Scientific 17104019) for 10-20 minutes at room temperature. Samples for scRNA-seq should meet the requirements of cell viability over 80% and aggregation rate below 20%. Construct single-cell RNA libraries using the 10×Genomics Chromium Next GEM Single Cell 3’ kit v3.1 (16rxns PN-1000268) and MGIDNBelab C RNA library V2.0. Sequence the libraries on the Illumina Novaseq Xplus and MGIDNBSEQ-T7.

[0068] Single-cell multi-omics ATAC + gene expression data preparation.

[0069] Digest the cells with 0.05% trypsin-EDTA and wash them three times with DPBS containing 0.04% BSA. Samples should meet the requirements of cell viability over 80% and aggregation rate below 20%. Construct single-cell RNA and ATAC libraries using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle (16rxns PN-1000283). Sequence the libraries on the Illumina Novaseq Xplus.

[0070] Bulk RNA sequencing analysis

[0071] To analyze the bulk RNA sequencing data, we used the Trim Galore v0.6.10 software to trim the adapter sequences. Subsequently, align the data to the mouse reference genome mm10 using STAR 2.7.10b. Generate the gene expression matrix using FeatureCounts v2.0.3. Calculate the differentially expressed genes and perform principal component analysis (PCA) using the DESeq2 v3.4.1 package. The criteria for genes to be considered significantly differentially expressed are: |log2(fold change)| > 0.5 and adjusted P-value < 0.05.

[0072] scRNA-seq analysis

[0073] To obtain the gene expression matrix, scRNA-seq FASTQ files were analyzed using one of the following tools: CellRanger v7.1.0: CellRanger official website; DNBC4tools v2.1.1: DNBC4tools documentation; For dimensionality reduction analysis, scRNA-seq data was normalized and integrated using the Seurat package v4.3.0 according to the standard procedure. The specific steps are as follows: Seurat package v4.3.0: Used for normalizing and integrating scRNA-seq data. The "RunHarmony" function of the Harmony package v0.1.1: Used to remove batch effects. UMAP (Uniform Manifold Approximation and Projection): Used to achieve dimensionality reduction. Pseudotime trajectory analysis was performed through the following steps: Import the data in Seurat into the Monocle package v2.26.0.

[0074] According to the standard procedure of Monocle, the "reduceDimension" and "orderCells" functions were used for dimensionality reduction and trajectory analysis. GO analysis was performed using the Metascape tool according to the provided instructions: Metascape: Metascape official website. Single-cell ATAC sequencing (scATAC-seq) and single-cell multi-omics ATAC+ gene data analysis To analyze scATAC-seq and single-cell multi-omics ATAC+ gene FASTQ files, we used the following tools respectively: CellRanger ATAC v2.1.0: Used to analyze scATAC-seq data. CellRanger ARC v2.0.2: Used to analyze single-cell multi-omics ATAC+ gene data. For quality control, data integration, and dimensionality reduction, we used the following software packages according to the standard procedure: Seurat v4.3.0: A widely used R package specifically for the analysis of single-cell RNA sequencing data, and can also be applied to the quality control and integration of ATAC-seq data. Signac v1.11.0: An R package specifically designed for single-cell chromatin accessibility data, which can be combined with Seurat for data processing and analysis. The specific steps are as follows: Quality control: Use Seurat and Signac to perform preliminary quality control on the data, including removing low-quality cells and low-coverage regions. Data integration: Integrate the data through Seurat and Signac to ensure that data from different batches can be effectively merged. Dimensionality reduction: Use the standard procedure of Seurat and Signac for dimensionality reduction to simplify the data and facilitate subsequent analysis. All of this data was downloaded and analyzed according to the same procedures as our data.

