Reagent combination or kit for constructing embryoid and use thereof

Abstract

Provided are a reagent combination or a kit for constructing an embryoid and the use thereof. The reagent combination or the kit can be used for inducing a stem cell to produce a blastocyst-like lineage precursor cell, a blastocyst-like cell and / or an embryoid, which are based on small-molecule induction and independent of transgenes, and have a single cell source. The produced blastocyst-like lineage precursor cell and blastocyst-like cell have a full blastocyst lineage, and are seed cells having balanced developmental competence and totipotency, which solves the problem of unbalanced cell maturation in the prior art. The produced embryoid is highly similar to natural embryos in terms of morphological characteristics and transcriptomic characteristics, has the capability of highly reproducing post-implantation embryonic development and / or gastrulation, which is beneficial for research on embryonic development in vitro. The method for inducing the stem cell to produce the blastocyst-like lineage precursor cell, the blastocyst-like cell and / or the embryoid by using the reagent combination or the kit is simple, and has high applicability and high embryoid construction efficiency.

Description

A reagent combination or kit for constructing a embryoid-like and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a reagent combination or kit for constructing an embryoid-like and application thereof. BACKGROUND

[0002] Constructing an embryo model in vitro with simulated natural embryo development has great biological significance and regenerative medicine research value. Due to the difficulty in obtaining natural embryo materials, ethical issues, and other problems, decoding the early life code urgently needs a reliable in vitro embryo model. At present, there are still many challenges in the research of in vitro embryo models, such as the problems of carrying transgenes by starting cells, low embryoid-like synthesis efficiency, and strong cell heterogeneity, which make it difficult to truly reproduce earlier embryonic development and lineage specification events in vitro.

[0003] In natural development of mammals, embryogenesis begins with the combination of sperm and egg to form a zygote and acquire totipotency. Mouse zygote development undergoes 2-cell, 4-cell, 8-cell cleavage to form a morula. The first cell lineage specification occurs after the morula tightens and acquires polarity, which marks the formation of the Trophectoderm (TE) and the Inner Cell Mass (ICM). Subsequently, the embryo develops to the 16-32 cell stage, and the TE of the outer layer of cells along the Na + -K + Ion pumps establish ion gradients transported to the intercellular space to form a blastocyst cavity, from which the second cell lineage specification occurs: the ICM further develops into the Epiblast (EPI) and the Primitive endoderm (PrE); by then, the three lineages of EPI, PrE, and TE are formed [1]. Among them, the EPI develops into the inner, middle, and outer three layers of the embryo to form an individual; while the PrE and TE develop into extraembryonic tissues to form the yolk sac and placenta, respectively, and the three are indispensable for the normal development of the body.

[0004] In recent years, the research of mouse embryonic development system based on mouse embryonic stem cells (ESC) has been greatly promoted (Figure 1). ESCs can self-assemble into organized structures such as blastoids, gastruloids and embryoids after induction in vitro [2-4]. Although this embryonic development system opens up a new way for early embryonic development and in vitro reproduction of the development process, it also faces many limitations. For example, the blastoids formed by combining mouse ESCs with trophoblast stem cells (TSC) only have EPI and TE, lack PrE lineage, and do not have the potential to develop into post-implantation embryos [5]. The gastruloids formed by applying exogenous stimulation to mouse ESCs can simulate asymmetric occurrence and body axis formation [2], but cannot completely simulate the signals and morphogenesis during body axis formation. The main reason is that the gastruloids lack the primitive endoderm (PrE) lineage, and the signals from the extraembryonic tissue are crucial for the development of epiblast (EPI) and the establishment of anterior-posterior axis [2]. In short, both blastoids and gastruloids have great defects and cannot form embryos with complete lineages.

[0005] Recently, in vitro embryonic-like system has broken through the limitations of previous ways and opened a new chapter of simulating post-implantation embryo development. Zernicka-Goetz team constructed pre-implantation and post-implantation embryo-like structures by co-culturing ESC with EPI differentiation potential, TSC and PE-derived extraembryonic endoderm stem cells (XEN), namely ETS synthetic embryos / embryoids (ETS-Embryoids)[6]. When the ETS embryoids developed to the primitive streak stage, the symmetry of the embryo was destroyed because there were not enough PE cells to develop into distal visceral endoderm (DVE) and anterior visceral endoderm (AVE). Jacob Hanna and Zernicka-Goetz team used iXEN cells (Dox-induced overexpression of PE key regulator Gata4 in ESC) instead of XEN and iTSC cells (Dox-induced overexpression of TSC key gene Cdx2) instead of TSC to form ETiX / EiTiX embryos, which made a great breakthrough[3-4]. ETiX / EiTiX system formed embryoids with better ability to simulate post-implantation development, which experienced peri-implantation and primitive streak stages, and had extraembryonic tissues[7]. These reports showed that the process of primitive streak formation and organogenesis in mammals could be reproduced in culture dishes, making it possible to reproduce the process of embryonic development. Jose Silva team found that by activating the STAT3 cell pathway through transgenic regulation of the STAT3 signaling pathway, embryonic stem cells (ESC) could have higher developmental potential, and a new type of cell: morula-like cells (MLC) could be obtained[8]. MLC has the ability to develop into all lineages in the embryo and extraembryonic tissues, and can effectively form blastoids and embryoids with certain developmental ability. However, the existing in vitro embryoid system has the following limitations: (1) it needs to mix cells of different lineages, including ESC, TSC and XEN or their substitutes, with very low efficiency; (2) TSC and XEN substitutes rely on transgenic activation of extraembryonic lineages, and the efficiency is still very low; (3) the most advanced mouse embryoids cannot reproduce the complete primitive streak movement, especially the specialized structures of endomesoderm, and lack the primitive streak morphology of natural embryos; (4) although it has multiple cell lineages, the proportion and maturity of cells with different fates are not balanced; (5) the heterogeneity of recombined cells results in an embryo formation rate of less than 0.5% (Fig. 1).

[0006] Therefore, constructing a cell with developmental totipotency based on small molecule induction, non-transgenic and single cell source, and constructing an in vitro embryonic-like system with high efficiency and closer to the natural embryonic development pathway will help to break through the limitations of previous methods, promote the development of this field, and reduce the difficulty of subsequent biomedical research and application. SUMMARY

[0007] The first aspect of the present application aims to provide a reagent combination or kit.

[0008] The second aspect of the present application aims to provide the use of the reagent combination or kit of the first aspect.

[0009] The third aspect of the present application aims to provide a method for inducing stem cells to generate blastocyst-like lineage precursor cells.

[0010] The fourth aspect of the present application aims to provide a method for inducing stem cells to generate blastocyst-like cells.

[0011] The fifth aspect of the present application aims to provide a method for constructing an embryonic-like embryo.

[0012] The sixth aspect of the present application aims to provide a blastocyst-like lineage precursor cell.

[0013] The seventh aspect of the present application aims to provide a blastocyst-like cell.

[0014] The eighth aspect of the present application aims to provide an embryonic-like embryo.

[0015] The ninth aspect of the present application aims to provide the use of the blastocyst-like lineage precursor cell of the sixth aspect, the blastocyst-like cell of the seventh aspect, and the embryonic-like embryo of the eighth aspect.

[0016] The tenth aspect of the present application aims to provide a product.

[0017] To achieve the above-mentioned objects, the technical solutions adopted by the present application are as follows:

[0018] The first aspect of the present application provides a reagent combination or kit, comprising a first culture medium.

[0019] The first culture medium is a basic culture medium containing a GSK-3 inhibitor, a STAT3 activator, a retinoic acid nuclear receptor (RAR) agonist, and a TGF-β receptor kinase inhibitor.

[0020] In some embodiments of the present application, the first culture medium consists of a GSK-3 inhibitor, a STAT3 activator, a retinoic acid nuclear receptor (RAR) agonist, a TGF-β receptor kinase inhibitor, and a basic culture medium.

[0021] In some embodiments of the application, the first medium is used to induce the stem cells to produce blastocyst-like lineage precursor cells.

[0022] In some embodiments of the application, the reagent combination or kit further comprises a second medium;

[0023] The second medium is a basal medium comprising a GSK-3 inhibitor, a retinoic acid nuclear receptor (RAR) agonist, and a TGF-β receptor kinase inhibitor.

[0024] In some embodiments of the application, the second medium consists of a GSK-3 inhibitor, a retinoic acid nuclear receptor (RAR) agonist, a TGF-β receptor kinase inhibitor, and a basal medium.

[0025] In some embodiments of the application, the second medium is used to induce the stem cells to produce blastocyst-like lineage precursor cells.

[0026] In some embodiments of the application, the GSK-3 inhibitor in the first medium and the GSK-3 inhibitor in the second medium are each independently selected from at least one of a GSK-3a inhibitor, a GSK-3b inhibitor; in some embodiments of the application, the GSK-3 inhibitor in the first medium and the GSK-3 inhibitor in the second medium are each independently selected from at least one of TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, CHIR-99021, L803-mts, BIO (6-bromo-indirubin-3’-oxime), AR-A014418, TDZD-8, 2-D08, IM-12, 1-Azakenpaullone (Indirubin); in some embodiments of the application, the GSK-3 inhibitor in the first medium and the GSK-3 inhibitor in the second medium are CHIR-99021.

[0027] In some embodiments of the application, the STAT3 activator in the first medium comprises at least one of Leukemia Inhibitory Factor (LIF) and Interleukin 6 (IL6); further Leukemia Inhibitory Factor (LIF); and further Mouse Leukemia Inhibitory Factor.

[0028] In some embodiments of the application, the retinoic acid nuclear receptor (RAR) agonist in the first medium, the second medium is each independently selected from at least one of AM580, all-trans retinoic acid, 9-cis retinoic acid, AC 261066, AC 55649, Adapalene, AM 80, BMS 753, BMS 961, CD 1530, CD 2314, CD 437, Ch55, Isotretinoin, Tazarotene, TTNTB, and EC19; in some embodiments of the application, the retinoic acid nuclear receptor (RAR) agonist in the first medium, the second medium is AM580.

[0029] In some embodiments of the application, the TGF-beta receptor kinase inhibitor in the first medium, the second medium is each independently selected from at least one of SB431542, A83-01, Galunisertib, SB525334, LY2109761, E616452, LY3200882, SB505124, PF06952229, SD208, ML347, R268712, Fresolimumab, ITD-1, AZ12601011, BIO-013077-01; in some embodiments of the application, the TGF-beta receptor kinase inhibitor in the first medium, the second medium is E616452.

[0030] In some embodiments of the application, the concentration of the GSK-3 inhibitor in the first medium is 1-15 mM; further 2.1-9.9 mM; more further 2.7-6.6 mM; still further 2.7-3.3 mM or 5.4-6.6 mM.

[0031] In some embodiments of the application, the concentration of the STAT3 activator in the first medium is 10-100 ng / mL; further 15-25 ng / mL; more further 18-22 ng / mL.

[0032] In some embodiments of the application, the concentration of the retinoic acid nuclear receptor (RAR) agonist in the first medium is 0.01-0.1 mM; further 0.02-0.08 mM; more further 0.04-0.06 mM.

[0033] In some embodiments of the application, the concentration of the TGF-beta receptor kinase inhibitor in the first medium is 1-20 mM; further 1-15 mM; more further 5-15 mM; still further 5-12 mM; more further 9-11 mM.

[0034] In some embodiments of the present application, the concentration of the GSK-3 inhibitor in the second medium is 1-15 μM; further 2.1-9.9 μM; more further 5.4-9.9 μM; still further 8.1-9.9 μM or 5.4-6.6 μM.

[0035] In some embodiments of the present application, the concentration of the retinoic acid nuclear receptor (RAR) agonist in the second medium is 0.01-0.1 μM; further 0.02-0.08 μM; more further 0.04-0.06 μM.

[0036] In some embodiments of the present application, the concentration of the TGF-β receptor kinase inhibitor in the second medium is 1-20 μM; further 1-15 μM; more further 5-15 μM; still further 5-12 μM; more further 9-11 μM.

[0037] In some embodiments of the present application, the reagent combination or kit comprises a first medium, or a first medium and a second medium, for inducing stem cells to generate blastocyst-like lineage precursor cells.

[0038] In some embodiments of the present application, the reagent combination or kit further comprises Instruction 1, which describes a method for inducing stem cells to generate blastocyst-like lineage precursor cells, when the reagent combination or kit is used for inducing stem cells to generate blastocyst-like lineage precursor cells.

[0039] In some embodiments of the present application, the method for inducing stem cells to generate blastocyst-like lineage precursor cells is the method of the third aspect of the present application.

[0040] In some embodiments of the present application, the reagent combination or kit further comprises a third medium;

[0041] The third medium is a basal medium containing fibroblast growth factor, a BMP4 signaling pathway activator, a TGF-β activator, a WNT signaling pathway inhibitor, a Lats kinase inhibitor, an anticoagulant, ascorbic acid or its derivatives, and an insulin-transferrin-selenium additive.

[0042] In some embodiments of the present application, the third medium is used for inducing blastocyst-like lineage precursor cells to generate blastocyst-like cells.

[0043] In some embodiments of the present application, the fibroblast growth factor (FGF) is at least one of FGF1-FGF23; more further FGF4 (preferably rhFGF4).

[0044] In some embodiments of the application, the activator of BMP4 signaling pathway in the third medium comprises at least one of BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoliquiritigenin, dihydroxydihydroguaiaretic acid, apigenin, and biochanin A; further is BMP4; further is hBMP4.

[0045] In some embodiments of the application, the activator of TGF-β in the third medium comprises at least one of TGF-β, Activin A; further is Activin A.

[0046] In some embodiments of the application, the inhibitor of WNT signaling pathway in the third medium comprises at least one of IWP4, IWP2, IWR-1, IWP1, IWP3, IWR-2, IWR-3, IWR-4, IWR-5, XAV939, DKK1, quercetin, ICG-001, pamoate, CCT031374, iCRT-3, iCRT-5, iCRT-14, CPG049090, NC043; further is XAV939.

[0047] In some embodiments of the application, the inhibitor of Lats kinase in the third medium comprises at least one of TRULI, GA-017, TDI-011536; further is TRULI.

[0048] In some embodiments of the application, the anticoagulant in the third medium comprises at least one of heparin, EDTA salt; further is heparin.

[0049] In some embodiments of the application, the ascorbic acid or its derivative in the third medium comprises at least one of ascorbic acid, calcium ascorbate, magnesium ascorbate, zinc ascorbate, potassium ascorbate, sodium ascorbate, dehydroascorbic acid, L-threonic acid, L-xylosic acid, L-lyxaric acid, L-ascorbyl monostearate, L-ascorbyl dipalmitate, L-ascorbyl 6-hexadecanoate, L-ascorbyl 2-phosphate, L-ascorbyl 3-phosphate, L-ascorbyl 2-sulfate; further is L-ascorbic acid-2-phosphate.

[0050] In some embodiments of the application, the concentration of the fibroblast growth factor in the third medium is 10-50 ng / mL; further is 15-30 ng / mL; further is 22-28 ng / mL.

[0051] In some embodiments of the present application, the concentration of the BMP4 signaling pathway activator in the third culture medium is 5-20 ng / mL; further 7-15 ng / mL; and more further 9-11 ng / mL.

