Reagent combination or kit for constructing embryoid and application of reagent combination or kit

Abstract

The invention belongs to the technical field of biology, and discloses a reagent combination or kit for constructing an embryoid and application of the reagent combination or kit. The reagent combination or the kit can be used for inducing stem cells to generate blastocyst-like pedigree precursor cells, blastocyst-like cells and / or embryoid-like cells, is based on small molecule induction, does not depend on transgenosis, and is single in cell source, and the generated blastocyst-like pedigree precursor cells and blastocyst-like cells have a whole blastocyst pedigree, and can be used for inducing stem cells to generate blastocyst-like pedigree precursor cells and blastocyst-like cells. The seed cells are balanced in developmental ability and have totipotency, and the problem of unbalanced cell maturation generated in the prior art is solved; the generated embryoid is highly similar to a natural embryo in morphological characteristics and transcriptome characteristics, has the capability of highly reproducing embryonic development and / or protointestinal movement after implantation, and is beneficial to in-vitro research of embryonic development; the reagent combination or the kit is used for inducing stem cells to generate blastocyst-like pedigree precursor cells, blastocyst-like cells and / or embryoid-like cells, and the method is simple, high in applicability and high in embryoid-like construction efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a reagent combination or kit for constructing embryo-like structures and its application. Background Technology

[0002] Constructing in vitro embryonic models that mimic natural embryonic development has significant biological implications and regenerative medicine research value. Due to the difficulty in obtaining natural embryonic materials and ethical concerns, reliable in vitro embryonic models are urgently needed to decode the early life code. Currently, research on in vitro embryonic models still faces many challenges, such as transgene-carrying starting cells, low embryonic-like synthesis efficiency, and strong cellular heterogeneity, making it difficult to realistically reproduce earlier embryonic development and lineage specialization events in vitro.

[0003] In natural mammalian development, embryogenesis begins with the union of sperm and egg to form a zygote and acquire totipotency. In mice, the zygote develops through 2-cell, 4-cell, and 8-cell cleavages to form a morula. The first cell lineage specialization occurs after the morula becomes compact and acquires polarity; this event marks the formation of the trophectoderm (TE) and inner cell mass (ICM). Subsequently, as the embryo develops to the 16-32 cell stage, the TE of the outer cells follows the Na+ lineage... + -K + The ion gradient established by the ion pump is transported to the intercellular space to form the blastocyst cavity, thereby causing a second cell lineage specialization: the ICM further develops into the epiblast (EPI) and the primitive endoderm (PrE); at this point, the three lineages of EPI, PrE, and TE are formed [1]. Among them, EPI develops into the three germ layers of the embryo: endoderm, mesoderm, and ectoderm, forming an individual; while PrE and TE develop into extraembryonic tissues, which then form the yolk sac and placenta, respectively. The interaction of the three is indispensable for the normal development of the organism.

[0004] In recent years, research on mouse embryonic development simulation systems based on mouse embryonic stem cells (ESCs) has made great progress. Figure 1). ESCs can self-organize into organized structures such as blastoids, gastruloids and embryoids after induction in vitro [2-4]. Although this embryonic development system opens up new ways 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 (TSCs) only have EPI and TE, lack the PrE lineage, and do not have the potential to develop into post-implantation embryos [5]. 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 being that gastruloids lack the primitive endoderm (PrE) lineage, and the signals of the extraembryonic tissue are crucial for the development of the epiblast (EPI) and the establishment of the 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, i.e. ETS synthetic embryos / embryoids, by co-culturing ESC with EPI differentiation potential, TSC-derived TSC and PE-derived Extraembryonic endoderm cells (XEN) stem cells[6]. When the ETS embryoids developed to the primitive streak movement period, 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 cells (iXEN) overexpressing PE key regulatory factor Gata4 in ESC induced by Dox instead of XEN, and cells (iTSC) overexpressing 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 period and primitive streak movement period, 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 STAT3 cell pathway through transgenic regulation of STAT3 signaling pathway, embryonic stem cells (ESC) could have higher developmental potential, and a new type of cells, morula-like cells (MLC), could be obtained efficiently[8]. MLC had the ability to develop into all lineages in the embryo and extraembryonic tissues, and could 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 comparable to 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 leads to an embryo formation rate of less than 0.5% Figure 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 a kit.

[0008] The second aspect of the present application aims to provide the use of the reagent combination or the 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] In order to achieve the above-mentioned purposes, the technical solution adopted by the present application is:

[0018] The first aspect of the present application provides a reagent combination or a 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] Preferably, 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] Preferably, the first medium is used to induce the stem cells to generate blastocyst-like lineage precursor cells.

[0022] Preferably, 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] Preferably, 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] Preferably, the second medium is used to induce the stem cells to generate blastocyst-like lineage precursor cells.

[0026] Preferably, the GSK-3 inhibitor in the first medium and 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 and 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 (6-bromo-indirubin-3’-oxime), AR-A014418, TDZD-8, 2-D08, IM-12, 1-Azakenpaullone (Indirubin); more preferably, the GSK-3 inhibitor in the first medium and the second medium is CHIR-99021.

[0027] Preferably, the STAT3 activator in the first medium comprises at least one of Leukemia Inhibitory Factor (LIF) and Interleukin 6 (IL6); further preferably, the STAT3 activator is Leukemia Inhibitory Factor (LIF); more preferably, the STAT3 activator is mouse Leukemia Inhibitory Factor.

[0028] Preferably, the retinoic acid nuclear receptor (RAR) agonist in the first medium, 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, AM80, BMS 753, BMS 961, CD 1530, CD 2314, CD 437, Ch55, Isotretinoin, Tazarotene, TTNTB, and EC19; further preferably, the retinoic acid nuclear receptor (RAR) agonist in the first medium, second medium is AM580.

[0029] Preferably, the TGF-β receptor kinase inhibitor in the first medium, 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; further preferably, the TGF-β receptor kinase inhibitor in the first medium, second medium is E616452.

[0030] Preferably, the concentration of the GSK-3 inhibitor in the first medium is 1-15 µM; further 2.1-9.9 µM; more further 2.7-6.6 µM; again further 2.7-3.3 or 5.4-6.6 µM.

[0031] Preferably, 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] Preferably, the concentration of the retinoic acid nuclear receptor (RAR) agonist in the first medium is 0.01-0.1 µM; further 0.02-0.08 µM; more further 0.04-0.06 µM.

[0033] Preferably, the concentration of the TGF-β receptor kinase inhibitor in the first medium is 1-20 µM; further 1-15 µM; more further 5-15 µM; again further 5-12 µM; more further 9-11 µM.

[0034] Preferably, 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 or 5.4-6.6 μM.

[0035] Preferably, 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] Preferably, 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] Preferably, the reagent combination or kit comprises a first medium, or a first medium and a second medium, for use in inducing stem cells to generate blastocyst-like lineage precursor cells.

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

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

[0040] Preferably, the reagent combination or kit further comprises a third medium;

[0041] 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 a derivative thereof, and an insulin-transferrin-selenium supplement.

[0042] Preferably, the third medium is used to induce blastocyst-like lineage precursor cells to generate blastocyst-like cells.

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

[0044] Preferably, the activator of BMP4 signaling pathway in the third culture medium comprises at least one of BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoliquiritigenin, dihydroxypropyl, apigenin, and biochanin A; further is BMP4; and more further is hBMP4.

[0045] Preferably, the activator of TGF-β in the third culture medium comprises at least one of TGF-β, Activin A; further is Activin A.

[0046] Preferably, the inhibitor of WNT signaling pathway in the third culture 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] Preferably, the inhibitor of Lats kinase in the third culture medium comprises at least one of TRULI, GA-017, TDI-011536; further is TRULI.

[0048] Preferably, the anticoagulant in the third culture medium comprises at least one of heparin, EDTA salt; further is heparin.