[0075] II. Results

[0076] Chemically induced BPSCs

[0077] To screen the culture conditions that promote the differentiation of pluripotent stem cells into trophoblast cells, we first established an expanded pluripotent stem cell (EPSC) line with Oct4-GFP and CDX2-mCherry fluorescent reporter proteins. After culturing in N2B27 basal medium supplemented with a chemical small molecule library for 3 days, we detected the fluorescence intensities of Oct4-GFP and CDX2-mCherry. Among the 1859 small molecules tested, 261 enhanced the fluorescence intensity of Oct4-GFP compared with the control group, but only 10 increased the fluorescence intensity of CDX2-mCherry. To our surprise, two small molecules, LY2090314 (LY) and AS1842856 (AS), promoted the expression of OCT4 and CDX2 simultaneously in a concentration-dependent manner. After further experiments, we found that 0.6 μM AS (66.03%) and 10 nM LY (53.67%) could induce a high proportion of double-positive cells of Oct4-GFP and CDX2-mCherry without cytotoxicity. When AS and LY (AL) were co-treated for 3 days, the proportion of CDX2 and OCT4 double-positive cells increased to 70.65%. ESCs also showed a significant response to AL treatment, with more than 60% of CDX2 and OCT4 double-positive cells. Notably, the AL-treated EPSCs produced dome-shaped clones similar to those of ESCs and EPSCs. Immunofluorescence results showed that OCT4 and CDX2 proteins were co-localized in the nucleus without obvious mutual exclusion. Nuclear staining and flow cytometry results showed that the proportion of cells co-expressing OCT4 and CDX2 proteins exceeded 70%.

[0078] Before the first cell fate determination, mouse embryos also showed co-expression of key transcription factors of different lineages. Single-cell sequencing data and protein detection showed that the key transcription factors Cdx2 and Oct4 were expressed at the 8-cell stage and highly expressed at the 16-cell stage. At the E3.5 blastocyst stage, these transcription factors were still co-expressed in the TE, and they were completely separated only at the late blastocyst stage (E4.5). Previous studies have shown that the outer cells of the morula can promote the formation of embryonic and extra-embryonic lineages, indicating that cells co-expressing CDX2 and OCT4 have bidirectional developmental potential. Then, we designated these AL-treated cells as bidirectional pluripotent stem cells (BPSCs).

[0079] BPSCs can be cultured long-term

[0080] Next, we investigated whether BPSCs could be maintained in vitro for a long time. Flow cytometry (FACS) analysis showed that after 10 passages, BPSCs retained up to 41.58% of CDX2 and OCT4 double-positive cells and maintained a dome-shaped colony morphology. Transcriptome analysis showed that long-term cultured BPSCs (BPSC-P3 or P4) were similar to short-term treated cells (BPSC-Day3, P0). To further explore the in vitro culture of BPSCs, we established BPSCs from embryos at the morula stage (16- to 32-cell). Two of the three cell lines (EBPSC-2 and EBPSC-5) showed normal karyotypes after 10 passages. The cells showed a high proportion (>50%) of double-positive cells for OCT4 and CDX2. To verify the developmental potential of BPSCs, we performed a tetraploid complementation assay. The results showed that EBPSCs could generate tetraploid complementation mice and develop normally to adulthood. These results indicate that this culture system can maintain BPSCs with the potential for whole embryo development for a long time.

[0081] In addition to LY, other GSK3 inhibitors can also promote the expression of Cdx2. To verify this, we tested CHIR99021, which is commonly used in ES cell medium. The results showed that when the concentration of CHIR99021 was increased to 10 μM (the concentration in normal ES cell medium is 3 μM), it promoted the expression of Cdx2, and its effect was comparable to that of 10 nM LY.

[0082] A unique transcriptional state

[0083] To investigate the composition of BPSCs, we collected scRNA-seq data and performed uniform manifold approximation and projection (UMAP) analysis. Similar to the FACS and immunofluorescence staining results, most BPSCs expressed Oct4 (89%), and approximately 48% of the cells expressed both Oct4 and Cdx2. Gene expression analysis showed that the expression of the trophectoderm-related gene Cdx2 in BPSCs was uniform. Although Oct4 and Cdx2 are typically mutually exclusive in pluripotent stem cells, their correlation in BPSCs and pre-implantation embryos has not been explored. We found that there was a negative correlation between Oct4 and Cdx2 from E3.5 to E5.5, but no obvious negative correlation at the 16-cell stage. BPSCs showed a weak negative correlation similar to that of 16-cell embryos. In BPSCs, Oct4 was strongly positively correlated with Sox2 and strongly negatively correlated with Fgf8, while Cdx2 was weakly correlated with Krt8 and Krt18. In addition, the correlation between Sox2:Cdx2 and Nanog:Cdx2 in BPSCs was more similar to that of embryos at the morula stage (16- to 32-cell) than to embryos after E3.5.