[0052] In some embodiments of the present application, the concentration of the TGF-β activator in the third culture medium is 10-40 ng / mL; further 15-30 ng / mL; and more further 18-22 ng / mL.

[0053] In some embodiments of the present application, the concentration of the WNT signaling pathway inhibitor in the third culture medium is 1.0-9.0 μM; further 2-5 μM; and more further 2.7-3.3 μM.

[0054] In some embodiments of the present application, the concentration of the Lats kinase inhibitor in the third culture medium is 1.0-5.0 μM; further 1.5-3 μM; and more further 1.8-2.2 μM.

[0055] In some embodiments of the present application, the concentration of the anticoagulant in the third culture medium is 0.5-2.0 μg / mL; further 0.8-1.5 μg / mL; and more further 0.9-1.1 μg / mL.

[0056] In some embodiments of the present application, the concentration of the ascorbic acid or its derivative in the third culture medium is 50-500 μM; further 100-250 μM; and more further 180-220 μM.

[0057] In some embodiments of the present application, the concentration of the insulin-transferrin-selenium additive in the third culture medium is 0.5×-2×; further 0.6×-1.5×; and more further 0.9×-1.1×.

[0058] In some embodiments of the present application, the reagent combination or kit comprises: the first culture medium and the third culture medium; or

[0059] the first culture medium, the second culture medium and the third culture medium;

[0060] The reagent combination or kit is used for inducing stem cells to produce blastocyst-like cells.

[0061] In some embodiments of the present application, when the reagent combination or kit is used for inducing stem cells to produce blastocyst-like cells, the reagent combination or kit further comprises Instruction 2, which describes a method for inducing stem cells to produce blastocyst-like cells.

[0062] In some embodiments of the present application, the method of inducing stem cells to produce blastocyst-like cells is the method of the fourth aspect of the present application.

[0063] In some embodiments of the present application, the reagent combination or kit further comprises a fourth medium;

[0064] The fourth medium is a basal medium of the third medium.

[0065] In some embodiments of the present application, the fourth medium is used for inducing blastocyst-like cells to produce embryonic-like structures.

[0066] In some embodiments of the present application, the reagent combination or kit comprises: a first medium, a third medium, and a fourth medium; or

[0067] a first medium, a second medium, a third medium, and a fourth medium;

[0068] The reagent combination or kit is used for inducing stem cells to produce embryonic-like structures.

[0069] In some embodiments of the present application, when the reagent combination or kit is used for inducing stem cells to produce embryonic-like structures, the reagent combination or kit further comprises a description 3, which describes a method of inducing stem cells to produce embryonic-like structures.

[0070] In some embodiments of the present application, the method of inducing stem cells to produce embryonic-like structures is the method of the fifth aspect of the present application (the method of inducing blastocyst-like cells to produce embryonic-like structures described in U2 does not comprise the steps of culturing and / or maturing the embryonic-like structures).

[0071] In some embodiments of the present application, the reagent combination or kit further comprises a fifth medium and a sixth medium;

[0072] The fifth medium is a basal medium comprising serum (preferably fetal bovine serum; more preferably FBS), glutamine (preferably L-glutamine; more preferably GlutaMax), insulin-transferrin-selenium-aminethanol, thyroid hormone receptor agonist, estradiol, progesterone, acetylcysteine, glucose, and antibiotics;

[0073] The sixth medium is a basal medium comprising serum, glutamine (preferably L-glutamine; more preferably GlutaMax), pyruvate or a salt thereof, buffer salt, glucose, and antibiotics.

[0074] In some embodiments of the present application, the fifth medium and the sixth medium are used for culturing and / or maturing the embryonic-like structures.

[0075] In some embodiments of the application, the thyroid hormone receptor agonist in the fifth medium comprises at least one of thyroxine (T4) or a salt thereof, triiodothyronine or a salt thereof; further triiodothyronine or a salt thereof; more further 3,3',5-triiodo-L-thyronine sodium salt.

[0076] In some embodiments of the application, the antibiotic in the fifth medium comprises at least one of amphotericin, nystatin, gentamicin, tetracycline, erythromycin, penicillin, streptomycin; further penicillin and streptomycin.

[0077] In some embodiments of the application, the concentration of the serum in the fifth medium is 10-40% by volume percentage; further 15-35%; more further 28-32%.

[0078] In some embodiments of the application, the concentration of the glutamine in the fifth medium is 0.5x-2x; further 0.7x-1.5x; more further 0.9x-1.1x.

[0079] In some embodiments of the application, the concentration of the insulin-transferrin-selenium-aminoethanol in the fifth medium is 0.5x-2x; further 0.7x-1.5x; more further 0.9x-1.1x.

[0080] In some embodiments of the application, the concentration of the thyroid hormone receptor agonist in the fifth medium is 50-200 nM; further 60-150 nM; more further 90-110 nM.

[0081] In some embodiments of the application, the concentration of the estradiol in the fifth medium is 5-11 nM; further 6-10 nM; more further 7.2-8.8 nM.

[0082] In some embodiments of the application, the concentration of the progesterone in the fifth medium is 100-300 ng / mL; further 150-270 ng / mL; more further 180-220 ng / mL.

[0083] In some embodiments of the application, the concentration of the acetylcysteine in the fifth medium is 5-45 μM; further 10-35 μM; more further 22.5-27.5 μM.

[0084] In some embodiments of the application, the concentration of the glucose in the fifth medium is 0.5-2 mg / mL; further 0.7-1.5 mg / mL; more further 0.9-1.1 mg / mL.

[0085] In some embodiments of the application, the concentration of the antibiotic in the fifth medium is 0.1%-2% by volume percentage; further 0.5%-1.7%; further 0.9%-1.1%.

[0086] In some embodiments of the application, the serum in the sixth medium comprises rat serum and human AB serum.

[0087] In some embodiments of the application, the volume ratio of the rat serum and the human AB serum is 60:(20-40); further 60:(24-36); further 60:(35-36).

[0088] In some embodiments of the application, the pyruvic acid or salt thereof in the sixth medium is sodium pyruvate.

[0089] In some embodiments of the application, the buffer salt in the sixth medium comprises at least one of phosphate, Tris, HEPES; further HEPES.

[0090] In some embodiments of the application, the antibiotic in the sixth medium comprises at least one of amphotericin, nystatin, gentamicin, tetracycline, erythromycin, penicillin, streptomycin; further penicillin and streptomycin.

[0091] In some embodiments of the application, the concentration of the serum in the sixth medium is 35%-95% by volume percentage; further 45%-89%; further 73%-84%.

[0092] In some embodiments of the application, the concentration of the rat serum in the sixth medium is 25%-60% by volume percentage; further 30%-55%; further 45%-52%.

[0093] In some embodiments of the application, the concentration of the human AB serum in the sixth medium is 10%-35% by volume percentage; further 15%-34%; further 28%-32%.

[0094] In some embodiments of the application, the concentration of the glutamine in the sixth medium is 0.5x-2x; further 0.7x-1.5x; further 0.9x-1.1x.

[0095] In some embodiments of the application, the concentration of the pyruvic acid or salt thereof in the sixth medium is 0.5x-2x; further 0.7x-1.5x; further 0.9x-1.1x.

[0096] In some embodiments of the present application, the concentration of the buffering salt in the sixth medium is 6-20 mM; further 8-16 mM; and further 10-12 mM.

[0097] In some embodiments of the present application, the concentration of the glucose in the sixth medium is 1-6 mg / mL; further 2-5 mg / mL; and further 3.6-4.4 mg / mL.

[0098] In some embodiments of the present application, the concentration of the antibiotic in the sixth medium is 0.1%-2% by volume percentage; further 0.5%-1.7%; and further 0.9%-1.1%.

[0099] In some embodiments of the present application, the reagent combination or kit comprises: the first medium, the third medium, the fourth medium, the fifth medium and the sixth medium; or

[0100] the first medium, the second medium, the third medium, the fourth medium, the fifth medium and the sixth medium;

[0101] The reagent combination or kit is used for inducing stem cells to produce embryoid-like and culturing and / or maturation of embryoid-like.

[0102] In some embodiments of the present application, when the reagent combination or kit is used for inducing stem cells to produce embryoid-like and culturing and / or maturation of embryoid-like, the reagent combination or kit further comprises instruction 4, which describes a method of inducing stem cells to produce embryoid-like and culturing and / or maturation of embryoid-like.

[0103] In some embodiments of the present application, the method of inducing stem cells to produce embryoid-like and culturing and / or maturation of embryoid-like is the method of the fifth aspect of the present application (the method of inducing blastocyst-like cells to produce embryoid-like in U2 further comprises a step of culturing and / or maturation of embryoid-like).

[0104] In some embodiments of the present application, the reagent combination or kit comprises any one or more of the first medium, the second medium, the third medium, the fourth medium, the fifth medium and the sixth medium (for example, the reagent combination or kit can comprise the third medium; or the reagent combination or kit can comprise the fourth medium and the fifth medium; or the reagent combination or kit can comprise the fourth medium, the fifth medium and the sixth medium); and the use of the reagent combination or kit corresponds to the medium.

[0105] In some embodiments of the application, the basal medium of the first medium, the second medium, the third medium, the fourth medium, the fifth medium, and the sixth medium is independently selected from at least one of IMDM (Iscove's Modified Dulbecco's Medium) medium, Neurobasal medium, Eagle's Basal Medium (BME) medium, MEM medium, DMEM medium, Ham's F-12 medium, RPMI1640 medium, Advanced RPMI 1640 medium, Advanced DF-12 (Advanced DMEM / F-12) medium, and DMEM / F12 medium; in some embodiments of the application, the basal medium of the first medium, the second medium, the third medium, the fourth medium, the fifth medium, and the sixth medium is independently selected from one of Neurobasal medium, DMEM medium, Advanced DF-12 (Advanced DMEM / F-12) medium, and DMEM / F12 medium.

[0106] In some embodiments of the application, the basal medium of the first medium and / or the second medium is DMEM / F12 medium and Neurobasal medium.

[0107] In some embodiments of the application, the volume ratio of DMEM / F12 medium and Neurobasal medium in the basal medium of the first medium and / or the second medium is independently selected from 1:(0.8-1.2); further selected from 1:(0.9-1.1).

[0108] In some embodiments of the application, the basal medium of the first medium and / or the second medium is a basal medium containing glutamine (preferably L-glutamine; more preferably GlutaMax), non-essential amino acids, B27, N2, and 2-mercaptoethanol.

[0109] In some embodiments of the application, the concentration of glutamine in the basal medium of the first medium and / or the second medium is 0.5x-1.5x; further 0.9x-1.1x.

[0110] In some embodiments of the application, the concentration of non-essential amino acids in the basal medium of the first medium and / or the second medium is 0.5x-1.5x; further 0.9x-1.1x.

[0111] In some embodiments of the application, the concentration of B27 in the basal medium of the first medium, and / or second medium is 0.5x-1.5x; further 0.9x-1.1x.

[0112] In some embodiments of the application, the concentration of N2 in the basal medium of the first medium, and / or second medium is 0.5x-1.5x; further 0.9x-1.1x.

[0113] In some embodiments of the application, the concentration of 2-mercaptoethanol in the basal medium of the first medium, and / or second medium is 0.05-1 mM; further 0.08-0.12 mM; still further 0.09-0.11 mM.

[0114] In some embodiments of the application, the basal medium of the third medium is DMEM medium.

[0115] In some embodiments of the application, the basal medium of the third medium is a basal medium comprising serum (preferably fetal bovine serum; more preferably FBS), pyruvate or a salt thereof (preferably sodium pyruvate), glutamine (preferably L-glutamine; more preferably GlutaMax), non-essential amino acids, 2-mercaptoethanol, and antibiotics (preferably comprising at least one of amphotericin, nystatin, gentamicin, tetracycline, erythromycin, penicillin, streptomycin; more preferably penicillin and streptomycin).

[0116] In some embodiments of the application, the concentration of serum in the basal medium of the third medium is 10%-30% by volume percentage; further 15%-25%; still further 18%-22%.

[0117] In some embodiments of the application, the concentration of pyruvate or a salt thereof in the basal medium of the third medium is 0.5x-2x; further 0.7x-1.5x; still further 0.9x-1.1x.

[0118] In some embodiments of the application, the concentration of glutamine in the basal medium of the third medium is 0.5x-2x; further 0.7x-1.5x; still further 0.9x-1.1x.

[0119] In some embodiments of the application, the concentration of non-essential amino acids in the basal medium of the third medium is 0.5x-2x; further 0.7x-1.5x; still further 0.9x-1.1x.

[0120] In some embodiments of the present application, the concentration of the 2-mercaptoethanol in the basal medium of the third medium is 0.05-0.15 mM; further 0.08-0.12 mM; still further 0.09-0.11 mM.

[0121] In some embodiments of the present application, the concentration of the antibiotic in the basal medium of the third medium is 0.1%-2% by volume percentage; further 0.5%-1.7%; still further 0.9%-1.1%.

[0122] In some embodiments of the present application, the basal medium of the fifth medium is Advanced DMEM / F12 medium.

[0123] In some embodiments of the present application, the basal medium of the sixth medium is DMEM medium (preferably low glucose, pyruvate-containing, glutamine-free, and phenol red-free DMEM medium).

[0124] In some embodiments of the present application, the stem cell is a stem cell with pluripotency.

[0125] In some embodiments of the present application, the stem cell with pluripotency comprises at least one of an embryonic stem cell, a parthenogenetic stem cell, an induced pluripotent stem cell, a mesenchymal stem cell, an adipose stem cell, and a cord blood stem cell; in some embodiments of the present application, the stem cell with pluripotency comprises an embryonic stem cell.

[0126] In some embodiments of the present application, the stem cell is derived from a mammal (e.g., a primate (e.g., a human, a chimpanzee, an ape), a rodent (e.g., a rat, a mouse, a guinea pig), a pet (e.g., a cat, a dog), a livestock (e.g., a horse, a cow, a sheep, a pig, a rabbit)).

[0127] In some embodiments of the present application, the stem cell is derived from a rodent; further derived from a murine; still further derived from a mouse.

[0128] In some embodiments of the present application, the stem cell is derived from a primate; further derived from a human.

[0129] In some embodiments of the present application, the stem cell is a human embryonic stem cell (hESC) (e.g., H1, H9) and / or a human induced pluripotent stem cell (hiPSC) (e.g., WC50, IMR90).

[0130] In some embodiments of the present application, the human embryonic stem cell is a commercialized human embryonic stem cell line.

[0131] In some embodiments of the present application, the human embryonic stem cells are stem cells isolated or obtained from human embryos that have not developed in vivo for 14 days or less after fertilization.

[0132] In a second aspect of the present application, there is provided use of the reagent combination or kit of the first aspect of the present application in any one of (1)-(10);

[0133] (1) preparing blastocyst-like lineage precursor cells; (2) preparing blastocyst-like cells; (3) preparing blastoids; (4) preparing a product for inducing stem cells to generate blastocyst-like lineage precursor cells; (5) preparing a product for inducing stem cells to generate blastocyst-like cells; (6) preparing a product for inducing stem cells to generate blastoids; (7) preparing primitive gut motility stage cells of blastoids; (8) preparing a product for inducing stem cells to generate primitive gut motility stage cells of blastoids; (9) preparing tissue (such as lung, pancreas, heart, intestine, liver, kidney, etc.) and / or organ precursor cells (such as lung precursor cells, pancreas precursor cells, heart-related precursor cells, intestine precursor cells, liver precursor cells, kidney precursor cells, etc.); (10) preparing a product for inducing stem cells to generate tissue (such as lung, pancreas, heart, intestine, liver, kidney, etc.) and / or organ precursor cells (such as lung precursor cells, pancreas precursor cells, heart-related precursor cells, intestine precursor cells, liver precursor cells, kidney precursor cells, etc.).