[0049] Preferably, the ascorbic acid or derivative thereof in the third culture 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] Preferably, the concentration of the fibroblast growth factor in the third culture medium is 10-50 ng / mL; further is 15-30 ng / mL; and more further is 22-28 ng / mL.

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

[0052] Preferably, 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] Preferably, 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] Preferably, 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] Preferably, 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] Preferably, 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] Preferably, 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] Preferably, 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] Preferably, 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] Preferably, the method for inducing stem cells to produce blastocyst-like cells is the method of the fourth aspect of the present application.

[0063] Preferably, the reagent combination or kit further comprises a fourth culture medium;

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

[0065] Preferably, the fourth culture medium is used for inducing blastocyst-like cells to produce embryoid-like cells.

[0066] Preferably, 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 embryoid-like.

[0069] Preferably, when the reagent combination or kit is used for inducing stem cells to produce embryoid-like, the reagent combination or kit further comprises instruction 3, which describes a method for inducing stem cells to produce embryoid-like.

[0070] Preferably, the method for inducing stem cells to produce embryoid-like is the method of the fifth aspect of the present application (the method for inducing blastocyst-like cells to produce embryoid-like described in U2 does not comprise the step of culturing and / or maturing the embryoid-like).

[0071] Preferably, 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] Preferably, the fifth medium and the sixth medium are used for culturing and / or maturing the embryoid-like.

[0075] Preferably, 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] Preferably, the antibiotics in the fifth medium comprise at least one of amphotericin, nystatin, gentamicin, tetracycline, erythromycin, penicillin, streptomycin; further penicillin and streptomycin.

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

[0078] Preferably, the concentration of the glutamine in the fifth medium is 0.5-2x; further 0.7-1.5x; and further 0.9-1.1x.

[0079] Preferably, the concentration of the insulin-transferrin-selenium-aminoethanol in the fifth medium is 0.5-2x; further 0.7-1.5x; and further 0.9-1.1x.

[0080] Preferably, the concentration of the thyroid hormone receptor agonist in the fifth medium is 50-200 nM; further 60-150 nM; and further 90-110 nM.

[0081] Preferably, the concentration of the estradiol in the fifth medium is 5-11 nM; further 6-10 nM; and further 7.2-8.8 nM.

[0082] Preferably, the concentration of the progesterone in the fifth medium is 100-300 ng / mL; further 150-270 ng / mL; and further 180-220 ng / mL.

[0083] Preferably, the concentration of the acetylcysteine in the fifth medium is 5-45 μM; further 10-35 μM; and further 22.5-27.5 μM.

[0084] Preferably, the concentration of the glucose in the fifth medium is 0.5-2 mg / mL; further 0.7-1.5 mg / mL; and further 0.9-1.1 mg / mL.

[0085] Preferably, the concentration of the antibiotic in the fifth medium is 0.1-2% by volume; further 0.5-1.7%; and further 0.9-1.1%.

[0086] Preferably, the serum in the sixth medium comprises rat serum and human AB serum.

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

[0088] Preferably, the pyruvate or salt thereof in the sixth medium is sodium pyruvate.

[0089] Preferably, the buffer salt in the sixth medium comprises at least one of phosphate, Tris, and HEPES; and further HEPES.

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

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

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

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

[0094] Preferably, the concentration of the glutamine in the sixth medium is 0.5-2x; further, 0.7-1.5x; still further, 0.9-1.1x.

[0095] Preferably, the concentration of the pyruvate or salt thereof in the sixth medium is 0.5-2x; further, 0.7-1.5x; still further, 0.9-1.1x.

[0096] Preferably, the concentration of the buffer salt in the sixth medium is 6-20 mM; further, 8-16 mM; still further, 10-12 mM.

[0097] Preferably, the concentration of the glucose in the sixth medium is 1-6 mg / mL; further, 2-5 mg / mL; still further, 3.6-4.4 mg / mL.

[0098] Preferably, the concentration of the antibiotic in the sixth medium is 0.1-2% by volume percentage; further, 0.5-1.7%; still further, 0.9-1.1%.

[0099] Preferably, 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 bodies and culturing and / or maturing the embryoid-like bodies.

[0102] Preferably, the reagent combination or kit for inducing stem cells to generate embryoid-like and culturing and / or maturation of embryoid-like further comprises an instruction 4, which describes a method of inducing stem cells to generate embryoid-like and culturing and / or maturation of embryoid-like.

[0103] Preferably, the method of inducing stem cells to generate 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 generate embryoid-like in U2 further comprises a step of culturing and / or maturation of embryoid-like).

[0104] Preferably, 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, the sixth medium (such as 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); the use of the reagent combination or kit corresponds to the medium.

[0105] Preferably, the base 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 RPMI 1640), Advanced DF-12 (Advanced DMEM / F-12) medium, DMEM / F12 medium; further preferably, the base 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, DMEM / F12 medium.

[0106] Preferably, the base medium of the first medium and / or the second medium is DMEM / F12 medium and Neurobasal medium.

[0107] Preferably, the volume ratio of DMEM / F12 medium and Neurobasal medium in the base medium of the first medium, and / or the second medium is each independently selected from 1:(0.8~1.2); further selected from 1:(0.9~1.1).

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

[0109] Preferably, the concentration of glutamine in the base medium of the first medium, and / or the second medium is 0.5~1.5x; further 0.9~1.1x.

[0110] Preferably, the concentration of non-essential amino acids in the base medium of the first medium, and / or the second medium is 0.5~1.5x; further 0.9~1.1x.

[0111] Preferably, the concentration of B27 in the base medium of the first medium, and / or the second medium is 0.5~1.5x; further 0.9~1.1x.

[0112] Preferably, the concentration of N2 in the base medium of the first medium, and / or the second medium is 0.5~1.5x; further 0.9~1.1x.

[0113] Preferably, the concentration of 2-mercaptoethanol in the base medium of the first medium, and / or the second medium is 0.05~1mM; further 0.08~0.12mM; more further 0.09~0.11mM.

[0114] Preferably, the base medium of the third medium is DMEM medium.

[0115] Preferably, the base medium of the third medium is a base medium containing 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] Preferably, the concentration of serum in the base medium of the third medium is 10~30% by volume percentage; further 15~25%; more further 18~22%.

[0117] Preferably, the concentration of the pyruvate or salt thereof in the basal medium of the third culture medium is 0.5-2x; further 0.7-1.5x; and more further 0.9-1.1x.

[0118] Preferably, the concentration of the glutamine in the basal medium of the third culture medium is 0.5-2x; further 0.7-1.5x; and more further 0.9-1.1x.

[0119] Preferably, the concentration of the non-essential amino acid in the basal medium of the third culture medium is 0.5-2x; further 0.7-1.5x; and more further 0.9-1.1x.

[0120] Preferably, the concentration of the 2-mercaptoethanol in the basal medium of the third culture medium is 0.05-0.15 mM; further 0.08-0.12 mM; and more further 0.09-0.11 mM.

[0121] Preferably, the concentration of the antibiotic in the basal medium of the third culture medium is 0.1-2% by volume percentage; further 0.5-1.7%; and more further 0.9-1.1%.

[0122] Preferably, the basal medium of the fifth culture medium is Advanced DMEM / F12 medium.

[0123] Preferably, the basal medium of the sixth culture medium is DMEM medium (preferably low glucose, pyruvate-containing, glutamine-free, and phenol red-free DMEM medium).

[0124] Preferably, the stem cell is a stem cell having multipotentiality.

[0125] Preferably, the stem cell having multipotentiality 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; further preferably, the stem cell having multipotentiality comprises an embryonic stem cell.

[0126] Preferably, 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] Preferably, the stem cell is derived from a rodent; further derived from a murine; and more further derived from a mouse.

[0128] Preferably, the stem cell is derived from a primate; further derived from a human.

[0129] Preferably, the stem cells are human embryonic stem cells (hESCs) (e.g. H1, H9) and / or human induced pluripotent stem cells (hiPSCs) (e.g. WC50, IMR90).