[0084] In addition, scATAC data combined with transcriptome analysis showed that the Cdx2 promoter region in the BPSCs genome was open regardless of whether Oct4 or Cdx2 was expressed. This indicates that although BPSCs are heterogeneous at the transcriptome and protein expression levels, their developmental potential may be consistent and they can be converted into each other. To prove this, we performed flow sorting and cultured single-positive (Oct4+ / Cdx2-) and double-positive (Oct4+ / Cdx2+) cells separately in AL medium (PO). The results showed that after one passage (P1), the cells reverted to a mixed state of single and double positives, and after the second passage (P2), the cell ratio was the same as that of the starting cells. In addition, when BPSCs derived from EPSCs were recultured in EPSCs medium (LCDM), most cells (96%) reverted to the single-positive state. To compare BPSCs with pre-implantation embryos, we analyzed single-cell RNA-seq data with published pre-implantation embryo data. The UMAP results showed that BPSCs were similar to morulae (16-cell) at the first cell fate determination stage. In contrast, ESCs and EPSCs were closer to ICM (E3.5) and Epi (E4.5). Further clustering analysis showed that BPSCs clustered together with 8-cell and 16-cell embryos.

[0085] BPSCs autonomously generate TE-like cells

[0086] Previous studies have shown that mouse pluripotent or totipotent stem cells cannot be rapidly converted into TSCs without ectopic expression of the TE core transcription factor CDX2. To investigate the developmental potential of BPSCs in vitro, we cultured BPSCs under serum medium conditions without induction factors. After 2 days, single cells positive for CDX2, OCT4, or GATA6 were detected in the autonomously differentiated clones. Contrary to the low conversion efficiency observed in EPSCs and ESCs, BPSCs significantly increased the yield of CDX2-positive cells to 40.36%. To explore the developmental state, we performed UMAP analysis of scRNA-seq data from BPSCs and EPSCs derived from 2 autonomously differentiated cells. Under the condition of comparative analysis with E4.5 blastocyst data, the data showed that TE- (32%), PrE- (8%), and Epi-like cells (42%) in the autonomously differentiated cells of BPSCs were mapped to the TE, PrE, and Epi of the E4.5 blastocyst respectively, and had robust lineage marker gene expression. In contrast, EPSCs only involved Epi- (97%) and PrE-like cells (3%), excluding TE-like cells.

[0087] Since BPSCs consist of single-positive (Oct4+ / Cdx2-) and double-positive (Oct4+ / Cdx2+) cells, it is unclear whether these two cell types differ in their differentiation directions. We screened out two types of cells from BPSCs and discarded PrE lineage cells using the PDGFRA antibody. An autonomous differentiation experiment was conducted on the two groups of cells, and the results showed that double-positive cells were the main source of TE lineage cells, single-positive cells rarely differentiated into TE lineage cells, and PrE lineage cells mainly differentiated from OCT4 single-positive cells. In addition, we also performed single-cell differentiation on the flow-sorted cells, and the results showed that double-positive cells produced few CDX2-positive clones without the TSC inducer (Fgf4 and heparin), while a large number were produced in the presence of added Fgf4. In addition, most clones contained only one cell type, indicating that BPSC immediately determined the differentiation direction at the single-cell level. The significant difference between the results of bulk cell differentiation and single-cell differentiation may be due to the communication between a large number of cells, and this signal can determine the fate of single-cell autonomous differentiation. This is consistent with the developmental trajectory of normal embryos, indicating that the differentiation of BPSCs follows the developmental paradigm of normal embryos.