[0134] In some embodiments of the present application, the stem cells are stem cells as defined in the first aspect of the present application.

[0135] In a third aspect of the present application, there is provided a method for inducing stem cells to generate blastocyst-like lineage precursor cells, comprising the step of using the reagent combination or kit of the first aspect of the present application.

[0136] In some embodiments of the present application, the method for inducing stem cells to generate blastocyst-like lineage precursor cells comprises S1 or S2:

[0137] S1: culturing the stem cells with the first medium in the reagent combination or kit of the first aspect of the present application;

[0138] S2: culturing the stem cells with the second medium in the reagent combination or kit of the first aspect of the present application for the first time, and then culturing the stem cells with the first medium in the reagent combination or kit of the first aspect of the present application for the second time.

[0139] In some embodiments of the present application, the culturing in S1 is for 36-108h; further for 42-78h; and more further for 54-66h.

[0140] In some embodiments of the present application, the time for the first culturing in S2 is 12-36h; further 18-30h; more further 22-26h.

[0141] In some embodiments of the present application, the time for the second culturing in S2 is 24-72h; further 24-48h; more further 32-40h.

[0142] In some embodiments of the present application, the culturing (including the culturing in S1, and the first and second culturing in S2) is adherent culturing; in some embodiments of the present application, the culturing is performed in a gelatin-treated culturing device.

[0143] In some embodiments of the present application, the conditions for the culturing (including the culturing in S1, and the first and second culturing in S2) are 33-40℃, 3-7% CO2; further 36-38℃, 4-6% CO2.

[0144] In some embodiments of the present application, the stem cell is the stem cell in the first aspect of the present application.

[0145] In a fourth aspect of the present application, a method for inducing stem cells to produce blastocyst-like cells is provided, comprising the step of using the reagent combination or the kit of the first aspect of the present application.

[0146] In some embodiments of the present application, the method for inducing stem cells to produce blastocyst-like cells comprises the following steps:

[0147] T1: inducing stem cells to produce blastocyst-like lineage precursor cells: the method for inducing stem cells to produce blastocyst-like lineage precursor cells is the method for inducing stem cells to produce blastocyst-like lineage precursor cells in the third aspect of the present application;

[0148] T2: inducing blastocyst-like lineage precursor cells to produce blastocyst-like cells.

[0149] In some embodiments of the present application, the method for inducing blastocyst-like lineage precursor cells to produce blastocyst-like cells in T2 comprises the following step: performing a third culturing of the blastocyst-like lineage precursor cells with the third culture medium in the reagent combination or the kit of the first aspect of the present application.

[0150] In some embodiments of the present application, the time for the third culturing is 24-48h; further 28-44h; more further 32-40h.

[0151] In some embodiments of the present application, the third culturing is a suspension culturing; in some embodiments of the present application, the third culturing is performed using Aggrewell plates; in some embodiments of the present application, the third culturing is performed using Aggrewell plates treated with an anti-adhesion rinse.

[0152] In some embodiments of the present application, the conditions of the third culturing are 33-40℃, 3-7% CO2; further, 36-38℃, 4-6% CO2.

[0153] In some embodiments of the present application, the stem cells are the stem cells of the first aspect of the present application.

[0154] In a fifth aspect of the present application, there is provided a method for inducing stem cells to produce embryoid-like structures, comprising the step of using the reagent combination or the kit of the first aspect of the present application.

[0155] In some embodiments of the present application, the method for inducing stem cells to produce embryoid-like structures comprises the following steps:

[0156] U1: inducing stem cells to produce blastocyst-like cells: the method for inducing stem cells to produce blastocyst-like cells is the method for inducing stem cells to produce blastocyst-like cells of the fourth aspect of the present application;

[0157] U2: inducing blastocyst-like cells to produce embryoid-like structures.

[0158] In some embodiments of the present application, the method for inducing blastocyst-like cells to produce embryoid-like structures of U2 comprises the following step: culturing the blastocyst-like cells for a fourth time with the fourth medium of the reagent combination or the kit of the first aspect of the present application.

[0159] In some embodiments of the present application, the fourth culturing is performed for 24-48h; further, 28-44h; more further, 32-40h.

[0160] In some embodiments of the present application, the fourth culturing is a suspension culturing; in some embodiments of the present application, the fourth culturing is performed using Aggrewell plates; in some embodiments of the present application, the fourth culturing is performed using Aggrewell plates treated with an anti-adhesion rinse.

[0161] In some embodiments of the present application, the conditions of the fourth culturing are 33-40℃, 3-7% CO2; further, 36-38℃, 4-6% CO2.

[0162] In some embodiments of the present application, the method for inducing the generation of embryoid-like from the blastocyst-like cells in U2 further comprises the steps of culturing and / or maturing the embryoid-like: sequentially culturing the embryoid-like obtained after the fourth culturing with the fifth medium in the reagent combination or kit of the first aspect of the present application for a fifth culturing, and a sixth culturing, and then culturing the embryoid-like with the sixth medium in the reagent combination or kit of the first aspect of the present application for a seventh culturing.

[0163] In some embodiments of the present application, the fifth culturing is performed for 12-36h; further for 20-28h; and more further for 22-26h.

[0164] In some embodiments of the present application, the sixth culturing is performed for 16-32h; further for 20-28h; and more further for 22-26h.

[0165] In some embodiments of the present application, the seventh culturing is performed for 16-32h; further for 20-28h; and more further for 22-26h.

[0166] In some embodiments of the present application, the fifth culturing is performed as a suspension culturing; in some embodiments of the present application, the fifth culturing is performed using Aggrewell plates; and in some embodiments of the present application, the fifth culturing is performed using Aggrewell plates treated with an anti-adhesion rinse.

[0167] In some embodiments of the present application, the sixth culturing is performed as a dynamic culturing; in some embodiments of the present application, the sixth culturing is performed using a suspension culturing device at 60-150rpm; and in some embodiments of the present application, the sixth culturing is performed using a suspension culturing device at 70-90rpm.

[0168] In some embodiments of the present application, the seventh culturing is performed as a dynamic culturing; in some embodiments of the present application, the seventh culturing is performed using a bioreactor at 30-100rpm; and in some embodiments of the present application, the seventh culturing is performed using a bioreactor at 40-60rpm.

[0169] In some embodiments of the present application, the conditions for the fifth culturing, the sixth culturing, and / or the seventh culturing are 33-40℃, 3-7% CO2; further 36-38℃, 4-6% CO2.

[0170] In some embodiments of the present application, the stem cells are the stem cells in the first aspect of the present application.

[0171] In a sixth aspect of the present application, there is provided a blastocyst-like lineage precursor cell obtained by the method of the third aspect of the present application.

[0172] In some embodiments of the present application, the blastocyst-like lineage precursor cell comprises 21.9-36.3% GATA6-positive cells; further 24.6-34.5%; still further 26.1-34.5%; yet further 28.4-31.9%.

[0173] In some embodiments of the present application, the blastocyst-like lineage precursor cell comprises 1.28-61.2% CDX2-positive cells; further 6.45-43.5%; still further 15.6-29.2%; yet further 26.8-29.2%.

[0174] In some embodiments of the present application, the blastocyst-like lineage precursor cell comprises 59.8-90.5% OCT4-positive cells; further 59.8-87.6%; still further 59.8-67.9%.

[0175] In some embodiments of the present application, the blastocyst-like lineage precursor cell does not comprise reproductive material.

[0176] In a seventh aspect of the present application, there is provided a blastocyst-like cell obtained by the method of the fourth aspect of the present application.

[0177] In some embodiments of the present application, the blastocyst-like cell comprises 15.8-37.49% primitive endoderm-like cell population; further 18.26-32.98%; still further 18.26-22.10%.

[0178] In some embodiments of the present application, the blastocyst-like cell comprises 7.26-41.99% trophoblast ectoderm-like cell population; further 16.87-25.00%; still further 20.87-23.48%.

[0179] In some embodiments of the present application, the blastocyst-like cell comprises 42.02-59.68% epiblast-like cell population; further 52.20-59.68%; still further 54.41-59.68%.

[0180] In some embodiments of the present application, the blastocyst-like cell does not comprise reproductive material.

[0181] In an eighth aspect of the present application, there is provided an embryoid obtained by the method of the fifth aspect of the present application.

[0182] In some embodiments of the application, the embryonic-like has the ability to recapitulate (preferably highly recapitulate) post-implantation embryo development and / or gastrulation movements.

[0183] In a ninth aspect of the application, there is provided a use of the blastocyst-like lineage precursor cells of the sixth aspect of the application, the blastocyst-like cells of the seventh aspect of the application and / or the embryonic-like of the eighth aspect of the application in any one of a1) to a8):

[0184] a1) preparing an embryo model;

[0185] a2) embryo (preferably early embryo) research;

[0186] a3) screening for key genes for embryo (preferably early embryo) development defects;

[0187] a4) investigating the effect of a knock-out library or over-expression library on embryo (preferably early embryo) development;

[0188] a5) testing drug safety;

[0189] a6) preparing gastrulation stage cells;

[0190] a7) preparing tissue (such as lung, pancreas, heart, intestine, liver, kidney, etc.) and / or organ precursor cells (such as lung precursor cells, pancreas precursor cells, heart-related precursor cells, intestine precursor cells, liver precursor cells, kidney precursor cells, etc.);

[0191] a8) preparing a product for any one of a1) to a7).

[0192] In some embodiments of the application, the product is a kit.

[0193] In a tenth aspect of the application, there is provided a product comprising: the blastocyst-like lineage precursor cells of the sixth aspect of the application, the blastocyst-like cells of the seventh aspect of the application and / or the embryonic-like of the eighth aspect of the application.

[0194] In some embodiments of the application, the product is for any one of a1) to a7) in the ninth aspect of the application.

[0195] In some embodiments of the application, the product is a kit.

[0196] The benefits of the application are:

[0197] The present application provides a reagent combination or kit, which can be used for inducing stem cells to generate blastocyst-like lineage precursor cells, blastocyst-like cells and / or blastoids, is based on small molecule induction, does not rely on transgenes, and has a single cell source, and the generated blastocyst-like lineage precursor cells, blastocyst-like cells have full blastocyst lineage, are seed cells with balanced developmental ability and totipotency, and solve the problem of uneven maturation of cells generated by the prior art; the generated blastoids are highly similar to natural embryos in morphological characteristics and transcriptome characteristics, have a high ability to reproduce post-implantation embryo development and / or primitive gut movement, and are beneficial to in vitro research on embryo development; the reagent combination or kit is simple in method for inducing stem cells to generate blastocyst-like lineage precursor cells, blastocyst-like cells and / or blastoids, has high applicability (is applicable to any stem cells), and has high efficiency in construction of blastoids (up to 30%, while the prior art is less than 0.5%). BRIEF DESCRIPTION OF DRAWINGS

[0198] FIG. 1 shows the developmental ability of an existing mouse in vitro embryo model and its limitations.

[0199] FIG. 2A-E shows the analysis of the embryonic stem cells induced by the kits of Examples 1-1 to 1-4, 2 to 7 in Examples 11-1 to 11-4, 12 to 17: A is the kit of Examples 1-1 to 1-4, 2 to 7, wherein the small molecules involved and the related abbreviations are noted: C (Chir99021) is a glycogen synthase kinase 3 inhibitor (GSK3i); L (mLif) is a murine leukemia inhibitory factor (mLIF) that can activate the JAK / STAT3 signaling pathway; 6 (E616452) is a selective inhibitor of transforming growth factor-beta receptor I (TGFp-Ri); A (AM580) is a retinoic acid nuclear receptor (RAR) agonist; B is the strategy of the blastocyst-like lineage precursor cells (denoted as Q0-a, Q0-b, Q0-c, Q0-d, respectively) induced by the kits of Examples 1-1 to 1-4 in Examples 11-1 to 11-4; C is the immunofluorescence analysis results of Q0-a, Q0-b, Q0-c, Q0-d (GATA6 is a marker protein of primitive endoderm (PrE), and CDX2 is a marker protein of Trophectoderm (TE)); D is the strategy of the blastocyst-like lineage precursor cells (Q1, Q2, Q3, Q4, Q5, Q6) induced by the kits of Examples 2 to 7 in Examples 12 to 17; E is the immunofluorescence analysis results of the blastocyst-like lineage precursor cells (denoted as Q1, Q2, Q3, Q4, Q5, Q6, respectively) induced by the kits of Examples 2 to 7 in Examples 12 to 17 (GATA6 is a marker protein of primitive endoderm (PrE), and CDX2 is a marker protein of Trophectoderm (TE)).

[0200] FIG. 3 shows the immunofluorescence results of the blastocyst-like lineage precursor cells (denoted as Q1, Q2, Q3, Q4, Q5, Q6, respectively) induced by the kits of Examples 2 to 7 in Examples 12 to 17 (red is the marker protein CDX2 of TE; purple is the marker protein GATA6 of PrE; green is the marker protein OCT4 of inner cell mass and epiblast; dark blue is the nuclear dye DAPI, and the scale bar is 50 pm).

[0201] FIG. 4A-B shows the expression of marker genes at different time points of the blastocyst-like lineage precursor cells (denoted as Q3, Q4, respectively) induced from embryonic stem cells using the kit of Example 4, 5 in Example 14, 15 (the internal reference gene is Actin, and the relative expression level of each gene is calculated using the method of ΔΔCt): wherein, A is the expression of marker genes at different time points of the blastocyst-like lineage precursor cells induced from embryonic stem cells in Example 14; B is the expression of marker genes at different time points of the blastocyst-like lineage precursor cells induced from embryonic stem cells in Example 15.

[0202] FIG. 5A-E shows the single-cell transcriptome analysis results of the blastocyst-like lineage precursor cells induced from embryonic stem cells using the kits of Example 2 to 7 in Example 12 to 17: wherein, A is the UMAP analysis plot of the blastocyst-like lineage precursor cells produced in Example 12 with mouse early embryo reference data using the nonlinear dimension reduction algorithm; B is the UMAP analysis plot of the blastocyst-like lineage precursor cells produced in Example 13 with mouse early embryo reference data using the nonlinear dimension reduction algorithm; C is the UMAP analysis plot of the mixture of the blastocyst-like lineage precursor cells produced in Example 14, 15 with mouse early embryo reference data using the nonlinear dimension reduction algorithm; D is the UMAP analysis plot of the blastocyst-like lineage precursor cells produced in Example 16 with mouse early embryo reference data using the nonlinear dimension reduction algorithm; E is the UMAP analysis plot of the blastocyst-like lineage precursor cells produced in Example 17 with mouse early embryo reference data using the nonlinear dimension reduction algorithm.