[0130] Preferably, the human embryonic stem cells are commercialized human embryonic stem cell lines.

[0131] Preferably, the human embryonic stem cells are stem cells isolated or obtained from human embryos that have not undergone in vivo development, within 14 days of 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) to (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 (e.g. lung, pancreas, heart, intestine, liver, kidney, etc.) and / or organ precursor cells (e.g. 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 (e.g. lung, pancreas, heart, intestine, liver, kidney, etc.) and / or organ precursor cells (e.g. lung precursor cells, pancreas precursor cells, heart-related precursor cells, intestine precursor cells, liver precursor cells, kidney precursor cells, etc.).

[0134] Preferably, the stem cells are stem cells of 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] Preferably, the method for inducing stem cells to generate blastocyst-like lineage precursor cells comprises S1 or S2:

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

[0138] S2: culturing 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 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] Preferably, the time for the culturing in S1 is 36-108h; further 42-78h; more further 54-66h.

[0140] Preferably, the time for the first culturing in S2 is 12-36h; further 18-30h; more further 22-26h.

[0141] Preferably, the time for the second culturing in S2 is 24-72h; further 24-48h; more further 32-40h.

[0142] Preferably, the culturing (including the culturing in S1, and the first and second culturing in S2) is adherent culturing; further preferably, the culturing is performed in a gelatin-treated culturing device.

[0143] Preferably, 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] Preferably, the stem cell is the stem cell of the first aspect of the present application.

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

[0146] Preferably, 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 of the third aspect of the present application;

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

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

[0150] Preferably, the time for the third culturing is 24-48h; further 28-44h; more further 32-40h.

[0151] Preferably, the third culturing is a suspension culturing; further preferably, the third culturing is performed using Aggrewell plates; more preferably, the third culturing is performed using Aggrewell plates treated with an anti-adhesion rinse.

[0152] Preferably, the third culturing is performed at 33-40°C, 3-7% CO2; further preferably, the third culturing is performed at 36-38°C, 4-6% CO2.

[0153] Preferably, the stem cell is the stem cell 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 bodies, comprising the step of using the reagent combination or kit of the first aspect of the present application.

[0155] Preferably, the method for inducing stem cells to produce embryoid-like bodies 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 bodies.

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

[0159] Preferably, the fourth culturing is performed for 24-48h; further preferably, the fourth culturing is performed for 28-44h; more preferably, the fourth culturing is performed for 32-40h.

[0160] Preferably, the fourth culturing is a suspension culturing; further preferably, the fourth culturing is performed using Aggrewell plates; more preferably, the fourth culturing is performed using Aggrewell plates treated with an anti-adhesion rinse.

[0161] Preferably, the fourth culturing is performed at 33-40°C, 3-7% CO2; further preferably, the fourth culturing is performed at 36-38°C, 4-6% CO2.

[0162] Preferably, the method for inducing blastocyst-like cells to produce embryoid-like bodies of U2 further comprises the steps of culturing and / or maturing the embryoid-like bodies: sequentially culturing the embryoid-like bodies obtained after the fourth culturing with the fifth medium of the reagent combination or kit of the first aspect of the present application for a fifth time, and with the sixth medium of the reagent combination or kit of the first aspect of the present application for a sixth time, and then culturing the embryoid-like bodies with the seventh medium of the reagent combination or kit of the first aspect of the present application for a seventh time.

[0163] Preferably, the time of the fifth culturing is 12-36h; further 20-28h; more further 22-26h.

[0164] Preferably, the time of the sixth culturing is 16-32h; further 20-28h; more further 22-26h.

[0165] Preferably, the time of the seventh culturing is 16-32h; further 20-28h; more further 22-26h.

[0166] Preferably, the fifth culturing is suspension culturing; further preferably, the fifth culturing is performed by Aggrewell plate; more further preferably, the fifth culturing is performed by Aggrewell plate treated by anti-adhesion rinsing solution.

[0167] Preferably, the sixth culturing is dynamic culturing; further preferably, the sixth culturing is performed by suspension culturing device at 60-150rpm; more further preferably, the sixth culturing is performed by suspension culturing device at 70-90rpm.

[0168] Preferably, the seventh culturing is dynamic culturing; further preferably, the seventh culturing is performed by bioreactor at 30-100rpm; more further preferably, the seventh culturing is performed by bioreactor at 40-60rpm.

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

[0170] Preferably, the stem cell is the stem cell in the first aspect of the present application.

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

[0172] Preferably, the content of GATA6 positive cells in the blastocyst-like lineage precursor cell is 21.9%-36.3%; further 24.6%-34.5%; more further 26.1%-34.5%; again further 28.4-31.9%.

[0173] Preferably, the content of CDX2 positive cells in the blastocyst-like lineage precursor cell is 1.28%-61.2%; further 6.45%-43.5%; more further 15.6%-29.2%; again further 26.8-29.2%.

[0174] Preferably, the content of OCT4 positive cells in the blastocyst-like lineage precursor cells is 59.8% to 90.5%; further 59.8% to 87.6%; and more further 59.8% to 67.9%.

[0175] Preferably, the blastocyst-like lineage precursor cells do not comprise reproductive material.

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

[0177] Preferably, the content of primitive endoderm-like cell population in the blastocyst-like cell is 15.8% to 37.49%; further 18.26% to 32.98%; and more further 18.26% to 22.10%.

[0178] Preferably, the content of trophoblast ectoderm-like cell population in the blastocyst-like cell is 7.26% to 41.99%; further 16.87% to 25.00%; and more further 20.87% to 23.48%.

[0179] Preferably, the content of epiblast-like cell population in the blastocyst-like cell is 42.02% to 59.68%; further 52.20% to 59.68%; and more further 54.41% to 59.68%.

[0180] Preferably, the blastocyst-like cell does not comprise reproductive material.

[0181] In an eighth aspect of the present application, an embryonic-like is provided, obtainable by the method of the fifth aspect of the present application.

[0182] Preferably, the embryonic-like has the ability to reproduce (preferably highly reproduce) post-implantation embryo development and / or primitive gut motility.

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

[0184] a1) preparing an embryo model;

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

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

[0187] a4) studying the effect of a knockout library or overexpression library on embryo (preferably early embryo) development;

[0188] a5) detecting drug safety;

[0189] a6) Preparation of gastrulation phase cells;

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

[0191] a8) Prepare a product, said product being used in any one of a1) to a7).

[0192] Preferably, the product is a reagent kit.

[0193] A tenth aspect of the present invention provides a product comprising: blastocyst-like lineage precursor cells of the sixth aspect of the present invention, blastocyst-like cells of the seventh aspect of the present invention, and / or embryo-like cells of the eighth aspect of the present invention.

[0194] Preferably, the product is used in any one of a1) to a7) of the ninth aspect of the present invention.

[0195] Preferably, the product is a reagent kit.

[0196] The beneficial effects of this invention are:

[0197] This invention provides a reagent combination or kit that can be used to induce stem cells to generate blastocyst-like lineage precursor cells, blastocyst-like cells, and / or embryo-like cells. It is based on small molecule induction, is not dependent on transgenes, and uses a single cell source. The generated blastocyst-like lineage precursor cells and blastocyst-like cells possess a complete blastocyst lineage, are seed cells with balanced developmental capacity and totipotency, and solve the problem of uneven cell maturation caused by existing technologies. The generated embryo-like cells are highly similar to natural embryos in morphological and transcriptomic characteristics, and have a high ability to reproduce post-implantation embryonic development and / or gastrulation, which is beneficial for in vitro studies of embryonic development. The method of inducing stem cells to generate blastocyst-like lineage precursor cells, blastocyst-like cells, and / or embryo-like cells using this reagent combination or kit is simple, highly applicable (any stem cell can be used), and has a high efficiency in constructing embryo-like cells (up to 30%, while existing technologies achieve less than 0.5%). Attached Figure Description

[0198] Figure 1 To demonstrate the developmental capabilities and limitations of existing mouse in vitro embryonic models.