[0088] Developmental potential of TE-like cells

[0089] To determine whether the TSC line could be generated, we treated BPSCs with a directed induction medium (TSM supplemented with Fgf4 and heparin) for 3 days and then detected them with CDX2-mCherry or CDCP1 (surface markers of TSCs). EPSC-derived BPSCs (EPSC-BPSC-TSMD3) and ESC-derived BPSCs (ESC-BPSC-TSMD3) both responded to TSM induction and produced up to 60.55% CDCP1-positive cells. CDX2-mCherry-positive or CDCP1-positive cells were collected and cultured with TSM for 3-4 days, and we successfully established the TSC line. Compared with embryo-derived TSCs, the BPSC-converted TSC lines BPSC-TSC (EPSC) and BPSC-TSC (ESC) both showed tight epithelial-like clones and expressed the TSC marker genes SOX2, TFAP2C, and CDX2.

[0090] To explore the differentiation potential of BPSCs in vivo, the differentiated cells (labeled with GFP) treated with TSM for 2 days were injected into 8-cell embryos and subjected to uterine transplantation. The results showed that 30-45% of E6.5 embryos exhibited obvious BPSCs chimerism (GFP positive) in the Epi, extraembryonic ectoderm (ExE), and ectoplacental cone (EPC). In contrast, only the Epi of the ESCs group showed GFP fluorescence signal. In addition, at the E12.5 stage chimeras, we detected that BPSCs contributed to both fetal and extraembryonic tissues (placenta and yolk sac). These findings indicate that BPSCs have extraordinary plasticity in development and the potential for embryonic and extraembryonic lineage development.

[0091] Construction of gastruloid-like structures

[0092] After demonstrating in vitro and in vivo that BPSC can effectively differentiate into embryonic and extraembryonic tissues, we attempted to determine whether BPSC can self-assemble into blastocyst-like structures. The results showed that single BPSC can form blastocyst-like structures and can also induce decidualization after uterine transplantation. Then, we tried to construct post-implantation embryo-like structures. After inducing BPSCs with TSM and PrE induction medium for 2 days, Epi-, TE-, and PrE-like cells appeared, and further embryo-like structure construction was carried out using an in vitro culture (IVC) system. After 4 days, solid spherical structures similar to E5.0 embryos appeared. These structures consisted of GATA6-positive cells (visceral endoderm-like cells) surrounded by OCT4-positive cells (epiblast-like cells) and CDX2-positive cells (extraembryonic ectoderm-like cells). One day later, the ectoderm-like structure rearranged to form a lumen-like structure similar to E5.5 embryos. Immunostaining showed that E5.5-like embryoid bodies correctly expressed lineage markers. We performed UMAP analysis on the scRNA-seq data of E5.5 embryoid bodies and published embryo data (E3.5-E6.5), and E5.5 embryoid bodies expressed the correct lineage marker genes.

[0093] To further explore the developmental potential, we cultured E5.5 embryoid bodies using an in vitro culture (EUCM) system. After 2 days, the embryoid bodies elongated and showed a cup-shaped structure. Comparable to natural E6.5-E7.5 embryos, the embryoid bodies showed an enlarged amniotic cavity, and the formation of the primitive streak (T marker) cells expanded at the posterior part of the Epi-like structure and the expression of Brachyury (T) represented gastrulation in the embryo. Single-cell transcriptome data showed that the E7.5-like structure was similar to the normal E7.5 embryo cell line.

[0094] Simulating neurulation

[0095] To evaluate the developmental potential of BPSC-derived embryoid bodies, we cultured the E7.5-like structures under roller culture conditions for two days and successfully obtained E8.5-like embryoid bodies. However, the efficiency was relatively low (<1%). Therefore, we changed the method: for the starting cells (EPSCs or ESCs), a part was transformed into BPSCs and then differentiated into TE-like and Epi-like cells. Another part was directly induced into PrE-like cells. Subsequently, we mixed these cells and seeded them in a microwell plate and cultured them under IVC conditions. On the 3rd day, all the aggregates in the well plate were transferred to an ultra-low attachment cell culture dish and cultured with shaking. By the 4th day, more than about 75% of the E5.5-like structures could be selected for further culture in EUCM. This efficiency was much higher than previously reported. Starting from the 5th day, morphologically normal embryos were subjected to roller culture until E8.5-like structures appeared on the 8th day, accompanied by regular early cardiac beats. Statistical analysis showed that after 8 days of culture, the number of E8.5-like structures exceeded 16% of all the aggregates on the 4th day.