[0203] FIG. 6A-G shows the single-cell transcriptome analysis results of the blastocyst-like cells produced in Example 18-1 to 18-7: wherein, A is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-2 with mouse early embryo reference data using the nonlinear dimension reduction algorithm; B is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-3 with mouse early embryo reference data using the nonlinear dimension reduction algorithm; C is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-4 with mouse early embryo reference data using the nonlinear dimension reduction algorithm; D is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-1 with mouse early embryo reference data using the nonlinear dimension reduction algorithm; E is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-5 with mouse early embryo reference data using the nonlinear dimension reduction algorithm; F is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-6 with mouse early embryo reference data using the nonlinear dimension reduction algorithm; G is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-7 with mouse early embryo reference data using the nonlinear dimension reduction algorithm.

[0204] FIGS. 7A-B show the results of single-cell transcriptome analysis of the blastoid cells obtained in Examples 18-1 to 18-7: where A is a UMAP analysis plot of the blastoid cells obtained in Examples 18-1 to 18-7 and mouse early embryo reference data, as well as the single-cell transcriptome expression levels of marker genes (GATA6 is a marker gene of primitive endoderm (PrE), and CDX2 is a marker gene of Trophectoderm (TE)); and B is the cell type proportion of the blastoid cells obtained in Examples 18-1 to 18-7.

[0205] FIGS. 8A-D show the expression of lineage marker genes and the results of immunofluorescence staining of the blastoid cells obtained in Example 18-1: where A is the single-cell transcriptome expression of lineage marker genes of the three cell populations (primitive endoderm-like cell population, Trophectoderm-like cell population, and epiblast-like cell population) of the blastoid cells obtained in Example 18-1; B is a plot of the results of immunofluorescence staining (KRT18 / PDGFRA / OCT4) of the blastoid cells obtained in Example 18-1; C is a plot of the results of immunofluorescence staining (KRT18 / CDX2 / OCT4) of the blastoid cells obtained in Example 18-1, two replicates; and D is a heatmap analysis of the three cell populations of the blastoid cells obtained in Example 18-1 and E4.5 natural embryos.

[0206] FIGS. 9A-H show results of inducing embryonic stem cells to generate embryoid-like structures using the kit of Example 8-1 in Example 18-1, wherein A is a schematic diagram of the process of inducing embryonic stem cells to generate embryoid-like structures (scale bar is 50 pm); B is a representative sample of Day 4.5 embryoid-like structures (ci-Embryoids) generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 (scale bar is 100 pm); C is a table comparing the efficiency of generating embryoid-like structures in Example 18-1 with reported EiTiX embryoid-like structures at different time periods; D is an immunohistochemical staining image of a representative sample of Day 4.5 embryoid-like structures (ci-Embryoids) generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 (scale bar is 100 pm); E is a graph of immunofluorescence staining results of a representative sample of Day 4.5 embryoid-like structures (ci-Embryoids) generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 (AP2y / SOX2 / OCT4); F is a graph of immunofluorescence staining results of a representative sample of Day 4.5 embryoid-like structures (ci-Embryoids) generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 (SOX2 / T / Bry) (scale bar is 100 pm); G is a graph of immunofluorescence staining results of a representative sample of Day 4.5 embryoid-like structures (ci-Embryoids) generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 (N-cad / T, N-cad / E-cad, T) (scale bar: 40 pm in the top panel and 10 pm in the bottom panel); and H is a graph of immunofluorescence staining results of a representative sample of Day 4.5 embryoid-like structures (ci-Embryoids) generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 (FOXA2 / GATA4) (scale bar is 100 pm).

[0207] FIG. 10 shows a UMAP analysis plot of single-cell transcriptome samples of Day 4.5 embryoid-like structures generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 and natural embryonic E7.5 samples in vivo in mice (generated from 10x Genomic scRNA-seq).

[0208] FIG. 11 shows single-cell transcriptome expression levels of marker genes of natural embryonic E7.5 samples in vivo in mice.

[0209] FIG. 12 shows single-cell transcriptome expression levels of marker genes of Day 4.5 embryoid-like structures generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1.

[0210] Fig. 13A-D show the universality of the kit and / or induction method of the present application (immunofluorescence staining of cells and their constructed embryoid bodies after induction of two other stem cell lines (C57BL / 6 mouse ICM-derived and ICR mouse background-derived stem cells) by Q3 and Q4 small molecule induction method, in addition to OG2 mouse embryonic stem cells): A is the immunofluorescence image of the blastocyst-like lineage precursor cells generated from the other cell line 1 (C57BL / 6 mouse-derived) stem cells after induction (the same method as in Examples 14, 15, except that the source of stem cells is different) (GATA6, CDX2 and SOX2 are the lineage marker proteins of the primitive endoderm-like cell population, the trophoblast ectoderm-like cell population and the epiblast-like cell population, respectively) (scale bar is 50 μm); B is the immunofluorescence image of the blastocyst-like lineage precursor cells generated from the other cell line 2 (ICR mouse-derived) stem cells after induction (the same method as in Examples 14, 15, except that the source of stem cells is different) (scale bar is 50 μm); C is the immunofluorescence image of the embryoid bodies constructed from the other cell line 1 (C57BL / 6 mouse-derived) after induction (i.e. the same method as in Examples 18-1, 18-4, except that the source of stem cells is different) at different days (CDX2 / GATA4 / OCT4; AP2y / SOX2) (scale bar is 50 μm); D is the immunofluorescence image of the embryoid bodies constructed from the other cell line 2 (ICR mouse-derived) after induction (i.e. the same method as in Examples 18-1, 18-4, except that the source of stem cells is different) at different days (CDX2 / GATA4 / OCT4; AP2y / SOX2) (scale bar is 50 μm). DETAILED DESCRIPTION

[0211] The present application will be further described in details by specific examples.

[0212] It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application.

[0213] Glossary:

[0214] In the present application, the term "blastocyst-like lineage precursor cells" refers to the cell population that has acquired the expression of key transcription factors GATA6, CDX2 and OCT4 of the three blastocyst lineages (primitive endoderm-like cells, trophoblast ectoderm-like cells and epiblast-like cells) after induction of stem cells (such as embryonic stem cells) by a specific small molecule combination, and the molecular level has not yet fully reached the molecular level of the blastocyst stage, but has the ability to mature into blastocyst-like three lineage cells.

[0215] In the present application, the term "blastocyst-like cell" refers to the cell molecular characteristics of three lineages (primitive endoderm-like cells, trophoblast ectoderm-like cells and epiblast-like cells) of E4.5 blastocyst stage embryos obtained after blastocyst-like lineage precursor cells are induced to mature by small molecule culture, and the cells have the ability to develop into all components of the embryonic-like embryo.

[0216] In the present application, the term "embryonic-like embryo", also known as "in vitro reconstructed embryo", refers to an embryo model that simulates the characteristics of an embryo and can be used to study the process of embryonic development, which is established by self-assembly of stem cells (such as embryonic stem cells).

[0217] In the present application, the term "intermediate cell" refers to a precursor cell population that is intermediate between the three lineages (primitive endoderm-like cells, trophoblast ectoderm-like cells and epiblast-like cells) of the blastocyst stage and has not yet been specific to any lineage at the cell molecular level after stem cells (such as embryonic stem cells) are induced by specific small molecule combination culture.

[0218] The experimental methods in the following examples not specified in the specific conditions are generally carried out according to the conventional conditions, or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the present examples, if not specifically stated, are reagents and materials obtained from commercial channels.

[0219] Some of the materials and experimental methods involved in the following examples / efficacy examples are as follows:

[0220] 1. Animals

[0221] All animal experiments were carried out in accordance with the Regulations on the Management of Laboratory Animals, approved by the Experimental Animal Management and Use Committee of the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, and animals suspected of having health problems before the start of the experiment were excluded. ICR mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and rats were purchased from Zhuhai Betareagent Biotechnology Co., Ltd. The mice were housed in a temperature-controlled room (22 ± 1°C) with a 12-hour light / dark cycle between 07:00 and 19:00, with free access to water and food. Euthanasia was performed by CO2 inhalation, and cervical dislocation was performed to ensure death after CO2 asphyxiation.

[0222] 2. Mouse embryonic stem cells and their maintenance culture

[0223] The OG2 mouse embryonic stem cell line was derived from the inner cell mass of a 3.5-day embryo of a 129 female mouse mated with an OG2 male mouse (B6;CBA-Tg(Pou5f1-EGFP)2Mnn / J). The other stem cell line 1 was derived from the inner cell mass of a 3.5-day embryo of a C57BL / 6 mouse mated with itself; and the stem cell line 2 was derived from the inner cell mass of a 3.5-day embryo of an ICR mouse mated with itself. The method for preparing the stem cell lines was described in [9]. The mice were purchased from the Jackson Laboratory.

[0224] The mouse embryonic stem cells were seeded on 1% gelatin (STEMCELL, 07903)-treated cell culture well plates and cultured with 2iLIF medium. The 2iLIF medium was a N2B27 base medium containing 20 ng / mL murine LIF (qKine, Qk018), 3 mM CHIR99021 (Selleck, S2924) and 1 mM PD0325901 (Selleck, S1036). The N2B27 base medium was a mixed medium containing 1x B27 (Gibco, 17504044), 1x N2 (Gibco, 17502048), 1x GlutaMAX (Gibco, 35050061), 1x MEM-NEAA (Gibco, 11140050), 0.1 mM 2-mercaptoethanol (Gibco, 21985023), which was obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1). The cell culture medium was refreshed every day and the cells were cultured at 37°C, 20% O2 and 5% CO2.

[0225] 3. Rat serum

[0226] The rat serum was prepared as follows: Prepare 3% isoflurane (RWD, R510-22-10) gas anesthetic and fill the box connected to the anesthetic apparatus with the gas at a flow rate of 3.0 L / min for 10 minutes. Transfer the prepared rat into the box for anesthesia. Gently stimulate the rat's paw with a pair of tweezers. If there is no response, the rat is fully anesthetized. Then cover the rat's nose with an inhalation mask connected to the gas anesthetic to ensure that the rat is in a deep anesthetized state during blood collection. Cut the skin and abdominal wall toward the forelimb with large scissors to further expose the posterior part of the abdominal cavity. Use a pair of tweezers to turn out the excess fat from the abdominal cavity. Use a pair of tweezers to separate the visceral fat in the midline of the rat's abdomen in a symmetrical direction to expose the abdominal aorta. Repeat the step to clean the fat in the longitudinal area of the abdominal aorta to clearly identify the bifurcation of the abdominal aorta. Carefully pick up the fat on the abdominal aorta and the bifurcation with a pair of tweezers to separate the fat from the target blood vessel.

[0227] Male rat abdominal aortic blood was collected using a 0.5 mm venous blood collection needle (SANLI, F0326-9-1) and a coagulation tube (BD Biosciences, 367955). After each collection, invert and mix the tube 6 to 7 times, then let it stand at room temperature for at least 30 minutes. When obvious stratification is observed, centrifuge at 2,000 x g for 15 minutes at room temperature. After centrifugation, carefully transfer the upper serum to a 15 mL centrifuge tube on a clean bench, and inactivate at 55°C for 45 minutes. Freeze with liquid nitrogen and store at -80°C, thaw before use and filter with a 0.45 μm (Millipore, HAWP04700) filter membrane.

[0228] 4. Immunofluorescence of cells and embryoid bodies

[0229] Cells were fixed with 4% PFA (Beyotime, P0099) for half an hour at room temperature, and the embryoid bodies were fixed with 4% PFA overnight at 4°C. After fixation, the samples were blocked with blocking solution (DPBS containing 5% v / v FBS, 2% v / v BSA, 0.3% v / v Trition X-100) for 1 hour, and the primary antibodies diluted with the blocking solution were added for overnight incubation at 4°C. The primary antibody information: GATA6 (Cell Signaling Technology, 5851); CDX2 (Biogenex, MU392A-UC); OCT4 (Cell Signaling Technology, 83932); T (Abeam, ab209665); SOX2 (Invitrogen eBioscience, 14-9811-82); AP2y (Santa Cruz, sc-8977); PDGRFA (Invitrogen eBioscience, PA5-16571); KRT18 (Sigma-Aldrich, SAB4501665); DKK1 (Santa Cruz, sc-374574); GATA4 (Invitrogen eBioscience, 14-9980-82); N-Cadherin (Invitrogen eBioscience, 33-3900); E-Cadherin (Invitrogen eBioscience, 14-3249-82); FOXA2 (Cell Signaling Technology, 8186S).

[0230] The next day, PBST (DPBS containing 0.05% v / v Tween-20) was used to wash three times, and Alexa Fluor secondary antibodies diluted with the blocking solution were added for incubation at room temperature for 1 hour. The corresponding secondary antibodies were purchased from Thermo Fisher Scientific, with the item numbers being 21202, 31573, 31570, 31572, 31571, 21472, and 21447, respectively. Finally, DAPI (Sigma, D9542) was added for nuclear staining, and PBST was used to wash three times for imaging.

[0231] For the embryoid bodies at 3.5 days and above, the transparentization treatment was performed after the staining was completed. The transparentization steps were gradient treatment with 25%, 50%, 75%, 95%, and 100% thioethanol (v / v, diluted in DPBS; Sigma, 166782).

[0232] Finally samples were observed and photographed by confocal microscope FV3000 (Olympus) and pictures were analyzed by software imageJ (NIH, USA).

[0233] 5. Cell flow cytometry analysis

[0234] Cells were digested by Accutase (Invitrogen, 00455556) and resuspended by appropriate medium. The steps of cell immunofluorescence were as follows: first, cells were blocked by blocking solution (DPBS without Ca 2+ and Mg 2+ containing 5% v / v FBS, 5% v / v NGS, 2% v / v BSA) for 20 minutes at room temperature. Then, primary antibodies (GATA6 (Cell Signaling Technology, 5851); CDX2 (Biogenex, MU392A-UC)) diluted by blocking solution were added and incubated for 1 hour at room temperature. After washing by DPBS (without Ca 2+ and Mg 2+ ) for three times, secondary antibodies (Thermo Fisher Scientific, 31571 and 31572 respectively) diluted by blocking solution were added and incubated for 30 minutes at room temperature. After washing by DPBS (without Ca 2+ and Mg 2+ ) for three times, cells were filtered by 40 pm filter (BD Biosciences, 352340) and analyzed by LSRFortessa X-20 (BD Biosciences). Finally, the results were analyzed by FlowJo software. FACS cell sorting was used to sort the target cell population for subsequent experiments by using instrument BD FACSAria III (BD Biosciences).

[0235] 6. RNA extraction and qPCR detection

[0236] Total RNA was extracted by Ultrapure RNA Kit (CWBIO, CW0581M) and 1 pg total RNA was used for reverse transcription according to the instructions of RevertAid TM First Strand cDNA Synthesis Kit (Thermo, K16225). qRT-PCR detection was performed by using TB Premix Ex Taq TM II (Takara, RR820A) kit, primers as shown in Table 1, and detected by QuantStudio 3 Real-Time PCR System (Applied Biosystems) after spotting. The relative change fold was calculated by ΔΔCt method, and the internal reference gene was Actin.