[0199] Figure 2Analysis of the induction of embryonic stem cells to generate blastocyst-like lineage precursor cells by the kits of Examples 1-1~1-4, 2~7 in Examples 11-1~11-4, 12~17: Wherein A is the small molecules involved in the kits of Examples 1-1~1-4, 2~7 and the related abbreviations are as follows: 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-β receptor I (TGFβ-Ri); A (AM580) is a retinoic acid nuclear receptor (RAR) agonist; B is the strategy for generating blastocyst-like lineage precursor cells (designated as Q0-a, Q0-b, Q0-c, Q0-d, respectively) from embryonic stem cells by the kits of Examples 1-1~1-4 in Examples 11-1~11-4; 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 for generating blastocyst-like lineage precursor cells (Q1, Q2, Q3, Q4, Q5, Q6) from embryonic stem cells by the kits of Examples 2~7 in Examples 12~17; E is the immunofluorescence analysis results of blastocyst-like lineage precursor cells (designated as Q1, Q2, Q3, Q4, Q5, Q6, respectively) generated from embryonic stem cells by the kits of Examples 2~7 in Examples 12~17 (GATA6 is a marker protein of primitive endoderm (PrE), and CDX2 is a marker protein of Trophectoderm (TE)).

[0200] Figure 3 Immunofluorescence results of blastocyst-like lineage precursor cells (designated as Q1, Q2, Q3, Q4, Q5, Q6, respectively) generated from embryonic stem cells by the kits of Examples 2~7 in Examples 12~17 (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 μm).

[0201] Figure 4The 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 Examples 14, 15 (the internal reference gene is Actin, and the relative expression level of each gene is calculated using the ΔΔCt method) are shown in A and B, respectively.

[0202] Figure 5 The single-cell transcriptome analysis results of the blastocyst-like lineage precursor cells induced from embryonic stem cells using the kit of Examples 2-7 in Examples 12-17 are shown in A-G, respectively. A is the UMAP analysis plot of the blastocyst-like lineage precursor cells produced in Example 12 and the reference data of mouse early embryos using a nonlinear dimension reduction algorithm; B is the UMAP analysis plot of the blastocyst-like lineage precursor cells produced in Example 13 and the reference data of mouse early embryos using a nonlinear dimension reduction algorithm; C is the UMAP analysis plot of the mixture of the blastocyst-like lineage precursor cells produced in Examples 14, 15 and the reference data of mouse early embryos using a nonlinear dimension reduction algorithm; D is the UMAP analysis plot of the blastocyst-like lineage precursor cells produced in Example 16 and the reference data of mouse early embryos using a nonlinear dimension reduction algorithm; E is the UMAP analysis plot of the blastocyst-like lineage precursor cells produced in Example 17 and the reference data of mouse early embryos using a nonlinear dimension reduction algorithm.

[0203] Figure 6 The single-cell transcriptome analysis results of the blastocyst-like cells produced in Examples 18-1-18-7 are shown in A-G, respectively. A is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-2 and the reference data of mouse early embryos using a nonlinear dimension reduction algorithm; B is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-3 and the reference data of mouse early embryos using a nonlinear dimension reduction algorithm; C is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-4 and the reference data of mouse early embryos using a nonlinear dimension reduction algorithm; D is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-1 and the reference data of mouse early embryos using a nonlinear dimension reduction algorithm; E is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-5 and the reference data of mouse early embryos using a nonlinear dimension reduction algorithm; F is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-6 and the reference data of mouse early embryos using a nonlinear dimension reduction algorithm; G is the UMAP analysis plot of the blastocyst-like cells produced in Example 18-7 and the reference data of mouse early embryos using a nonlinear dimension reduction algorithm.

[0204] Figure 7Single-cell transcriptome analysis results of the blastoid cells obtained in Examples 18-1 to 18-7: A, UMAP analysis plot of the blastoid cells obtained in Examples 18-1 to 18-7 and mouse early embryo reference data by nonlinear dimension reduction algorithm, and single-cell transcriptome expression level of marker genes (GATA6 is a marker gene of primitive endoderm (PrE), and CDX2 is a marker gene of Trophectoderm (TE)); B, cell type proportion of the blastoid cells obtained in Examples 18-1 to 18-7.

[0205] Figure 8 Expression of lineage marker genes and immunofluorescence staining results of the blastoid cells obtained in Example 18-1: A, single-cell transcriptome expression of lineage marker genes of the three cell groups (primitive endoderm-like cell group, Trophectoderm-like cell group and epiblast-like cell group) of the blastoid cells obtained in Example 18-1; B, immunofluorescence staining result plot (KRT18 / PDGFRA / OCT4) of the blastoid cells obtained in Example 18-1; C, immunofluorescence staining result plot (KRT18 / CDX2 / OCT4) of the blastoid cells obtained in Example 18-1, two repeats; D, heat map analysis of the three cell groups of the blastoid cells obtained in Example 18-1 and E4.5 natural embryos.

[0206] Figure 9Results of inducing embryonic stem cells to generate ci-Embryoids using the kit of Example 8-1 in Example 18-1 are shown: A is a schematic diagram of the process of inducing embryonic stem cells to generate ci-Embryoids (scale bar is 50 pm); B is a representative sample of ci-Embryoids (scale bar is 100 pm) generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 on Day 4.5; C is a table comparing the efficiency of ci-Embryoids generated in Example 18-1 with reported EiTiX ci-Embryoids at different stages; D is an immunohistochemical staining image of a representative sample of ci-Embryoids (scale bar is 100 pm) generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 on Day 4.5; E is a result image of immunofluorescence staining of a representative sample of ci-Embryoids (AP2y / SOX2 / OCT4) generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 on Day 4.5; F is a result image of immunofluorescence staining of a representative sample of ci-Embryoids (SOX2 / T / Bry) generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 on Day 4.5 (scale bar is 100 pm); G is a result image of immunofluorescence staining of a representative sample of ci-Embryoids (N-cad / T, N-cad / E-cad, T) generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 on Day 4.5 (scale bar is 40 pm in the upper panel and 10 pm in the lower panel); H is a result image of immunofluorescence staining of a representative sample of ci-Embryoids (FOXA2 / GATA4) generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 on Day 4.5 (scale bar is 100 pm).

[0207] Figure 10 UMAP analysis of single-cell transcriptome samples of ci-Embryoids generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 on Day 4.5 and samples of natural mouse embryos E7.5 in vivo (generated from 10x Genomic scRNA-seq) is shown.

[0208] Figure 11 Single-cell transcriptome expression levels of marker genes of samples of natural mouse embryos E7.5 in vivo are shown.

[0209] Figure 12 Single-cell transcriptome expression levels of marker genes of ci-Embryoids generated from embryonic stem cells using the kit of Example 8-1 in Example 18-1 on Day 4.5 are shown.

[0210] Figure 13 Demonstration of the universality of the kit and / or induction method of the present application (immunofluorescence staining of cells induced by Q3 and Q4 small molecules and constructed into embryoid bodies using two other stem cell lines (stem cells derived from C57BL / 6 mouse inner cell mass and ICR mouse background) other than OG2 mouse embryonic stem cells): A is an immunofluorescence image of blastocyst-like lineage precursor cells generated by induction of stem cells of other cell line 1 (C57BL / 6 mouse origin) (the same method as in Examples 14 and 15, except that the source of stem cells is different) (GATA6, CDX2 and SOX2 are lineage marker proteins of primitive endoderm-like cell population, trophoblast ectoderm-like cell population and epiblast-like cell population, respectively) (scale bar is 50 μm); B is an immunofluorescence image of blastocyst-like lineage precursor cells generated by induction of stem cells of other cell line 2 (ICR mouse origin) (the same method as in Examples 14 and 15, except that the source of stem cells is different) (scale bar is 50 μm); C is an immunofluorescence image of embryoid bodies constructed by induction of other cell line 1 (C57BL / 6 mouse origin) (i.e. the same method as in Examples 18-1 and 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 an immunofluorescence image of embryoid bodies constructed by induction of other cell line 2 (ICR mouse origin) (i.e. the same method as in Examples 18-1 and 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 the examples are only used for illustrating the present application but not for limiting the scope of the present application.