[0096] The D8 embryoid bodies developed amnion-like and yolk sac-like structures. In addition, on the yolk sac membrane, areas similar to blood islands could be observed, accompanied by red pigmentation. After dissection, we observed that the D8 embryoid bodies had structures such as head folds, beating hearts, allantois, and foregut. The morphology was between E8.5 and E8.75 of normal embryos cultured in EUCM. To determine the integrity of the cell lineages in the embryoid bodies, we performed scRNA-seq. The results showed that the D8 embryoid bodies were highly similar to E8.5-E9.0 embryos and had a relatively complete cell line. We also performed statistical analysis on the cell proportions of the main lineages (excluding the yolk sac and allantois), and the results showed that only a few lineages had inconsistent proportions with normal embryos.

[0097] Canonical tissue development pattern

[0098] To evaluate the structural integrity of the D8-like embryos, we performed immunofluorescence detection using various lineage markers. Initially, we studied the development of the nervous system. The results showed that the D8-like embryos were similar to normal E8.75 embryos. In the D8-like embryos, neural folds formed in the head region, and the neural tube formed in the back and gradually extended towards the tail. The expression levels of the neural lineage markers SOX1 and SOX2 were highly expressed in the head and lowly expressed in the tail. From the images of the three-dimensional reconstructed tissue structures, it could be clearly observed that the neural tube near the tail had closed. The notochord was clearly labeled by protein T located at the base of the neural tube. The enrichment of protein T in the tail region also indicated the emergence of the tail bud. Through scRNA-seq analysis, the brain could be further divided into the forebrain, midbrain, hindbrain, and floor plate.

[0099] The D8 embryoid bodies have shown regular heartbeats, and the staining of the cardiac marker myosin heavy chain II makes the early cardiac structure located in the anterior part of the embryo clearly visible. According to the expression of Handl, Tbxl, Tbx5, Nkx2-5, Tlxl, and Isll in the scRNA-seq data, the heart can be divided into cardiac mesoderm, the first heart field, and the second heart field. Somites give rise to skeletal muscle, blood vessels, and skin. At the neural tube stage, they are arranged in a plexiform structure on both sides of the neural tube. Immunofluorescence staining of HOBX4 shows that the D8 embryoid bodies have obvious paired somite structures. Surprisingly, using a cell line with a ΔPE-Oct4-GFP reporter system, we successfully detected the presence of primordial germ cells (PGCs) in the D8 embryos. The same result was observed in the scRNA-seq data. At this stage, the PGCs are confined near the allantois and are migrating anteriorly. The scRNA-seq data indicate that the PGCs of the D8 embryoid bodies are similar to those of natural embryos and highly express Dnd1, Dppa3, and Ifitm3. The above findings suggest that our embryoid body model effectively reproduces the key events of natural embryo development, highlighting its important potential for simulating the early embryo development process.

[0100] Example 2

[0101] EPSCs can efficiently (30%-40%) generate primitive state PrE-like cells (GATA6 / PDGFRα+OCT4+) under the induction of CRFA medium (N2B27 basal medium supplemented with 3 μM CHIR99021, 10 nM RA, 100 ng / ml FGF4, 1 mM 8Br-cAMP), while ESCs can only generate approximately 15% under the same conditions ( Figure 1B). Therefore, we need to improve the existing induction conditions and enhance the induction efficiency. Thus, we found several small molecules (1-Azakenpaullone, CHIR99021, CD1531, TTNPB) and added them to the N2B27 basal medium, and conducted combined tests using the OCT4-GFP reporter cell line. Test1: 2.5 μM 1-Azakenpaullone, 0.5 μM CD1530, 100 ng / ml FGF4, 1 mM 8Br-cAMP; Test2: 2.5 μM 1-Azakenpaullone, 0.2 μM TTNPB, 100 ng / ml FGF4, 1 mM 8Br-cAMP; Test3: 7.5 μM 1-Azakenpaullone, 0.5 μM CD1530, 100 ng / ml FGF4, 1 mM 8Br-cAMP; Test4: 7.5 μM 1-Azakenpaullone, 0.2 μM TTNPB, 100 ng / ml FGF4, 1 mM 8Br-cAMP. The results showed that the second combination had the highest conversion efficiency, reaching more than twice that of the traditional induction condition CRFA in the ESC group. It should be noted that 1-Azakenpaullone (1-azakenpaullone), TTNPB (aromatic retinoic acid), FGF4 (fibroblast growth factor 4), 8Br-cAMP (8-bromo-adenosine-3',5'-cyclic monophosphate sodium)