[0237] Table 1 qPCR primers

[0238] 7. Preparation of single cell suspension and sequencing

[0239] The cells induced in the above examples were digested with Accutase. The ci-embryoids induced in the above examples on Day 4.5 were digested with 100 μL of 10 U / mL papain (Worthington Biochemicals, LS003126) at 37 °C for 15 min. The dissociated cells were washed twice with pre-cooled DPBS (without Ca 2+ and Mg 2+ ) and filtered with a 40 μm filter (BD Biosciences, 352340) to obtain a single cell suspension, and loaded into a 10x Genomics Chromium system at a concentration of 16,000 single cells per sample using Single Cell 3’ Reagent Kits V3.1. The library was sequenced on an MGISEQ 2000 (MGI Tech) system in PE 100 mode.

[0240] 8. Preprocessing of single cell RNA sequencing data

[0241] The reference genome was constructed following the guideline provided by 10x Genomics official website (https: / / support.10xgenomics.com / single-cell-gene-expression / software / pipelines / latest / using / tutorial_mr) and all single-cell RNA sequencing data were analyzed accordingly. The pre-constructed genome sequence and gene annotation file were downloaded from Ensembl database version GRCm39 104. The raw data was first quality controlled using fastp (v.0.21.0, https: / / github.com / OpenGene / fastp) with default parameters, and then the quality controlled data was aligned, annotated, PCR duplicates removed and gene expression quantified using STAR (v.2.7.9a) following the standard STARsolo analysis pipeline (https: / / github.com / alexdobin / STAR / blob / master / docs / STARsolo.md).

[0242] 9. Public single-cell RNA sequencing data

[0243] The public data such as GSE45719

[0010] , GSE84892

[0011] , GSE109071

[0012] , GSE100597

[0013] , GSE123046

[0014] and E-MTAB-6967

[0015] (mouse embryonic stages from zygote to E7.5, which contains all known cell lineages within this stage) were downloaded and pre-processed using the same method as the data generated by this study, or adjusted according to the instructions of the authors of the data source article for use.

[0244] 10. Downstream analysis of single-cell RNA sequencing data

[0245] Linear dimensionality reduction and principal component analysis (PCA). Further analysis was performed in R 4.2.2 using Seurat software (v.4.3.0). Based on the preliminary assessment of each cell quality control indicator, cells with less than 4000 detected genes (Smart-seq2 data) or 2000 (10x Genomics data) or greater than or equal to 5% mitochondrial gene percentage were removed from the screening, and only genes detected in at least 3 cells were retained. The filtered sequencing data was sequentially log-normalized, centered and scaled using the NormalizeData and ScaleData functions, and the top 2000 highly variable genes were selected using the FindVariableFeatures function. Principal component analysis (PCA) was applied to the scaled data using the RunPCA function in Seurat and default parameters based on the selected highly variable genes. The PCA coordinates were then used as input for the graph-based clustering method.

[0246] 11. Batch effect correction and integration of multi-origins Smart-seq2 and 10x Genomics single-cell RNA sequencing data

[0247] To compare the ci-embryoid induced in the examples at Day 4.5 and the public natural embryo dataset, the standard canonical correlation analysis (CCA) method was used (Figure 10), following the standard pipeline provided by Seurat official website (https: / / satijalab.org / seurat / articles / integration_introduction.html). To integrate the public data from multiple origins of different cell lineages and developmental stages (Figures 5, 6), the inventors used the fastMNN method, and treated each dataset from different experiments as a batch, and each batch contained at least one cell population shared with other cells. The inventors used the computeSumFactors function in the scran package (v.1.20.1) to log-normalize the data, and then used the multiBatchNorm function in the batchchelor package (v.1.8.1) to scale-normalize across batches. The log-normalized and batch effect corrected datasets were then integrated using the fastMNN method, which was implemented through SeuratWrappers (v.0.3.0, https: / / github.com / satijalab / seurat-wrappers). The MNN low-dimensional coordinates were then used for graph-based clustering and visualization.

[0248] 12. Visualization using graph-based clustering and Uniform Manifold Approximation and Projection (UMAP)

[0249] Based on the dimensionality-reduced and batch effect-corrected data, the inventors constructed a shared nearest neighbor (SNN) graph using the FindNeighbors function, and then partitioned it using the Louvain algorithm implemented in the FindClusters function, with resolution ranges selected according to library size and data heterogeneity. Finally, the inventors visualized the data using the RunUMAP function.

[0250] Example 1-1 A kit for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0251] A kit for inducing stem cells to generate blastocyst-like lineage precursor cells, comprising: a first culture medium; the first culture medium is an N2B27 base medium (CL6A) containing 1 μM CHIR99021 (Selleck, S2924), 10 ng / mL murine leukemia inhibitory factor (murine LIF, qKine, Qk018), 1 μM E616452 (Selleck, S7223), and 0.01 μM AM580 (Selleck, S2933).

[0252] Wherein, the N2B27 base medium is a mixed culture medium containing 1 × B27 (Gibco, 17504044), 1 × N2 (Gibco, 17502048), 1 × GlutaMAX (Gibco, 35050061), 1 × MEM NEAA (Gibco, 11140050), and 0.1 mM 2-mercaptoethanol (Gibco, 21985023), and the mixed culture medium is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0253] Example 1-2 A kit for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0254] A kit for inducing stem cells to produce blastocyst-like lineage precursor cells, comprising: a first medium; the first medium is N2B27 base medium (CL6A) containing 3 μM CHIR99021 (Selleck, S2924), 20 ng / mL murine LIF (qKine, Qk018), 10 μM E616452 (Selleck, S7223) and 0.05 μM AM580 (Selleck, S2933).

[0255] wherein the N2B27 base medium is a mixed medium containing 1×B27 (Gibco, 17504044), 1×N2 (Gibco, 17502048), 1×GlutaMAX (Gibco, 35050061), 1×MEM NEAA (Gibco, 11140050) and 0.1 mM 2-mercaptoethanol (Gibco, 21985023), and the mixed medium is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0256] Example 1-3 A kit for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0257] A kit for inducing stem cells to produce blastocyst-like lineage precursor cells, comprising: a first medium; the first medium is N2B27 base medium (CL6A) containing 6 μM CHIR99021 (Selleck, S2924), 20 ng / mL murine LIF (qKine, Qk018), 10 μM E616452 (Selleck, S7223) and 0.05 μM AM580 (Selleck, S2933).

[0258] wherein the N2B27 base medium is a mixed medium containing 1×B27 (Gibco, 17504044), 1×N2 (Gibco, 17502048), 1×GlutaMAX (Gibco, 35050061), 1×MEM NEAA (Gibco, 11140050) and 0.1 mM 2-mercaptoethanol (Gibco, 21985023), and the mixed medium is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0259] Embodiment 1-4 A kit for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0260] A kit for inducing stem cells to produce blastocyst-like lineage precursor cells, comprising: a first culture medium; the first culture medium is N2B27 basic culture medium (CL6A) containing 15 μM CHIR99021 (Selleck, S2924), 100 ng / mL murine leukemia inhibitory factor (murine LIF, qKine, Qk018), 15 μM E616452 (Selleck, S7223) and 0.1 μM AM580 (Selleck, S2933).

[0261] Wherein, the N2B27 basic culture medium is a mixed culture medium containing 1×B27 (Gibco, 17504044), 1×N2 (Gibco, 17502048), 1×GlutaMAX (Gibco, 35050061), 1×MEM NEAA (Gibco, 11140050) and 0.1 mM 2-mercaptoethanol (Gibco, 21985023), and the mixed culture medium is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0262] Embodiment 2 A kit for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0263] A kit for inducing stem cells to produce blastocyst-like lineage precursor cells, comprising: a first culture medium and a second culture medium; wherein the first culture medium is N2B27 basic culture medium (CL6A) containing 1 μM CHIR99021, 10 ng / mL murine leukemia inhibitory factor, 1 μM E616452 and 0.01 μM AM580; and the second culture medium is N2B27 basic culture medium (C6A) containing 1 μM CHIR99021, 1 μM E616452 and 0.01 μM AM580.

[0264] N2B27 base medium is a mixed medium containing 1x B27 (Gibco, 17504044), 1x N2 (Gibco, 17502048), 1x GlutaMAX (Gibco, 35050061), 1x MEM NEAA (Gibco, 11140050) and 0.1 mM 2-mercaptoethanol (Gibco, 21985023), which is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0265] Example 3 A kit for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0266] A kit for inducing stem cells to generate blastocyst-like lineage precursor cells, comprising: a first culture medium and a second culture medium; wherein the first culture medium is N2B27 base medium (CL6A) containing 3 μM CHIR99021, 20 ng / mL mouse leukemia inhibitory factor, 5 μM E616452 and 0.02 μM AM580; and the second culture medium is N2B27 base medium (C6A) containing 3 μM CHIR99021, 5 μM E616452 and 0.02 μM AM580.

[0267] N2B27 base medium is a mixed medium containing 1x B27 (Gibco, 17504044), 1x N2 (Gibco, 17502048), 1x GlutaMAX (Gibco, 35050061), 1x MEM NEAA (Gibco, 11140050) and 0.1 mM 2-mercaptoethanol (Gibco, 21985023), which is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0268] Example 4 A kit for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0269] A kit for inducing stem cells to produce blastocyst-like lineage precursor cells, comprising: a first medium and a second medium; wherein the first medium is N2B27 base medium (CL6A) containing 6 μM CHIR99021, 20 ng / mL mouse leukemia inhibitory factor, 10 μM E616452 and 0.05 μM AM580; and the second medium is N2B27 base medium (C6A) containing 6 μM CHIR99021, 10 μM E616452 and 0.05 μM AM580.

[0270] The N2B27 base medium is a mixed medium containing 1×B27 (Gibco, 17504044), 1×N2 (Gibco, 17502048), 1×GlutaMAX (Gibco, 35050061), 1×MEM NEAA (Gibco, 11140050) and 0.1 mM 2-mercaptoethanol (Gibco, 21985023), and is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0271] Example 5 A kit for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0272] A kit for inducing stem cells to produce blastocyst-like lineage precursor cells, comprising: a first medium and a second medium; wherein the first medium is N2B27 base medium (CL6A) containing 3 μM CHIR99021, 20 ng / mL mouse leukemia inhibitory factor, 10 μM E616452 and 0.05 μM AM580; and the second medium is N2B27 base medium (C6A) containing 9 μM CHIR99021, 10 μM E616452 and 0.05 μM AM580.

[0273] The N2B27 base medium is a mixed medium containing 1×B27 (Gibco, 17504044), 1×N2 (Gibco, 17502048), 1×GlutaMAX (Gibco, 35050061), 1×MEM NEAA (Gibco, 11140050) and 0.1 mM 2-mercaptoethanol (Gibco, 21985023), and is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0274] Embodiment 6 A kit for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0275] A kit for inducing stem cells to produce blastocyst-like lineage precursor cells, comprising: a first culture medium and a second culture medium; wherein the first culture medium is N2B27 basic medium (CL6A) containing 9 μM CHIR99021, 50 ng / mL mouse leukemia inhibitory factor, 12 μM E616452 and 0.08 μM AM580; and the second culture medium is N2B27 basic medium (C6A) containing 9 μM CHIR99021, 12 μM E616452 and 0.08 μM AM580.

[0276] The N2B27 basic medium is a mixed culture medium containing 1 × B27 (Gibco, 17504044), 1 × N2 (Gibco, 17502048), 1 × GlutaMAX (Gibco, 35050061), 1 × MEM NEAA (Gibco, 11140050) and 0.1 mM 2-mercaptoethanol (Gibco, 21985023), and the mixed culture medium is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0277] Embodiment 7 A kit for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0278] A kit for inducing stem cells to produce blastocyst-like lineage precursor cells, comprising: a first culture medium and a second culture medium; wherein the first culture medium is N2B27 basic medium (CL6A) containing 15 μM CHIR99021, 100 ng / mL mouse leukemia inhibitory factor, 15 μM E616452 and 0.1 μM AM580; and the second culture medium is N2B27 basic medium (C6A) containing 15 μM CHIR99021, 15 μM E616452 and 0.1 μM AM580.

[0279] N2B27 base medium is a mixed medium containing 1x B27 (Gibco, 17504044), 1x N2 (Gibco, 17502048), 1x GlutaMAX (Gibco, 35050061), 1x MEM NEAA (Gibco, 11140050) and 0.1 mM 2-mercaptoethanol (Gibco, 21985023), which is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0280] Example 8-1 A kit for constructing a blastoid

[0281] A kit for constructing a blastoid comprises: a first medium, a second medium, a third medium, a fourth medium, a fifth medium, and a sixth medium; wherein the first medium and the second medium are the first medium and the second medium in Example 5, which are used for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells; the third medium is a FC base medium (ciBP medium) containing 25 ng / mL rhFGF4 (R&D, 235F4), 10 ng / mL hBMP4 (Miltenyi, 130111167), 20 ng / mL Activin A (PeproTech, 12014E), 3 mM XAV939 (Sigma, X3004), 2 mM TRULI (Selleck, E1061), 1 pg / mL Heparin (Sigma, H3149), 200 mM L-ascorbic acid 2-phosphate (Sigma, A8960) and 1x insulin-transferrin-selenium supplement (ITS-G, Gibco, 41400045), which is used for inducing blastocyst-like lineage precursor cells to generate blastocyst-like cells.

[0282] The fourth medium is a FC base medium, which is used for inducing blastocyst-like cells to generate a blastoid.

[0283] The fifth medium is an Advanced DMEM / F12 medium (Gibco, 12634028) [4, 13] containing 30% (v / v) FBS (Carpicorn scientific, FBS52A), 1x GlutaMax (Gibco, 35050038), 1x Insulin-Transferrin-Selenium-Aminoethanol (ITS-X, Gibco, 51500056), 100 nM T3 (3,3',5-Triiodo-L-thyronine sodium salt, sigma, T6397), 8 nM β-estradiol (Sigma, E8875), 200 ng / mL progesterone (Sigma, P0130), 25 μΜ N-acetyl-L-cysteine (Sigma, C7880), 1 mg / mL D-glucose (sigma, G8644), and 1% (v / v) penicillin / streptomycin (Gibco, 15140122) (IVC1 medium).

[0284] The sixth medium is a DMEM medium (low glucose, pyruvate, no glutamine, no phenol red, Gibco, 11880) [4, 13] containing 50% (v / v) rat serum (self-made), 30% (v / v) human serum AB (Off the Clot, Gemini, 100-318), 1x GlutaMax (Gibco, 35050038), 1x sodium pyruvate (Gibco, 11360039), 11 mM HEPES (Gibco, 15630130), 4 mg / mL D-glucose (sigma, G8644), and 1% (v / v) penicillin / streptomycin (Gibco, 15140122) (IVC2 medium).

[0285] The fifth medium, and the sixth medium are used for culture and / or maturation of blastoids.

[0286] The FC base medium is DMEM medium (Gibco, 41966052) containing 20% (v / v) FBS (Carpicorn scientific, FBS-52A), 1x sodium pyruvate (Gibco, 11360039), 1x GlutaMAX (Gibco, 35050061), 1x MEM non-essential amino acids (Gibco, 11140050), 0.1 mM 2-mercaptoethanol (Gibco, 21985023), and 1% (v / v) penicillin / streptomycin (Gibco, 15140122) [4].