[0213] Glossary:

[0214] In the present application, "blastocyst-like lineage precursor cells" refer to a cell population that has acquired expression of key transcription factors GATA6, CDX2 and OCT4 of 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 specific small molecule combinations. The molecular level of the cell population has not yet fully reached that of the blastocyst stage, but it has the ability to mature into blastocyst-like three lineage cells.

[0215] In this invention, "blastocyst-like cells" refers to the cellular and molecular characteristics of three lineages (primitive endoderm-like cells, trophectoderm-like cells, and epiblast-like cells) of the E4.5 blastocyst stage embryo after the blastocyst-like lineage precursor cells are induced to mature through small molecule culture. They have the ability to develop into all components of an embryo-like embryo.

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

[0217] In this invention, "intermediate cells" refers to a population of precursor cells (such as embryonic stem cells) whose molecular expression characteristics at the cellular level are intermediate between those of the three lineages (primordial endoderm-like cells, trophectoderm-like cells, and epiblast-like cells) during the blastocyst stage, and which have not yet been specific to any particular lineage, after being induced by a specific combination of small molecules.

[0218] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0219] The following examples / effects involve some of the materials and experimental methods:

[0220] 1. Animals

[0221] All animal experiments were conducted in accordance with the "Regulations on the Management of Laboratory Animals" and approved by the Laboratory Animal Management and Use Committee of the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. Animals that appeared unhealthy 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 Beston Biotechnology Co., Ltd. Rats were housed in a temperature-controlled room (22±1℃) with a 12-hour light / dark cycle from 07:00 to 19:00, with free access to water and food. Euthanasia was performed using the CO2 inhalation method, and cervical dislocation was performed to ensure death by CO2 asphyxiation.

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

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

[0224] Mouse embryonic stem cells were seeded on 1% gelatin (STEMCELL, 07903)-treated cell culture well plates and cultured with 2iLIF medium. 2iLIF medium was N2B27 basal medium containing 20 ng / mL murine LIF (qKine, Qk018), 3 mM CHIR99021 (Selleck, S2924) and 1 mM PD0325901 (Selleck, S1036). N2B27 basal medium was a mixed medium containing 1x B27 (Gibco, 17504044), 1x B27 (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). Cell culture medium was refreshed every day and 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 6 to 7 times, and then let stand at room temperature for at least 30 minutes. When obvious stratification is observed, centrifuge at room temperature at 2,000 x g for 15 minutes. 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. Before use, thaw 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 embryoid bodies were fixed with 4% PFA overnight at 4°C. After fixation, 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 primary antibodies diluted with blocking solution were added for overnight incubation at 4°C. The information of primary antibodies: 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 blocking solution were added for 1 hour incubation at room temperature. The corresponding secondary antibodies were purchased from Thermo Fisher Scientific, part numbers: 21202, 31573, 31570, 31572, 31571, 21472, 21447. Finally, DAPI (Sigma, D9542) was added to stain the nucleus, and PBST was used to wash three times for imaging.

[0231] For the embryoid bodies at 3.5 days and above, a transparentization process was performed after staining. The transparentization process was performed by using 25%, 50%, 75%, 95%, and 100% of thioethanol (v / v, diluted in DPBS; Sigma, 166782) for gradient treatment.

[0232] Finally samples were observed and photographed by confocal microscope FV3000 (Olympus) and analyzed by 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, 31572) 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 of total RNA was used for reverse transcription according to the instructions of RevertAid TM FirstStrand cDNA Synthesis Kit (Thermo, K16225). qRT-PCR detection was performed by using TB Premix Ex Taq TM II (Takara, RR820A) kit, and the primers are shown in Table 1. After spotting, the samples were detected by QuantStudio 3 Real-Time PCR System (Applied Biosystems). The relative change fold was calculated by AACt method, and the internal reference gene was Actin.

[0237] Table 1 qPCR primers

[0238]

[0239]

[0240] 7. Single cell suspension preparation and sequencing

[0241] The cells induced in the Examples were digested with Accutase. The ci-embryoids induced in the Examples at 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 using Single Cell 3’ Reagent Kits V3.1 at a concentration of 16,000 single cells per sample. The library was sequenced on an MGISEQ 2000 (MGI Tech) system in PE 100 mode.

[0242] 8. Single cell RNA sequencing data preprocessing

[0243] The reference genome was constructed following the guidelines provided by the 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 according to the reference genome (https: / / support.10xgenomics.com / single-cell-gene-expression / software / pipelines / latest / using / tutorial_mr). The pre-constructed genome sequence and gene annotation file used were downloaded from the Ensembl database of GRCm39 version 104. First, the raw data was quality controlled using fastp (v.0.21.0, https: / / github.com / OpenGene / fastp) with default parameters, and then the data after quality control was aligned, annotated, PCR duplicate removed and gene expression quantified using STAR (v.2.7.9a) following the standard STARsolo analysis process (https: / / github.com / alexdobin / STAR / blob / master / docs / STARsolo.md).

[0244] 9. Public single-cell RNA sequencing data

[0245] Public data such as GSE45719

[10] , GSE84892

[11] , GSE109071

[12] , GSE100597

[13] , GSE123046

[14] and E-MTAB-6967

[15] (mouse embryonic stages from zygote to E7.5, containing all known cell lineages within this stage) were downloaded and pre-processed using the same methods as the data generated in this study, or adjusted post-processing according to the instructions of the authors of the data source articles.

[0246] 10. Downstream analysis of single-cell RNA sequencing data

[0247] Linear dimensionality reduction and principal component analysis (PCA). Further analysis was performed in R 4.2.2 using Seurat software (v.4.3.0). According to 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 more than or equal to 5% mitochondrial gene percentage were filtered out, 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.

[0248] 11. Batch effect correction and integration of multi-source Smart-seq2 and 10x Genomics single-cell RNA sequencing data

[0249] To compare the induced ci-embryoids at Day4.5 in the examples with the public natural embryo datasets, the standard canonical correlation analysis (CCA) method ( Figure 10 ) was used, following the standard procedure provided by the Seurat website (https: / / satijalab.org / seurat / articles / integration_introduction.html). To integrate multiple sources of public data from different cell lineages and developmental stages ( Figure 5 、 6), the inventors used the fastMNN method and treated each dataset from different experiments as one batch, and each batch contained at least one cell population shared with other batches. 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 dataset after log-normalization and batch effect correction was 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.

[0250] 12. Visualization using graph-based clustering and Uniform Manifold Approximation and Projection (UMAP)

[0251] Based on the data after dimensionality reduction and batch effect correction, the inventors used the FindNeighbors function to construct a shared nearest neighbor (SNN) graph, and then used the Louvain algorithm implemented by the FindClusters function to partition it, with the resolution range selected according to the library size and data heterogeneity. Finally, the inventors used the RunUMAP function to visualize the data using UMAP.

[0252] Example 1-1 A kit for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0253] A kit for inducing stem cells to produce blastocyst-like lineage precursor cells, comprising: a first culture medium; the first culture medium is 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);

[0254] The N2B27 base medium contains 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). The mixed medium is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0255] Example 1-2 A kit for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0256] 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 3 mM CHIR99021 (Selleck, S2924), 20 ng / mL murine LIF (qKine, Qk018), 10 mM E616452 (Selleck, S7223), and 0.05 mM AM580 (Selleck, S2933).

[0257] The N2B27 base medium contains 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). The mixed medium is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0258] Example 1-3 A kit for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0259] 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);

[0260] 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.