[0102] We established a proliferation culture condition that allows for the long-term culture of PrELC and maintains the characteristics of its primitive endoderm-like (PrE) cells ( Figure 2 A-C). After treatment with the PrE induction medium, ESCs can also be used to culture a stable PrELC line through the proliferation medium, and the traits are the same as those of the cells transformed from EPSC Figure 2 A and B). The formula of the proliferation medium is N2B27 basal medium supplemented with 10 μM CHIR99021, 50 ng FGF4, and 2 μM (S)-(+)-dimethylene maleate (Tocris, 1425). After long-term culture observation, PrELC can maintain a high genomic replication activity without affecting its stability Figure 2 D and E), the proliferation rate of PrELC is similar to that of ESCs and much higher than that of XEN, showing good cell activity Figure 2 F).

[0103] Subsequently, we detected the transcriptome of PrELC. The results showed that compared with ESCs, PrELC highly expressed the marker genes of the PrE lineage, downregulated the expression of pluripotency genes Nanog, Sox2, and Klf2, while maintaining the expression of another part of pluripotency genes Oct4 and Klf4 ( Figure 3 A and B). GO analysis showed that the upregulated genes in PrELC were mainly enriched in aspects such as protein secretion, ion transport, extracellular matrix organization, and cell migration, which was highly consistent with the functions of PrE lineage cells ( Figure 3 C). Further, we performed PCA analysis on different cells and embryonic tissues. The results showed that PrELC was significantly different from ESCs, E4.5 EPI, E5.5 EPI, and E5.5 EXE in terms of overall expression levels, but was closer to PrESCs and tended to develop in the VE direction ( Figure 3 D). GSEA data showed that both PrELC and XEN were enriched in genes specifically expressed in PrE, but differently, XEN did not express the genes co-expressed by PrELC and ES, including some pluripotency genes ( Figure 3 E and F). The comparison with XEN cells demonstrated that PrELC possessed the molecular characteristics of early PrE lineage.

[0104] In addition to transcriptional verification, we also conducted chimeric experiments to verify the developmental potential of PrELC in vivo. To demonstrate the substitution of PrELC for in vivo PrE cells, we first treated E2.5 embryos with the MEK kinase inhibitor PD0325901 (hereinafter referred to as PD03) for 48 hours to induce PrE lineage defects, and then injected PrELC into the blastocoel ( Figure 4 A), followed by uterine transplantation. Through immunofluorescence staining, we could observe that E4.5 embryos treated with PD03 had fewer PrE cells and hardly produced normal embryos after transplantation ( Figure 4 B). In contrast, embryos injected with PrELC could successfully develop to E7.5 and E12.5, and their morphology was similar to that of normal embryos ( Figure 4 C and D). Fluorescence detection results showed that there was extensive chimerism in both the VE and PE of E7.5, and obvious fluorescence signals were also detected in the yolk sac of E12.5 ( Figure 4 C and D). To further verify the chimerism between PrELC and normal embryos, we used in vitro fertilized embryos as recipients and conducted cell injection experiments at E4.5. The results showed that the VE of E7.5 embryos after implantation also had a high chimerism, and the chimerism efficiency exceeded 50% ( Figure 4 E and F). The above results indicate that PrELC has the complete developmental ability of extraembryonic endoderm and can be used for the differentiation research of PrE lineage cells.