[0287] Example 8-2 A kit for constructing a synthetic embryo

[0288] A kit for constructing a synthetic embryo, which is the same as the kit for constructing a synthetic embryo of Example 8-1, except that the first medium and the second medium are the first medium and the second medium of Example 2.

[0289] Example 8-3 A kit for constructing a synthetic embryo

[0290] A kit for constructing a synthetic embryo, which is the same as the kit for constructing a synthetic embryo of Example 8-1, except that the first medium and the second medium are the first medium and the second medium of Example 3.

[0291] Example 8-4 A kit for constructing a synthetic embryo

[0292] A kit for constructing a synthetic embryo, which is the same as the kit for constructing a synthetic embryo of Example 8-1, except that the first medium and the second medium are the first medium and the second medium of Example 4.

[0293] Example 8-5 A kit for constructing a synthetic embryo

[0294] A kit for constructing a synthetic embryo, which is the same as the kit for constructing a synthetic embryo of Example 8-1, except that the first medium and the second medium are the first medium and the second medium of Example 6.

[0295] Example 8-6 A kit for constructing a synthetic embryo

[0296] A kit for constructing a synthetic embryo, which is the same as the kit for constructing a synthetic embryo of Example 8-1, except that the first medium and the second medium are the first medium and the second medium of Example 7.

[0297] Embodiment 9 A kit for constructing a blastoid embryo

[0298] A kit for constructing a blastoid embryo, comprising: a first medium, a second medium, a third medium, a fourth medium, a fifth medium, and a sixth medium; wherein the first medium and the second medium are the first medium and the second medium in Embodiment 2, which are used for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells.

[0299] The third medium is FC basal medium containing 10 ng / mL rhFGF4 (R&D, 235F4), 5 ng / mL hBMP4 (Miltenyi, 130111167), 10 ng / mL Activin A (PeproTech, 12014E), 1 mM XAV939 (Sigma, X3004), 1 mM TRULI (Selleck, E1061), 0.5 pg / mL Heparin (Sigma, H3149), 50 mM L-ascorbic acid 2-phosphate (Sigma, A8960), and 0.5x insulin-transferrin-selenium supplement (ITS-G, Gibco, 41400045), which is used for inducing blastocyst-like lineage precursor cells to generate blastocyst-like cells.

[0300] The fourth medium is FC basal medium, which is used for inducing blastocyst-like cells to generate a blastoid embryo.

[0301] The fifth medium is an Advanced DMEM / F12 medium (Gibco, 12634028) (IVC1 medium) containing 10% (v / v) FBS (Carpicorn scientific, FBS52A), 0.5 mM GlutaMax (Gibco, 35050038), 0.5x insulin-transferrin-selenium-aminoethanol (ITS-X, Gibco, 51500056), 50 nM T3 (3,3',5-Triiodo-L-thyronine sodium salt, sigma, T6397), 5 nM β-estradiol (Sigma, E8875), 100 ng / mL progesterone (Sigma, P0130), 5 μΜ N-acetyl-L-cysteine (Sigma, C7880), 0.5 mg / mL D-glucose (sigma, G8644), and 0.1% (v / v) penicillin / streptomycin (Gibco, 15140122).

[0302] The sixth medium is a DMEM medium (low glucose, pyruvate, no glutamine, no phenol red, Gibco, 11880) (IVC2 medium) containing 25% (v / v) rat serum (self-made), 10% (v / v) human serum AB (Off the Clot, Gemini, 100-318), 0.5x GlutaMax (Gibco, 35050038), 0.5x sodium pyruvate (Gibco, 11360039), 6 mM HEPES (Gibco, 15630130), 1 mg / mL D-glucose (sigma, G8644), and 0.1% (v / v) penicillin / streptomycin (Gibco, 15140122).

[0303] The fifth medium, and the sixth medium are used for culture and / or maturation of the embryoid.

[0304] The FC base medium is DMEM medium (Gibco, 41966052) containing 20% (v / v) FBS (Carpicorn scientific, FBS-52A), 1x sodium pyruvate (Gibco, 11360039), 1x GlutaMAX (Gibco, 35050061), 1x MEM non-essential amino acids (Gibco, 11140050), 0.1 mM 2-mercaptoethanol (Gibco, 21985023), and 1% (v / v) penicillin / streptomycin (Gibco, 15140122) [4].

[0305] Example 10 A kit for constructing a blastoid

[0306] A kit for constructing a blastoid, comprising: a first medium, a second medium, a third medium, a fourth medium, a fifth medium, and a sixth medium; wherein the first medium and the second medium are the first medium and the second medium in Example 7, which are used to induce mouse embryonic stem cells to generate blastocyst-like lineage precursor cells.

[0307] The third medium is FC base medium containing 50 ng / mL rhFGF4 (R&D, 235F4), 20 ng / mL hBMP4 (Miltenyi, 130111167), 40 ng / mL Activin A (PeproTech, 12014E), 9 mM XAV939 (Sigma, X3004), 5 mM TRULI (Selleck, E1061), 2 pg / mL Heparin (Sigma, H3149), 500 mM L-ascorbic acid 2-phosphate (Sigma, A8960), and 2x insulin-transferrin-selenium additive (ITS-G, Gibco, 41400045), which is used to induce blastocyst-like lineage precursor cells to generate blastocyst-like cells.

[0308] The fourth medium is FC base medium, which is used to induce blastocyst-like cells to generate a blastoid.

[0309] The fifth medium is an Advanced DMEM / F12 medium (Gibco, 12634028) (IVC1 medium) containing 40 (v / v) FBS (Carpicorn scientific, FBS52A), 2x GlutaMax (Gibco, 35050038), 2x Insulin-Transferrin-Selenium-Aminoethanol (ITS-X, Gibco, 51500056), 200 nM T3 (3,3',5-Triiodo-L-thyronine sodium salt, sigma, T6397), 11 nM β-estradiol (Sigma, E8875), 300 ng / mL progesterone (Sigma, P0130), 45 μM N-acetyl-L-cysteine (Sigma, C7880), 2 mg / mL D-glucose (sigma, G8644), and 2% (v / v) penicillin / streptomycin (Gibco, 15140122).

[0310] The sixth medium is a DMEM medium (low glucose, pyruvate, no glutamine, no phenol red, Gibco, 11880) (IVC2 medium) containing 60% (v / v) rat serum (self-made), 35% (v / v) human serum AB (Off the Clot, Gemini, 100-318), 2x GlutaMax (Gibco, 35050038), 2x sodium pyruvate (Gibco, 11360039), 20 mM HEPES (Gibco, 15630130), 6 mg / mL D-glucose (sigma, G8644), and 2% (v / v) penicillin / streptomycin (Gibco, 15140122).

[0311] The fifth medium, and the sixth medium are used for culture and / or maturation of parablastula.

[0312] FC base medium is DMEM medium (Gibco, 41966052) containing 20% (v / v) FBS (Carpicorn scientific, FBS-52A), 1x sodium pyruvate (Gibco, 11360039), 1x GlutaMAX (Gibco, 35050061), 1x MEM non-essential amino acids (Gibco, 11140050), 0.1 mM 2-mercaptoethanol (Gibco, 21985023), and 1% (v / v) penicillin / streptomycin (Gibco, 15140122) [4].

[0313] Example 11-1 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0314] A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells comprises the steps of using the kit of Example 1-1, in particular as follows:

[0315] OG2 mouse embryonic stem cells (mESCs) are seeded at a density of 2x10 5 cells / well in 6-well plates (Greiner, 657160) treated with 0.1% (w / v) gelatine (STEMCELL, 07903) with 2 mL of the first medium in Example 1-1, and cultured at 37°C, 5% CO2 for 108h.

[0316] Example 11-2 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0317] A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as that of Example 11-1, except that the kit of Example 1-2 is used, and the culture time is 60h.

[0318] Example 11-3 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0319] A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as that of Example 11-1, except that the kit of Example 1-3 is used, and the culture time is 60h.

[0320] Example 11-4 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0321] A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells, identical to the method of Example 11-1, except that the kit of Example 1-4 is used, and the culture time is 36 h.

[0322] Example 12 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0323] A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells, comprising the step of using the kit of Example 2, as follows:

[0324] OG2 mouse embryonic stem cells (mESCs) are seeded at a density of 2 x 10 5 OG2 mouse embryonic stem cells (mESCs) are seeded at a density of 2 x 10

[0325] Example 13 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0326] A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells, identical to Example 12, except that the kit of Example 2 is replaced by the kit of Example 3, and the culture time for the second culture medium is 30 h, and the culture time for the first culture medium is 48 h.

[0327] Example 14 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0328] A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells, identical to Example 12, except that the kit of Example 2 is replaced by the kit of Example 4, and the culture time for the second culture medium is 24 h, and the culture time for the first culture medium is 36 h.

[0329] Example 15 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0330] A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells, identical to Example 12, except that the kit of Example 2 is replaced by the kit of Example 5, and the culture time for the second culture medium is 24 h, and the culture time for the first culture medium is 36 h.

[0331] Example 16 A method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0332] A method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells, which is identical to Example 12, except that the kit of Example 2 is replaced by the kit of Example 6, the culture time of the second culture medium is 18 h, and the culture time of the first culture medium is 24 h.

[0333] Example 17 A method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0334] A method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells, which is identical to Example 12, except that the kit of Example 2 is replaced by the kit of Example 7, the culture time of the second culture medium is 12 h, and the culture time of the first culture medium is 24 h.

[0335] Example 18-1 A method for constructing a blastocyst-like embryo

[0336] A method for constructing a blastocyst-like embryo, comprising the steps of using the kit of Example 8-1, which is as follows:

[0337] (1) Inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells: the method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is identical to that of Example 15;

[0338] (2) Inducing blastocyst-like lineage precursor cells to produce blastocyst-like cells (ciBPCs): the cells obtained in step (1) are digested using Accutase (Invitrogen, 00455556), and after centrifugation, the cells are resuspended in DMEM medium (Gibco, A1443001) and counted; at the same time, an Aggrewell400 plate (Stemcell, 34415) treated with Anti-Adherence Rinsing Solution (Stemcell, 7010) is prepared; the cells are inoculated into the Aggrewell400 at a density of 3 x 10 4 cells / well, 1.5 mL of the third culture medium of Example 8-1 is added, and the cells are cultured at 37°C, 5% CO2 for 36 h;

[0339] (3) Inducing ciBPCs to produce ci-Embryoids: remove the third medium (denoted as Day 0 of ci-Embryoids induction and culture), and wash twice with 1 mL of the fourth medium in Example 8-1, then add 1 mL of the fourth medium in Example 8-1, and culture at 37°C, 5% CO2 for 1.5 days (36h) (end of culture is Day 1.5); remove the fourth medium, add 1.5 mL of the fifth medium in Example 8-1, and culture at 37°C, 5% CO2 for 1 day (24h) (end of culture is Day 2.5); gently resuspend the ci-Embryoids in the Aggrewell 400 and transfer to a suspension culture six-well plate (Greiner, 657185), add 4 mL of the fifth medium, and continue to culture on a shaker (Zilang, ZCLY180N, shaker setting parameters: 80 rpm, 37°C, 5% CO2) for 1 day (24h) (end of culture is Day 3.5); transfer the ci-Embryoids to a 5 mL bioreactor (bioreactor, ABLE Biott, BWV-S005A), add 3 mL of the sixth medium in Example 8-1, and continue to culture on a bioreactor magnetic stirring system (6-ch) base plate (Bioreactor Magnetic Stir System-6ch. Base plate, ABLE Biott, BWS-S03N0S-6B) at 37°C, 5% CO2 for 1 day (24h) (end of culture is Day 4.5), and set the system speed to 50 rpm.

[0340] Example 18-2 A method of constructing ci-Embryoids

[0341] A method of constructing ci-Embryoids, comprising the steps of using the kit of Example 8-2, which is the same as the method of Example 18-1, except that the kit of Example 8-1 is replaced by the kit of Example 8-2, and the method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as the method of Example 12, and the culture time of the third medium is 24h.

[0342] Example 18-3 A method of constructing ci-Embryoids

[0343] A method of constructing ci-Embryoids, comprising the steps of using the kit of Example 8-3, which is the same as the method of Example 18-1, except that the kit of Example 8-1 is replaced by the kit of Example 8-3, and the method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as the method of Example 13, and the culture time of the third medium is 32h.

[0344] Example 18-4 A method of constructing ci-Embryoids

[0345] A method of constructing a synthetic embryo comprising the steps of using the kit of Example 8-4, which is the same as the method of Example 18-1, except that the kit of Example 8-1 is replaced by the kit of Example 8-4, the method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as the method of Example 14, and the incubation time of the third medium is 36 h.

[0346] Example 18-5 A method of constructing a synthetic embryo

[0347] A method of constructing a synthetic embryo comprising the steps of using the kit of Example 8-4, which is the same as the method of Example 18-1, except that the kit of Example 8-1 is replaced by the kit of Example 8-4, the method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as the method of Example 14, and the incubation time of the third medium is 48 h.

[0348] Example 18-6 A method of constructing a synthetic embryo

[0349] A method of constructing a synthetic embryo comprising the steps of using the kit of Example 8-5, which is the same as the method of Example 18-1, except that the kit of Example 8-1 is replaced by the kit of Example 8-5, the method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as the method of Example 16, and the incubation time of the third medium is 40 h.

[0350] Example 18-7 A method of constructing a synthetic embryo

[0351] A method of constructing a synthetic embryo comprising the steps of using the kit of Example 8-6, which is the same as the method of Example 18-1, except that the kit of Example 8-1 is replaced by the kit of Example 8-6, the method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as the method of Example 17, and the incubation time of the third medium is 48 h.

[0352] Example 19 A method of constructing a synthetic embryo

[0353] A method of constructing a synthetic embryo comprising the steps of using the kit of Example 9, which is the same as the method of Example 18-1, except that the kit of Example 8-1 is replaced by the kit of Example 9.

[0354] Example 20 A method of constructing a synthetic embryo

[0355] A method of constructing a synthetic embryo comprising the steps of using the kit of Example 10, which is the same as the method of Example 18-1, except that the kit of Example 8-1 is replaced by the kit of Example 10.

[0356] Example 1-1 A method for culturing mouse embryonic stem cells

[0357] A method for culturing mouse embryonic stem cells, which is identical to Example 11-1, except that the first medium of Example 1-1 is replaced with 2iLIF medium.