[0261] Example 1-4 A kit for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0262] 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);

[0263] 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.

[0264] Embodiment 2 A kit for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0265] A kit for inducing stem cells to generate blastocyst-like lineage precursor cells, comprising: a first culture medium and a second culture medium;

[0266] The first culture medium is N2B27 base medium (CL6A) containing 1 μM CHIR99021, 10 ng / mL mouse leukemia inhibitory factor, 1 μM E616452 and 0.01 μM AM580;

[0267] The second culture medium is N2B27 base medium (C6A) containing 1 μM CHIR99021, 1 μM E616452 and 0.01 μM AM580;

[0268] 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), which is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0269] Embodiment 3 A kit for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0270] A kit for inducing stem cells to generate blastocyst-like lineage precursor cells, comprising: a first culture medium and a second culture medium;

[0271] The first culture medium is N2B27 base medium (CL6A) containing 1 μM CHIR99021, 10 ng / mL mouse leukemia inhibitory factor, 1 μM E616452 and 0.01 μM AM580;

[0272] The second culture medium is N2B27 base medium (C6A) containing 1 μM CHIR99021, 1 μM E616452 and 0.01 μM AM580;

[0273] 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.

[0274] Example 4 A kit for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0275] A kit for inducing stem cells to generate blastocyst-like lineage precursor cells, comprising: a first medium and a second medium;

[0276] 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;

[0277] The second medium is N2B27 base medium (C6A) containing 6 μM CHIR99021, 10 μM E616452 and 0.05 μM AM580;

[0278] 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.

[0279] Example 5 A kit for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0280] A kit for inducing stem cells to generate blastocyst-like lineage precursor cells, comprising: a first medium and a second medium;

[0281] The first culture medium is an N2B27 basic culture medium (CL6A) containing 3 μM CHIR99021, 20 ng / mL mouse leukemia inhibitory factor, 10 μM E616452, and 0.05 μM AM580;

[0282] The second culture medium is an N2B27 basic culture medium (C6A) containing 9 μM CHIR99021, 10 μM E616452, and 0.05 μM AM580;

[0283] 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 is obtained by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0284] Example 6 A kit for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0285] A kit for inducing stem cells to generate blastocyst-like lineage precursor cells, comprising: a first culture medium and a second culture medium;

[0286] The first culture medium is an N2B27 basic culture medium (CL6A) containing 9 μM CHIR99021, 50 ng / mL mouse leukemia inhibitory factor, 12 μM E616452, and 0.08 μM AM580;

[0287] The second culture medium is an N2B27 basic culture medium (C6A) containing 9 μM CHIR99021, 12 μM E616452, and 0.08 μM AM580;

[0288] The 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.

[0289] Example 7 A kit for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0290] A kit for inducing stem cells to generate blastocyst-like lineage precursor cells, comprising: a first medium and a second medium;

[0291] The first medium is an N2B27 base medium (CL6A) containing 15 μM CHIR99021, 100 ng / mL mouse leukemia inhibitory factor, 15 μM E616452, and 0.1 μM AM580;

[0292] The second medium is an N2B27 base medium (C6A) containing 15 μM CHIR99021, 15 μM E616452, and 0.1 μM AM580;

[0293] The 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.

[0294] Example 8-1 A kit for constructing a blastocyst-like embryo

[0295] A kit for constructing a blastocyst-like embryo, comprising: a first medium, a second medium, a third medium, a fourth medium, a fifth medium, and a sixth medium;

[0296] 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;

[0297] The third medium is 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 mg / 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;

[0298] The fourth medium is FC base medium;

[0299] The fourth medium is used for inducing blastocyst-like cells to generate embryoid-like;

[0300] The fifth medium is Advanced DMEM / F12 medium (Gibco, 12634028) [4, 13] (IVC1 medium) 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 mM N-acetyl-L-cysteine (Sigma, C7880), 1 mg / mL D-glucose (sigma, G8644), and 1% (v / v) penicillin / streptomycin (Gibco, 15140122), which is used for inducing blastocyst-like cells to generate embryoid-like;

[0301] The sixth culture medium is a DMEM medium (low glucose, pyruvate, no glutamine, no phenol red, Gibco, 11880) [4, 13] containing 50% (v / v) rat serum (made in house), 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);

[0302] The fifth culture medium, and the sixth culture medium are used for culture and / or maturation of the parablastula;

[0303] The FC base medium is a DMEM medium (Gibco, 41966052) [4] 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).

[0304] Example 8-2 A kit for constructing a parablastula

[0305] A kit for constructing a parablastula, which is the same as the kit for constructing a parablastula of Example 8-1, except that the first culture medium and the second culture medium are the first culture medium and the second culture medium of Example 2.

[0306] Example 8-3 A kit for constructing a parablastula

[0307] A kit for constructing a class embryo, which is the same as the kit for constructing a class embryo of Example 8-1, except that the first medium and the second medium are the first medium and the second medium in Example 3.

[0308] Example 8-4 A kit for constructing a class embryo

[0309] A kit for constructing a class embryo, which is the same as the kit for constructing a class embryo of Example 8-1, except that the first medium and the second medium are the first medium and the second medium in Example 4.

[0310] Example 8-5 A kit for constructing a class embryo

[0311] A kit for constructing a class embryo, which is the same as the kit for constructing a class embryo of Example 8-1, except that the first medium and the second medium are the first medium and the second medium in Example 6.

[0312] Example 8-6 A kit for constructing a class embryo

[0313] A kit for constructing a class embryo, which is the same as the kit for constructing a class embryo of Example 8-1, except that the first medium and the second medium are the first medium and the second medium in Example 7.

[0314] Example 9 A kit for constructing a class embryo

[0315] A kit for constructing a class embryo, comprising: a first medium, a second medium, a third medium, a fourth medium, a fifth medium, and a sixth medium;

[0316] wherein the first medium and the second medium are the first medium and the second medium in Example 2, which are used for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells;

[0317] 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 mg / 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 to induce blastocyst-like lineage precursor cells to generate blastocyst-like cells;

[0318] The fourth medium is FC basal medium;

[0319] The fourth medium is used to induce blastocyst-like cells to generate embryoid-like;

[0320] The fifth medium is Advanced DMEM / F12 medium (Gibco, 12634028) containing 10% (v / v) FBS (Carpicorn scientific, FBS52A), 0.5 mM GlutaMax (Gibco, 35050038), 0.5x insulin-transferrin-selenium-aminomethanol (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 mM N-acetyl-L-cysteine (Sigma, C7880), 0.5 mg / mL D-glucose (sigma, G8644), and 0.1% (v / v) penicillin / streptomycin (Gibco, 15140122) (IVC1 medium);

[0321] The sixth culture medium was DMEM containing 25% (v / v) rat serum (made in house), 10% (v / v) human serum AB (Off the Clot, Gemini, 100-318), 0.5×GlutaMax (Gibco, 35050038), 0.5×sodium pyruvate (Gibco, 11360039), 6mM HEPES (Gibco, 15630130), 1mg / mL D-glucose (Sigma, G8644), and 0.1% (v / v) penicillin / streptomycin (Gibco, 15140122). This medium was low in glucose, contained pyruvate, and was glutamine-free and phenol-free. red), Gibco, 11880 (IVC2 medium);

[0322] The fifth and sixth culture media are used for the culture and / or maturation of embryo-like organisms;

[0323] The FC basal medium was DMEM medium (Gibco, 41966052) containing 20% ​​(v / v) FBS (Carpicorn Scientific, FBS-52A), 1× sodium pyruvate (Gibco, 11360039), 1× GlutaMAX (Gibco, 35050061), 1× MEM non-essential amino acids (Gibco, 11140050), 0.1 mM 2-mercaptoethanol (Gibco, 21985023) and 1% (v / v) penicillin / streptomycin (Gibco, 15140122) [4].