[0105] References

[0106] 1 Bassalert, C., Valverde-Estrella, L. & Chazaud, C. Primitive Endoderm Differentiation: From Specification to Epithelialization. Curr Top Dev Biol 128, 81-104 (2018). https: / / doi.org / 10.1016 / bs.ctdb.2017.12.001

[0107] 2 Filimonow, K. & de la Fuente, R. Specification and role of extraembryonic endoderm lineages in the periimplantation mouse embryo. Theriogenology 180, 189-206 (2022). https: / / doi.org / 10.1016 / j.theriogenology.2021.12.021

[0108] 3 Mole, M. A., Weberling, A. & Zernicka-Goetz, M. Comparative analysis of human and mouse development: From zygote to pre-gastrulation. Gastrulation: From Embryonic Pattern to Form 136, 113-+(2020). https: / / doi.org / 10.1016 / bs.ctdb.2019.10.002

[0109] 4 Canu, G. & Ruhrberg, C. First blood: the endothelial origins of hematopoietic progenitors. Angiogenesis 24, 199-211 (2021). https: / / doi.org / 10.1007 / s10456-021-09783-9

[0110] 5 Soares-da-Silva, F. et al. Yolk sac, but not hematopoietic stem cell-derived progenitors, sustain erythropoiesis throughout murine embryonic life. J Exp Med 218(2021). https: / / doi.org / 10.1084 / jem.20201729

[0111] 6 Coucouvanis, E. & Martin, G. R. BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo. Development 126, 535 - 546(1999).

[0112] 7 Kunath, T. et al. Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts. Development 132, 1649 - 1661(2005). https: / / doi.org / 10.1242 / dev.01715

[0113] 8 Zhong, Y. et al. Isolation of primitive mouse extraembryonic endoderm (pXEN) stem cell lines. Stem Cell Res 30, 100 - 112(2018). https: / / doi.org / 10.1016 / j.scr.2018.05.008

[0114] 9 Anderson, K. G. V. et al. Insulin fine-tunes self-renewal pathways governing naive pluripotency and extra-embryonic endoderm. Nat Cell Biol 19, 1164 - 1177(2017). https: / / doi.org / 10.1038 / ncb3617

[0115] 10 Ohinata, Y. et al. Establishment of mouse stem cells that can recapitulate the developmental potential of primitive endoderm. Science 375, 574 - 578 (2022). https: / / doi.org / 10.1126 / science.aay3325

[0116] 11 Vrij, E. J. et al. A pendulum of induction between the epiblast and extra - embryonic endoderm supports post - implantation progression. Development 149(2022). https: / / doi.org / ARTN dev192310

[0117] 10.1242 / dev.192310

[0118] The above - mentioned are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A pharmaceutical composition for improving induction efficiency, characterized in that, The described pharmaceutical composition consists of 1-Azakenpaullone, B, FGF4, and 8Br-cAMP, where B is CD1530 or TTNPB, 1-Azakenpaullone is 2.5 - 7.5 μM, B is 0.2 - 0.5 μM, FGF4 is 100 ng / ml, and 8Br-cAMP is 1 mM.

2. The pharmaceutical composition according to claim 1, wherein The described pharmaceutical composition consists of 2.5 μM 1-Azakenpaullone, 0.2 μM TTNPB, 100 ng / ml FGF4, and 1 mM 8Br-cAMP.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The described pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

4. Use of the pharmaceutical composition according to any one of claims 1 - 3 in the preparation of a culture medium product for enhancing the efficient transformation of embryonic stem cells or extended pluripotent stem cells.

5. Use of the pharmaceutical composition according to any one of claims 1 - 3 in the preparation of a culture medium product for enhancing the transformation and establishment of PrE lineage cells.

6. An N2B27 medium for improving induction efficiency, characterized in that, The N2B27 culture medium is added with the pharmaceutical composition, which consists of 1-Azakenpaullone, B, FGF4, and 8Br-cAMP, where B is CD1530 or TTNPB, 1-Azakenpaullone is 2.5 - 7.5 μM, B is 0.2 - 0.5 μM, FGF4 is 100 ng / ml, and 8Br-cAMP is 1 mM.

7. The culture medium according to claim 6, characterized in that, The described pharmaceutical composition consists of 2.5 μM 1-Azakenpaullone, 0.2 μM TTNPB, 100 ng / ml FGF4, and 1 mM 8Br-cAMP.

8. Use of the culture medium according to claim 6 or 7 in enhancing the efficient transformation of embryonic stem cells or extended pluripotent stem cells.

9. Use of the culture medium according to claim 6 or 7 in enhancing the transformation and establishment of PrE lineage cells.

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