[0358] Effect Example

[0359] 1. Cell flow cytometry analysis was performed on the blastocyst-like lineage precursor cells (denoted as Q0-a, Q0-b, Q0-c, Q0-d, Q1, Q2, Q3, Q4, Q5, Q6) obtained from Examples 11-1 to 11-4, 12 to 17 and mouse embryonic stem cells (ESC) obtained from Comparative Example 1, and the results are shown in Figure 2: in the mouse embryonic stem cells (ESC) obtained from Comparative Example 1, 0.52% were GATA6 positive cells, i.e. PrE lineage precursor cells, 0.088% were CDX2 positive cells, i.e. TE lineage precursor cells, and 98.9% were OCT4 positive cells, i.e. EPI-like cells; in the cells obtained from Example 11-1 (Q0-a), 35.3% were GATA6 positive cells, i.e. PrE lineage precursor cells, 5.11% were CDX2 positive cells, i.e. TE lineage precursor cells, and 68.2% were OCT4 positive cells, i.e. EPI-like cells; in the cells obtained from Example 11-2 (Q0-b), 36.3% were GATA6 positive cells, i.e. PrE lineage precursor cells, 1.28% were CDX2 positive cells, i.e. TE lineage precursor cells, and 90.5% were OCT4 positive cells, i.e. EPI-like cells; in the cells obtained from Example 11-3 (Q0-c), 22.8% were GATA6 positive cells, i.e. PrE lineage precursor cells, 56.3% were CDX2 positive cells, i.e. TE lineage precursor cells, and 61.2% were OCT4 positive cells, i.e. EPI-like cells; in the cells obtained from Example 11-4 (Q0-d), 21.9% were GATA6 positive cells, i.e. PrE lineage precursor cells, 61.2% were CDX2 positive cells, i.e. TE lineage precursor cells, and 60.6% were OCT4 positive cells, i.e. EPI-like cells (B and C in Figure 2); in the cells obtained from Example 12 (Q1), 34.3% were GATA6 positive cells, i.e. PrE lineage precursor cells, 6.45% were CDX2 positive cells, i.e. TE lineage precursor cells, and 67.5% were OCT4 positive cells, i.e. EPI-like cells; in the cells obtained from Example 13 (Q2), 34.5% were GATA6 positive cells, i.e. PrE lineage precursor cells, 15.6% were CDX2 positive cells, i.e. TE lineage precursor cells, and 87.6% were OCT4 positive cells, i.e. EPI-like cells; in the cells obtained from Example 14 (Q3), 31.9% were GATA6 positive cells, i.e. PrE lineage precursor cells, 26.8% were CDX2 positive cells, i.e. TE lineage precursor cells, and 67.9% were OCT4 positive cells, i.e. EPI-like cells; in the cells obtained from Example 15 (Q4), 26.1% were GATA6 positive cells, i.e. PrE lineage precursor cells, 29.2% were CDX2 positive cells, i.e. TE lineage precursor cells, and 67.1% were OCT4 positive cells, i.e. EPI-like cells; in the cells obtained from Example 16 (Q5), 28.4% were GATA6 positive cells, i.e. PrE lineage precursor cells, 26.8% were CDX2 positive cells, i.e. TE lineage precursor cells, and 59.8% were OCT4 positive cells, i.e. EPI-like cells; in the cells obtained in Example 17 (Q6): 24.6% were GATA6 positive cells, i.e. PrE lineage precursor cells, 43.5% were CDX2 positive cells, i.e. TE lineage precursor cells, and 64.7% were OCT4 positive cells, i.e. EPI-like cells (D and E in Figure 2); also, the distribution of GATA6 positive cells and CDX2 positive cells in the total cell population was analyzed together, and it was observed that Q0-a, Q0-b, Q0-c, Q0-d, Q1, Q2, Q3, Q4, Q5, Q6 all had a high activation of GATA6, while the activation of CDX2 was less in Q0-a, Q0-b, Q1, too high in Q0-c, Q0-d, Q6, and similar in Q3, Q4, Q5 to the distribution of GATA6 positive cells (C and E in Figure 2); it was observed that the kits of Examples 1-1 to 1-4, 2 to 7 allowed the induction of the generation of PrE lineage precursor cells, TE lineage precursor cells, and EPI-like cells from embryonic stem cells, obtaining total blastocyst lineage precursor cells. Examples 11-1 to 11-4 allowed obtaining all the blastocyst-like lineage precursor cells, however the proportion of the three blastocyst-like lineage cell populations was imbalanced, with Q0-a, Q0-b having less CDX2 positive cells, and Q0-c, Q0-d having a higher proportion of CDX2 positive cells.

[0360] 2. Immunofluorescence experiments were performed on the blastocyst-like lineage precursor cells (denoted as Q1, Q2, Q3, Q4, Q5, Q6) obtained in Examples 12 to 17 and mouse embryonic stem cells (ESC) obtained in Comparative Example 1, and the results are shown in Figure 3: the ESC cells exhibited a round shape and expressed only the EPI marker protein OCT4; the Q1, Q2, Q3, Q4, Q5 and Q6 cells were detected to express different amounts of the TE marker protein CDX2 and the PrE lineage marker protein GATA6; among them, Q3, Q4 and Q5 exhibited a relatively balanced activation ratio of GATA6 and CDX2 in both immunofluorescence analysis and immunofluorescence analysis, and the cell morphology was relatively healthy (Figure 2E and Figure 3). It can be seen that the blastocyst-like lineage precursor cells obtained in Examples 14 to 16 have balanced three blastocyst-like lineage cells (Figure 2E and Figure 3). The cells cultured for different times (0, 12, 24, 36, 48 and 60 h) in Examples 14 and 15 (denoted as Q3 and Q4, respectively) were taken for RNA extraction and qPCR detection; the results showed that the trophoblast ectoderm marker genes (Gata3, Hand1 and Id2) gradually increased, among which Cdx2 had a peak expression at 24-36 h; the primitive endoderm marker genes (Gata6, Gata4, Sox17 and Foxa2) showed a gradient increase trend with time; in addition, the primitive state marker genes Nanog and Sox2 were down-regulated, while the primordial state genes Otx2 and Fgf5 did not increase, which indicated that the cells were not developing in the direction of differentiation, but from the epiblast lineage cells, the trophoblast ectoderm lineage and the primitive endoderm (Figure 4).

[0361] 3. Single-cell transcriptome analysis (single-cell suspension preparation and sequencing, single-cell RNA sequencing data preprocessing, single-cell RNA sequencing data downstream analysis, multi-source Smart-seq2 and 10x Genomics single-cell RNA sequencing data batch effect correction and integration, visualization using graph-based clustering and Uniform Manifold Approximation and Projection (UMAP), in sequence) was performed on the blastocyst-like lineage precursor cells obtained in Examples 12 to 17 (corresponding to Q1 (108h), Q2 (78h), Q3 (60h), Q4 (60h), Q5 (42h) and Q6 (36h) in Figure 5, respectively, Q3&Q4 (60h) in Figure 5 represents a mixture of blastocyst-like lineage precursor cells obtained in Example 14 (Q3 (60h)) and Example 15 (Q4 (60h)), and blastocyst-like cells obtained in Examples 18-1 to 18-7 (corresponding to Q4-ciBP (36h), Q1-ciBP (24h), Q2-ciBP (32h), Q3-ciBP (36h), Q3-ciBP (48h), Q5-ciBP (40h), Q6-ciBP (48h) in Figures 6, 7, respectively) using the blastocyst-like lineage precursor cells obtained in Example 18-1, step (1) (i.e. the blastocyst-like lineage precursor cells obtained in Example 15), and the blastocyst-like cells obtained in step (2), RNA extraction and qPCR detection, and immunofluorescence experiments were performed, and the results are shown in Figures 5 to 8: the blastocyst-like precursor cells obtained by induction using small molecules Q1 (108h), Q2 (78h), Q3 (60h), Q4 (60h), Q5 (42h) and Q6 (36h) can be seen to have four main clusters in comparison with early embryonic reference data: epiblast-like precursor cells, primitive endoderm-like precursor cells, trophoectoderm-like precursor cells and intermediate cells (Figure 5). Among them, the epiblast-like precursor cells are located between E3.5 inner cell mass and E4.5 epiblast cells, have a certain degree of reprogramming, and belong to epiblast-like precursor cells; the primitive endoderm-like precursor cells are located between E4.5 primitive endoderm cells and E3.5 inner cell mass, have the potential to develop into PrE, and are precursors of the cells; the trophoectoderm-like precursor cells are located between epiblast and trophoectoderm cells, and are trophoectoderm precursor cells; and the intermediate cells have the characteristics of two or three lineage precursors, and are therefore intermediate cells (Figure 5).The blastocyst-like lineage precursor cells after culture by ciBP (Q4-ciBP (36h), Q1-ciBP (24h), Q2-ciBP (32h), Q3-ciBP (36h), Q3-ciBP (48h), Q5-ciBP (40h), Q6-ciBP (48h)) obtained three lineage cells with lineage specification: epiblast-like cells, primitive endoderm-like cells and trophoblast extraembryonic-like cells; the three groups of cells were clustered with E4.5 EPI, E4.5 PrE, E4.5 TE respectively (Figure 6). Gene dimension reduction analysis chart showed that the blastocyst-like precursor cells after ciBP specification were significantly grouped and obtained three blastocyst lineage cells with different proportions: epiblast-like cells, primitive endoderm-like cells and trophoblast extraembryonic-like cells (Figure 7). It can be seen that the cells obtained by further induction by the third medium have similar properties to the three lineage cells of natural embryo E4.5 blastocyst, and the obtained blastocyst-like cells have the characteristics of three lineage cells of epiblast, primitive endoderm and trophoblast extraembryonic (analyzed at the level of single cell transcriptome, protein and RNA). The results of blastocyst-like lineage precursor cells obtained in step (1) and blastocyst-like cells obtained in step (2) in Examples 19 and 20 are similar to those in Examples 12 to 17 and 18-1 to 18-7.

[0362] The blastomere-like precursor cells of Q4 (60h) (Example 15) were subjected to single cell level transcriptome analysis alignment with the blastomere-like cells of Q4-ciBP (36h) (Example 18-1), and the expression of epiblast marker genes (Nanog, Sox2, Pou5f1, Igfbp2, Fgf4, Tdgf1), primitive endoderm marker genes (Gata6, Gata4, Sox17, Foxa2, Cubn, Srgn), and trophoblast marker genes (Cited, Rhox6, Rhox9, Id2, Krt8, Krt18) were detected. After maturation in ciBP third medium, the blastomere-like cells of Q4-ciBP (36h) expressed significantly higher levels of marker genes than the blastomere-like precursor cells of Q4 (60h); it was shown that ciBP has the function of maturing and differentiating the blastomere-like precursor cells (Figure 8A). Immunofluorescence staining also showed the expression of proteins specific to blastomere-like lineage (KRT18 / PDGFRa / OCT4) (Figure 8B). In addition, the trophoblast cells in the blastocyst stage are heterogeneous, and there are polar trophoblast cells (polar TE) and mural trophoblast cells (mural TE); in the trophoblast-like cells in the present application, the markers of the two groups of cells (CDX2 and KRT18) were also found (Figure 8B and C). The heat map analysis of the three cell groups of the blastomere-like precursor cells induced by 96-hour small molecules and E4.5 natural embryos showed that they had very similar molecular characteristics (Figure 8D). It was shown that the blastomere-like lineage cells have similar cell characteristics to the E4.5 blastocyst lineage cells at the single cell transcriptome level, protein level, and RNA level.

[0363] 4. Day 1.5, Day 2.5, Day 3.5 and Day 4.5 ci-Embryoids obtained from step (3) in Example 18-1 were subjected to microscopic observation, immunohistochemical staining, immunofluorescence staining, single-cell transcriptome analysis (in turn, single-cell suspension preparation and sequencing, single-cell RNA sequencing data preprocessing, single-cell RNA sequencing data downstream analysis, multi-source Smart-seq2 and 10x Genomics single-cell RNA sequencing data batch effect correction and integration, visualization using graph-based clustering and Uniform Manifold Approximation and Projection (UMAP)). Known blastoid cells have full-lineage primitive cell characteristics and developmental totipotency, in order to further verify their developmental totipotency and construct in vitro synthetic embryos, study their lineage differentiation and primitive gut movement potential, and establish a ciBPC-derived ci-Embryoids in vitro reconstituted embryo system. First, ciBPCs were induced by FC, IVC1, and IVC2 at regular intervals, combined with static and dynamic culture modes, to obtain ci-Embryoids (A in FIG. 9). The observation of ci-Embryoids development is as follows: ciBPCs aggregated into a mass after overnight, and Rosette-to-lumen structure changes began to occur at Day 2.5, with a layer of cells forming on the outside of the embryo, and cells derived from the primitive endoderm (GATA6 marker) wrapping the TE (CDX2 marker) and EPI cells (OCT4 marker) of the ci-Embryoids; at Day 3.5, the ci-Embryoids aggregate was similar in shape to the E5.5-6.5 natural embryo, showing a clear cup structure; at Day 4.5, the embryo continued to develop, forming an expanded amniotic cavity, and successfully forming an asymmetric structure, and primitive streak tissue (T expression on one side of the primitive streak marker, and DKK1 of the AVE marker) appeared (A in FIG. 9). In the Day 4.5 ci-Embryoids, all embryo characteristics were observed that mimic the primitive gut movement period of natural embryos (B in FIG. 9), which is much higher than the efficiency of less than 0.5% in previous work [4], reaching an efficiency of 31.6% (C in FIG. 9).Immunohistochemistry Immunohistochemistry staining was performed on representative embryos, and the results showed that the embryos had ectoplacental cone (EPC), extra-embryonic ectoderm (EXE), chorion (Ch), amnion (Am), primitive streak (PS), definitive endoderm (DE), epiblast, extraembryonic endoderm, extraembryonic ectoderm, mesoderm, primitive streak (PS), visceral endoderm, and other characteristics of the primitive gut stage (Fig. 9D); not only did they have primitive streak and epithelial-mesenchymal transition (T / E-Cad / N-Cad) to form endoderm, ectoderm, and mesoderm (FOXA2 / GATA4 / OCT4), but also had the ability to implant ectoplacental cone and extra-embryonic ectoderm and mesoderm (AP2y / OCT4 / Sox2). The in vitro synthetic embryo system was feasible (Fig. 9E-H). As can be seen, the blastocyst-like cells (ciBPCs) were induced to produce embryoid-like (ci-Embryoids) by the fourth, fifth, and sixth culture media, and the embryoid-like was cultured and / or matured, efficiently and highly reducedly simulating the development of the pre- and post-implantation embryos (Fig. 9A), and constructing a complete post-primitive gut movement embryo (Fig. 9A, B): not only did they have the development of endoderm, mesoderm, and ectoderm, the formation of primitive streak, epithelial-mesenchymal transition, and other characteristics of the primitive gut, but also had the development of the trophoblast ectoderm to the extra-embryonic ectoderm and ectoplacental cone (Fig. 9D-H).

[0364] Single cell sequencing further confirmed that the ci-Embryoids constructed by the ciBPCs obtained in the present application (Example 18-1) were highly similar to natural embryos at the E7.5 stage in vivo, and had different lineages developed at this stage: ectoderm, gut, extraembryonic mesoderm, epiblast ectoderm, tail side ectoderm, primary mesoderm, amniotic villus mesoderm, embryonic endoderm, mixed mesoderm, blood and endothelial progenitor cells, preprimitive streak, extraembryonic endoderm, apical wall endoderm, extraembryonic ectoderm, primitive streak and adjacent ectoderm, tail neural ectoderm, anterior neural ectoderm, visceral mesoderm, paraxial mesoderm A / B, notochord, definitive endoderm, etc. (Figure 10). In addition, the ci-Embryoids constructed by the ciBPCs had correct expression patterns (Figures 10-12): not only mesoderm (Mesp1, Snail, T, Mixl1), endoderm (Dkk1 / Cer1 / Foxa2), ectoderm (Noto / Nkx1-2 / Pax6 / Hoxa1) developed correctly; but also had trophoblast ectoderm development to extraembryonic ectoderm and placental cone (Tfap2c, Sox2, Id2, etc.) (Figures 11-12). The results of the Day4.5-like embryos (ci-Embryoids) obtained in step (3) in Examples 18-2 to 18-7, 19, 20 were similar to those in Example 18-1.