[0324] Example 10: A kit for constructing embryo-like structures

[0325] A kit for constructing embryo-like structures, comprising: a first culture medium, a second culture medium, a third culture medium, a fourth culture medium, a fifth culture medium, and a sixth culture medium;

[0326] The first culture medium and the second culture medium are the first culture medium and the second culture medium in Example 7, which are used to induce mouse embryonic stem cells to produce blastocyst-like lineage precursor cells.

[0327] The third medium is FC basal 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 mg / 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;

[0328] The fourth medium is FC basal medium;

[0329] The fourth medium is used to induce blastocyst-like cells to generate embryoid-like;

[0330] The fifth medium is Advanced DMEM / F12 medium (Gibco, 12634028) (IVC1 medium) containing 40 (v / v) FBS (Carpicorn scientific, FBS52A), 2x GlutaMax (Gibco, 35050038), 2x Insulin-transferrin-selenium-aminethanol (ITS-X, Gibco, 51500056), 200 nM T3 (3,3',5-Triiodo-L-thyronine sodium salt, sigma, T6397), 11 nM beta-estradiol (Sigma, E8875), 300 ng / mL progesterone (Sigma, P0130), 45 mM N-acetyl-L-cysteine (Sigma, C7880), 2 mg / mL D-glucose (sigma, G8644), and 2% (v / v) penicillin / streptomycin (Gibco, 15140122);

[0331] The sixth culture medium is a DMEM medium (low glucose, pyruvate, no glutamine, no phenol red, Gibco, 11880) containing 60% (v / v) rat serum (made in house), 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) (IVC2 medium);

[0332] The fifth culture medium, and the sixth culture medium are used for culture and / or maturation of blastoids;

[0333] The FC base medium is a 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].

[0334] Example 11-1 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0335] 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, specifically as follows:

[0336] At 2x10 5OG2 mouse embryonic stem cells (mESCs) were seeded at a density of cells / well in 6-well plates (Greiner, 657160) treated with 0.1% (w / v) gelatin (STEMCELL, 07903), and 2 mL of the first culture medium from Example 1-1 was added. The plates were then incubated at 37°C and 5% CO2 for 108 h.

[0337] Example 11-2 A method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0338] A method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as the method in Examples 11-1, except that the kit in Examples 1-2 is used and the culture time is 60 hours.

[0339] Example 11-3 A method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0340] A method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as the method in Examples 11-1, except that the kits in Examples 1-3 are used and the culture time is 60 hours.

[0341] Example 11-4 A method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0342] A method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as the method in Examples 11-1, except that the kits in Examples 1-4 are used and the culture time is 36 hours.

[0343] Example 12: A method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0344] A method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells, comprising the steps of using the kit of Example 2, as detailed below:

[0345] With 2×10 5 OG2 mouse embryonic stem cells (mESCs) were seeded at a density of 0.1% (w / v) gelatin (STEMCELL, 07903) in 6-well plates (Greiner, 657160). 2 mL of the second culture medium from Example 2 was added, and the plates were cultured at 37°C and 5% CO2 for 36 h. Then, 2 mL of the first culture medium from Example 2 was added, and the plates were cultured at 37°C and 5% CO2 for 72 h.

[0346] Example 13 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0347] 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 with the kit of Example 3, the incubation time of the second medium is 30 h, and the incubation time of the first medium is 48 h.

[0348] Example 14 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0349] 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 with the kit of Example 4, the incubation time of the second medium is 24 h, and the incubation time of the first medium is 36 h.

[0350] Example 15 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0351] 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 with the kit of Example 5, the incubation time of the second medium is 24 h, and the incubation time of the first medium is 36 h.

[0352] Example 16 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0353] 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 with the kit of Example 6, the incubation time of the second medium is 18 h, and the incubation time of the first medium is 24 h.

[0354] Example 17 A method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0355] 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 with the kit of Example 7, the incubation time of the second medium is 12 h, and the incubation time of the first medium is 24 h.

[0356] Example 18-1 A method of constructing a blastoid

[0357] A method of constructing a blastoid, comprising the steps of using the kit of Example 8-1, as follows:

[0358] (1) Inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells: the method of inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells is the same as that of Example 15;

[0359] (2) Inducing blastocyst-like lineage precursor cells to generate blastocyst-like cells (ciBPCs): the cells obtained in step (1) were digested using Accutase (Invitrogen, 00455556), and after centrifugation, the cells were 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) was prepared; the cells were inoculated into the Aggrewell400 at a density of 3x10 4 cells / well, 1.5 mL of the third medium in Example 8-1 was added, and the cells were cultured at 37°C, 5% CO2 for 36 h;

[0360] (3) Inducing blastocyst-like cells ciBPCs to generate ci-Embryoids: the third medium was removed (referred to as Day0 of ci-Embryoid induction and culture), and after being washed twice with the fourth medium, 1 mL of the fourth medium in Example 8-1 was added, and the cells were cultured at 37°C, 5% CO2 for 1.5 days (36 h) (the end of the culture was Day1.5); the fourth medium was removed, 1.5 mL of the fifth medium in Example 8-1 was added, and the cells were cultured at 37°C, 5% CO2 for 1 day (24 h) (the end of the culture was Day2.5); the ci-Embryoids in the Aggrewell400 were gently resuspended and transferred to a suspension culture six-well plate (Greiner, 657185), 4 mL of the fifth medium was added, and the cells were cultured on a shaker (ZCLY180N, shaker setting parameters: 80 rpm, 37°C, 5% CO2) for another day (24 h) (the end of the culture was Day3.5); the ci-Embryoids were transferred to a 5 mL bioreactor (bioreactor, ABLEBiott, BWV-S005A), 3 mL of the sixth medium in Example 8-1 was added, and the cells were cultured at 37°C, 5% CO2 for another day (24 h) on the base plate of the bioreactor magnetic stirring system (6-ch) (Bioreactor Magnetic Stir System-6ch.Base plate, ABLE Biott, BWS-S03N0S-6B) (the end of the culture was Day4.5), and the system was set to rotate at 50 rpm.

[0361] Example 18-2 A method for constructing ci-Embryoids

[0362] A method for constructing a synthetic embryo, 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 with the kit of Example 8-2, the method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells is the same as the method of Example 12, and the culture time of the third medium is 24 h.

[0363] Example 18-3 A method for constructing a synthetic embryo

[0364] A method for constructing a synthetic embryo, 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 with the kit of Example 8-3, the method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells is the same as the method of Example 13, and the culture time of the third medium is 32 h.

[0365] Example 18-4 A method for constructing a synthetic embryo

[0366] A method for 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 with the kit of Example 8-4, the method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells is the same as the method of Example 14, and the culture time of the third medium is 36 h.

[0367] Example 18-5 A method for constructing a synthetic embryo

[0368] A method for 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 with the kit of Example 8-4, the method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells is the same as the method of Example 14, and the culture time of the third medium is 36 h.

[0369] Example 18-6 A method for constructing a synthetic embryo

[0370] A method for 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 with the kit of Example 8-5, the method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells is the same as the method of Example 16, and the culture time of the third medium is 40 h.

[0371] Example 18-7 A method for constructing a synthetic embryo

[0372] A method for constructing a blastoid 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 with the kit of Example 8-6, and the method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as that of Example 17, and the culture time of the third medium is 48 h.

[0373] Example 19 A method for constructing a blastoid embryo

[0374] A method for constructing a blastoid 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 with the kit of Example 9.

[0375] Example 20 A method for constructing a blastoid embryo

[0376] A method for constructing a blastoid 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 with the kit of Example 10.

[0377] Comparative Example 1 A method for culturing mouse embryonic stem cells

[0378] A method for culturing mouse embryonic stem cells, which is the same as Example 11-1, except that the first culture medium of Example 1-1 is replaced with 2iLIF culture medium.