[0365] The kits and / or induction methods in the present application have universality, and the use of other sources of embryonic stem cells (stem cell line 1: C57BL / 6 source; stem cell line 2: ICR source) and the use of Q3, Q4 for stem cell culture induction to obtain blastocyst-like precursor cells (i.e. the same as in Examples 14, 15, with the only difference being the source of stem cells) also have good levels of activation of trophoblast ectoderm marker protein CDX2 and primitive endoderm marker protein GATA6 (A and B in Figure 13). Furthermore, after the ciBP culture is specialized into blastocyst-like cells, 3.5-day-like embryo culture is performed (i.e. the same as in Examples 18-1, 18-4, with the only difference being the source of stem cells), and the gradual development of the like embryo from 1.5 days to 2.5 days to 3.5 days can be seen, simulating important events such as perivitelline, egg cylinder formation, primitive streak initial formation, etc. (C and D in Figure 13). It can be seen that different stem cell lines have good induction ability and the ability to form like embryos, and the universality of the present application can be seen.

[0366] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and shall be included in the protection scope of the present application.

[0367] References

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[0369] 2. Veenvliet, J.V., et al., Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites. 2020. 370(6522): p. eaba4937.

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[0372] 5. Rivron, N.C., et al., Blastocyst-like structures generated solely from stem cells. 2018. 557(7703): p. 106-111.

[0373] 6. Harrison, S.E., et al., Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro. Science, 2017. 356(6334).

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Claims

1. A reagent combination or kit comprising a first medium; the first medium is a basal medium comprising a GSK-3 inhibitor, a STAT3 activator, a retinoic acid nuclear receptor agonist, and a TGF-β receptor kinase inhibitor.

2. The reagent combination or kit of claim 1, wherein: the reagent combination or kit further comprises a second medium; the second medium is a basal medium comprising a GSK-3 inhibitor, a retinoic acid nuclear receptor agonist, and a TGF-β receptor kinase inhibitor; preferably, the GSK-3 inhibitor in the first medium, the second medium is each independently selected from at least one of a GSK-3a inhibitor, a GSK-3b inhibitor; further preferably, the GSK-3 inhibitor in the first medium, the second medium is each independently selected from at least one of TWS119, NP031112, SB216763, CHIR-98014, AZD2858, AZD1080, SB415286, LY2090314, CHIR-99021, L803-mts, BIO, AR-A014418, TDZD-8, 2-D08, IM-12, 1-azakenpaullone; preferably, the STAT3 activator in the first medium comprises at least one of Leukemia inhibitory factor and Interleukin 6; preferably, the retinoic acid nuclear receptor agonist in the first medium, the second medium is each independently selected from at least one of AM580, All-trans retinoic acid, 9-cis retinoic acid, AC 261066, AC 55649, Adapalene, AM 80, BMS 753, BMS 961, CD 1530, CD 2314, CD 437, Ch55, Isotretinoin, Tazarotene, TTNTB, and EC19; preferably, the TGF-β receptor kinase inhibitor in the first medium, the second medium is each independently selected from at least one of SB431542, A83-01, Galunisertib, SB525334, LY2109761, E616452, LY3200882, SB505124, PF06952229, SD208, ML347, R268712, Fresolimumab, ITD-1, AZ12601011, BIO-013077-01.

3. The reagent combination or kit of any one of claims 1-2, wherein: the reagent combination or kit further comprises a third medium; the third medium is a basal medium comprising a fibroblast growth factor, a BMP4 signaling pathway activator, a TGF-β activator, a WNT signaling pathway inhibitor, a Lats kinase inhibitor, an anticoagulant, ascorbic acid or its derivatives, and an insulin-transferrin-selenium supplement; preferably, the fibroblast growth factor (FGF) is at least one of FGF1-FGF23. Preferably, the activator of BMP4 signaling pathway in the third medium comprises at least one of BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoliquiritigenin, dihydroxypropyl, apigenin and biochanin A; Preferably, the activator of TGF-β in the third medium comprises at least one of TGF-β, Activin A; Preferably, the inhibitor of WNT signaling pathway in the third medium comprises at least one of IWP4, IWP2, IWR-1, IWP1, IWP3, IWR-2, IWR-3, IWR-4, IWR-5, XAV939, DKK1, quercetin, ICG-001, pamoate, CCT031374, iCRT-3, iCRT-5, iCRT-14, CPG049090, NC043; Preferably, the inhibitor of Lats kinase in the third medium comprises at least one of TRULI, GA-017, TDI-011536; Preferably, the anticoagulant in the third medium comprises at least one of heparin, EDTA salt; Preferably, the ascorbic acid or derivative thereof in the third medium comprises at least one of ascorbic acid, calcium ascorbate, magnesium ascorbate, zinc ascorbate, potassium ascorbate, sodium ascorbate, dehydroascorbic acid, L-threonic acid, L-xylosic acid, L-lyxaric acid, L-ascorbyl monostearate, L-ascorbyl dipalmitate, L-ascorbyl 6-hexadecanoate, L-ascorbyl 2-phosphate, L-ascorbyl 3-phosphate, L-ascorbyl 2-sulfate.

4. The reagent combination or kit of any one of claims 1-3, wherein: the reagent combination or kit further comprises a fourth medium; the fourth medium is a basal medium of the third medium; Preferably, the reagent combination or kit further comprises a fifth medium and a sixth medium; the fifth medium is a basal medium containing serum, glutamine, insulin-transferrin-selenium-aminethanol, thyroid hormone receptor agonist, estradiol, progesterone, acetylcysteine, glucose and antibiotics; the sixth medium is a basal medium containing serum, glutamine, pyruvic acid or salt thereof, buffer salt, glucose and antibiotics; Preferably, the thyroid hormone receptor agonist in the fifth medium comprises at least one of thyroxine or salt thereof, triiodothyronine or salt thereof.

5. The reagent combination or kit of any one of claims 1-4, wherein: the concentration of the GSK-3 inhibitor in the first medium is 1-15 μM; Preferably, the concentration of the STAT3 activator in the first medium is 10-100 ng / mL; Preferably, the concentration of the retinoic acid nuclear receptor agonist in the first medium is 0.01-0.1 μM; Preferably, the concentration of the TGF-beta receptor kinase inhibitor in the first medium is 1-20 μM; Preferably, the concentration of the GSK-3 inhibitor in the second medium is 1-15 μM; Preferably, the concentration of the retinoic acid nuclear receptor agonist in the second medium is 0.01-0.1 μM; Preferably, the concentration of the TGF-beta receptor kinase inhibitor in the second medium is 1-20 μM; Preferably, the concentration of the fibroblast growth factor in the third medium is 10-50 ng / mL; Preferably, the concentration of the BMP4 signaling pathway activator in the third medium is 5-20 ng / mL; Preferably, the concentration of the TGF-beta activator in the third medium is 10-40 ng / mL; Preferably, the concentration of the WNT signaling pathway inhibitor in the third medium is 1-9 μM; Preferably, the concentration of the Lats kinase inhibitor in the third medium is 1-5 μM; Preferably, the concentration of the anticoagulant in the third medium is 0.5-2.0 μg / mL; Preferably, the concentration of the ascorbic acid or its derivative in the third medium is 50-500 μM; Preferably, the concentration of the insulin-transferrin-selenium additive in the third medium is 0.5x-2x; Preferably, the concentration of the serum in the fifth medium is 10%-40% by volume percentage; Preferably, the concentration of the glutamine in the fifth medium is 0.5x-2x; Preferably, the concentration of the insulin-transferrin-selenium-aminoethanol in the fifth medium is 0.5x-2x; Preferably, the concentration of the thyroid hormone receptor agonist in the fifth medium is 50-200 nM; Preferably, the concentration of the estradiol in the fifth medium is 5-11 nM; Preferably, the concentration of the progesterone in the fifth medium is 100-300 ng / mL; Preferably, the concentration of the acetylcysteine in the fifth medium is 5-45 μM; Preferably, the concentration of the glucose in the fifth medium is 0.5-2 mg / mL; Preferably, the concentration of the antibiotic in the fifth medium is 0.1%-2% by volume percentage; Preferably, the concentration of the serum in the sixth medium is 35%-95% by volume percentage; Preferably, the concentration of the glutamine in the sixth medium is 0.5x-2x; Preferably, the concentration of the pyruvic acid or its salt in the sixth medium is 0.5x-2x; Preferably, the concentration of the buffer salt in the sixth medium is 6-20 mM; Preferably, the concentration of the glucose in the sixth medium is 1-6 mg / mL; Preferably, the concentration of the antibiotic in the sixth medium is 0.1%-2% by volume percentage; Preferably, the basal medium of the first medium, the second medium, the third medium, the fourth medium, the fifth medium, and the sixth medium is independently selected from at least one of IMDM medium, Neurobasal medium, Eagle's Basal Medium medium, MEM medium, DMEM medium, Ham's F-12 medium, RPMI1640 medium, Advanced RPMI 1640 medium, Advanced DF-12 medium, and DMEM / F12 medium; Preferably, the basal medium of the first medium and / or the second medium is DMEM / F12 medium and Neurobasal medium; Preferably, the basal medium of the first medium and / or the second medium is a basal medium containing glutamine, non-essential amino acids, B27, N2 (N-2 Supplement), and 2-mercaptoethanol; Preferably, the basal medium of the third medium is DMEM medium; Preferably, the basal medium of the third medium is a basal medium containing serum, pyruvate or a salt thereof, glutamine, non-essential amino acids, 2-mercaptoethanol, and antibiotics; Preferably, the basal medium of the fifth medium is Advanced DMEM / F12 medium; Preferably, the basal medium of the sixth medium is DMEM medium.

6. Use of the reagent combination or kit of any one of claims 1-5 in any one of (1)-(10); (1) preparing blastocyst-like lineage precursor cells; (2) preparing blastocyst-like cells; (3) preparing blastoids; (4) preparing a product for inducing stem cells to produce blastocyst-like lineage precursor cells; (5) preparing a product for inducing stem cells to produce blastocyst-like cells; (6) preparing a product for inducing stem cells to produce blastoids; (7) preparing primitive gut motility stage cells of blastoids; (8) preparing a product for inducing stem cells to produce primitive gut motility stage cells of blastoids; (9) preparing tissue and / or organ precursor cells; and (10) preparing a product for inducing stem cells to produce tissue and / or organ precursor cells; Preferably, the stem cells are stem cells with pluripotency; Preferably, the stem cells with pluripotency comprise at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and cord blood stem cells.

7. A method for inducing stem cells to produce blastocyst-like lineage precursor cells, comprising the step of using the reagent combination or kit of any one of claims 1-5.

8. The method of claim 7, wherein: the method for inducing stem cells to produce blastocyst-like lineage precursor cells comprises S1 or S2: S1: culturing stem cells with the first medium in the reagent combination or kit of any one of claims 1-5; S2: culturing the stem cells in a second medium of the reagent combination or kit of any one of claims 2-5, followed by culturing the stem cells in a first medium of the reagent combination or kit of any one of claims 1-5; Preferably, the culturing in S1 is for 36-108 h; Preferably, the first culturing in S2 is for 12-36 h; Preferably, the second culturing in S2 is for 24-72 h; Preferably, the culturing in S1, the first culturing in S2 and / or the second culturing in S2 is adherent culturing.

9. A method of inducing stem cells to produce blastocyst-like cells, comprising the step of using the reagent combination or kit of any one of claims 1-5; Preferably, the method of inducing stem cells to produce blastocyst-like cells comprises the steps of: T1 : inducing stem cells to produce blastocyst-like lineage precursor cells: the method of inducing stem cells to produce blastocyst-like lineage precursor cells is the method of claim 7 or 8; T2: inducing blastocyst-like lineage precursor cells to produce blastocyst-like cells.

10. The method of claim 9, wherein: the method of inducing blastocyst-like lineage precursor cells to produce blastocyst-like cells in T2 comprises the step of culturing the blastocyst-like lineage precursor cells in a third medium of the reagent combination or kit of any one of claims 3-5; Preferably, the third culturing is for 24-48 h; Preferably, the third culturing is in suspension.

11. A method of inducing stem cells to produce embryoid-like, comprising the step of using the reagent combination or kit of any one of claims 1-5; Preferably, the method of inducing stem cells to produce embryoid-like comprises the steps of: U1 : inducing stem cells to produce blastocyst-like cells: the method of inducing stem cells to produce blastocyst-like cells is the method of claim 9 or 10; U2: inducing blastocyst-like cells to produce embryoid-like.

12. The method of claim 11, wherein: the method of inducing blastocyst-like cells to produce embryoid-like in U2 comprises the step of culturing the blastocyst-like cells in a fourth medium of the reagent combination or kit of any one of claims 4-5; Preferably, the fourth culturing is for 24-48 h; Preferably, the fourth culturing is in suspension; Preferably, the method of inducing blastocyst-like cells to produce embryoid-like in U2 further comprises the steps of culturing and / or maturing the embryoid-like: sequentially culturing the embryoid-like obtained after the fourth culturing in a fifth medium of the reagent combination or kit of any one of claims 4-5, and in a sixth medium of the reagent combination or kit of any one of claims 4-5, followed by culturing the embryoid-like in a seventh medium of the reagent combination or kit of any one of claims 4-5; Preferably, the fifth culturing is for 12-36 h; Preferably, the sixth culturing is for 16-32 h; Preferably, the seventh culturing is for 16-32 h; Preferably, the fifth culturing is in suspension; Preferably, the sixth culturing is a dynamic culturing. Preferably, the seventh culturing is a dynamic culturing.

13. Any one of d1) - d3) biological material: d1) a biological material, which is blastocyst-like lineage precursor cells, obtained by the method of claim 7 or 8; d2) a biological material, which is blastocyst-like cells, obtained by the method of claim 9 or 10; d3) a biological material, which is a blastoid, obtained by the method of claim 11 or 12; Preferably, the content of GATA6 positive cells in the blastocyst-like lineage precursor cells is 21.9% - 36.3%; Preferably, the content of CDX2 positive cells in the blastocyst-like lineage precursor cells is 1.28% - 61.2%; Preferably, the content of OCT4 positive cells in the blastocyst-like lineage precursor cells is 59.8% - 90.5%; Preferably, the content of primitive endoderm-like cell population in the blastocyst-like cells is 15.8% - 37.49%; Preferably, the content of trophoectoderm-like cell population in the blastocyst-like cells is 7.26% - 41.99%; Preferably, the content of epiblast-like cell population in the blastocyst-like cells is 42.02% - 59.68%; Preferably, the blastoid has the ability to reproduce post-implantation embryo development and / or primitive gut movement.

14. Use of at least one of the biological materials of d1) - d3) of claim 13 in any one of a1) - a8): a1) preparing an embryo model; a2) embryo research; a3) screening key genes for embryo development defects; a4) studying the effect of a knockout library or overexpression library on embryo development; a5) detecting drug safety; a6) preparing primitive gut movement period cells; a7) preparing tissue, and / or organ precursor cells; a8) preparing products for any one of a1) - a7).

15. A product comprising at least one of the biological materials of d1) - d3) of claim 13.

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