[0379] Effects of Examples

[0380] 1. Cell flow analysis was performed on the blastocyst-like lineage precursor cells (designated Q0-a, Q0-b, Q0-c, Q0-d, Q1, Q2, Q3, Q4, Q5, Q6, in order) obtained in Examples 11-1 to 11-4, 12 to 17, and mouse embryonic stem cells (ESCs) obtained in Comparative Example 1, and the results are shown in Table 1. Figure 2Shown: in mouse embryonic stem cells (ESCs) obtained from Comparative Example 1 : 0.52% GATA6 positive cells, i.e. PrE lineage precursor cells, 0.088% CDX2 positive cells, i.e. TE lineage precursor cells, 98.9% OCT4 positive cells, i.e. EPI-like cells; in cells obtained from Example 11-1 (Q0-a): 35.3% GATA6 positive cells, i.e. PrE lineage precursor cells, 5.11% CDX2 positive cells, i.e. TE lineage precursor cells, 68.2% OCT4 positive cells, i.e. EPI-like cells; in cells obtained from Example 11-2 (Q0-b): 36.3% GATA6 positive cells, i.e. PrE lineage precursor cells, 1.28% CDX2 positive cells, i.e. TE lineage precursor cells, 90.5% OCT4 positive cells, i.e. EPI-like cells; in cells obtained from Example 11-3 (Q0-c): 22.8% GATA6 positive cells, i.e. PrE lineage precursor cells, 56.3% CDX2 positive cells, i.e. TE lineage precursor cells, 61.2% OCT4 positive cells, i.e. EPI-like cells; in cells obtained from Example 11-4 (Q0-d): 21.9% GATA6 positive cells, i.e. PrE lineage precursor cells, 61.2% CDX2 positive cells, i.e. TE lineage precursor cells, 60.6% OCT4 positive cells, i.e. EPI-like cells Figure 2Example 12 (Q1) : 34.3% of GATA6 positive cells, i.e. PrE lineage precursor cells, 6.45% of CDX2 positive cells, i.e. TE lineage precursor cells, 67.5% of OCT4 positive cells, i.e. EPI-like cells; Example 13 (Q2) : 34.5% of GATA6 positive cells, i.e. PrE lineage precursor cells, 15.6% of CDX2 positive cells, i.e. TE lineage precursor cells, 87.6% of OCT4 positive cells, i.e. EPI-like cells; Example 14 (Q3) : 31.9% of GATA6 positive cells, i.e. PrE lineage precursor cells, 26.8% of CDX2 positive cells, i.e. TE lineage precursor cells, 67.9% of OCT4 positive cells, i.e. EPI-like cells; Example 15 (Q4) : 26.1% of GATA6 positive cells, i.e. PrE lineage precursor cells, 29.2% of CDX2 positive cells, i.e. TE lineage precursor cells, 67.1% of OCT4 positive cells, i.e. EPI-like cells; Example 16 (Q5) : 28.4% of GATA6 positive cells, i.e. PrE lineage precursor cells, 26.8% of CDX2 positive cells, i.e. TE lineage precursor cells, 59.8% of OCT4 positive cells, i.e. EPI-like cells; Example 17 (Q6) : 24.6% of GATA6 positive cells, i.e. PrE lineage precursor cells, 43.5% of CDX2 positive cells, i.e. TE lineage precursor cells, 64.7% of OCT4 positive cells, i.e. EPI-like cells Figure 2 Examples 1-1 to 1-4, 2-7, and the same can be seen in the analysis of the distribution of GATA6 positive cells and CDX2 positive cells in the total cell population, Q0-a, Q0-b, Q0-c, Q0-d, Q1, Q2, Q3, Q4, Q5, Q6 all have high GATA6 activation, while the activation of CDX2 is less in Q0-a, Q0-b, Q1, too much in Q0-c, Q0-d, Q6, and similar distribution of CDX2 and GATA6 positive cells in Q3, Q4, Q5 Figure 2 Examples 1-1 to 1-4, 2-7, and the same can be seen in the analysis of the distribution of GATA6 positive cells and CDX2 positive cells in the total cell population, Q0-a, Q0-b, Q0-c, Q0-d, Q1, Q2, Q3, Q4, Q5, Q6 all have high GATA6 activation, while the activation of CDX2 is less in Q0-a, Q0-b, Q1, too much in Q0-c, Q0-d, Q6, and similar distribution of CDX2 and GATA6 positive cells in Q3, Q4, Q5

[0381] 2. Immunofluorescence analysis was performed on the blastocyst-like lineage precursor cells (denoted as Q1, Q2, Q3, Q4, Q5, Q6) obtained from Examples 12-17 and mouse embryonic stem cells (ESC) obtained from Comparative Example 1, and the results are shown in Figure 2. 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, the Q3, Q4, and Q5 cells exhibited a relatively balanced activation ratio of GATA6 and CDX2 in both the immunofluorescence analysis and the immunofluorescence analysis, and the cell morphology was relatively healthy. Figure 3 Figure 2 Figure 3 It can be seen that the blastocyst-like lineage precursor cells obtained from Examples 14-16 have balanced three blastocyst-like lineage cells. Figure 2 Figure 3 RNA extraction and qPCR detection were performed on 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); 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 over time; in addition, the primitive state marker genes Nanog and Sox2 were down-regulated, and the primordial state genes Otx2 and Fgf5 did not increase, which indicated that the cells were not developing in the differentiation direction, but were stimulating the trophoblast ectoderm lineage and primitive endoderm. Figure 4

[0382] 3. The blastocyst-like lineage precursor cells obtained from Examples 12-17 (corresponding to Q1 (108 h), Q2 (78 h), Q3 (60 h), Q4 (60 h), Q5 (42 h), and Q6 (36 h), respectively), Comparative Example 1 (Q3 & Q4 (60 h) represents a mixture of the blastocyst-like lineage precursor cells obtained from Example 14 (Q3 (60 h)) and Example 15 (Q4 (60 h))), and Examples 18-1-18-7 (corresponding to Figure 5 Figure 5 Figure 6 7 ​​​​​​​Single-cell transcriptome analysis (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)) was performed on the blastocyst-like cells obtained in Example 18-1 step (1) (i.e., the blastocyst-like lineage precursor cells obtained in Example 15), step (2) (i.e., the blastocyst-like cells obtained in Example 18-1 step (1)), RNA extraction and qPCR detection, and immunofluorescence experiments were performed on the blastocyst-like cells obtained in Example 18-1 step (1) (i.e., the blastocyst-like cells obtained in Example 18-1 step (1)), and the results are shown in FIGS. 18-1 to 18-3. Figures 5-8 As shown in FIG. 18-1, the blastocyst-like precursor cells obtained by induction of small molecules Q1 (108h), Q2 (78h), Q3 (60h), Q4 (60h), Q5 (42h), and Q6 (36h) can be seen in four main clusters: epiblast-like precursor cells, primitive endoderm-like precursor cells, trophoectoderm-like precursor cells, and intermediate cells, in comparison with early embryonic reference data. Figure 5 Among them, the epiblast-like precursor cells are located between the E3.5 inner cell mass and the 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 the E4.5 primitive endoderm cells and the 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 the epiblast and the trophoectoderm cells, and are trophoectoderm precursor cells; and the intermediate cells have the characteristics of two or three lineage precursors, and are intermediate cells. Figure 5 After the blastocyst-like lineage precursor cells were cultured by ciBP (Q4-ciBP (36h), Q1-ciBP (24h), Q2-ciBP (32h), Q3-ciBP (36h), Q3-ciBP (48h), Q5-ciBP (40h), Q6-ciBP (48h)), three lineage cells with lineage specialization were obtained: epiblast-like cells, primitive endoderm-like cells, and trophoectoderm-like cells, which were clustered with E4.5 EPI, E4.5 PrE, and E4.5 TE, respectively. Figure 6 Gene dimensionality reduction analysis chart, after the blastocyst-like precursor cells were specialized by ciBP, significant clustering was shown and different proportions of three blastocyst lineage cells were obtained: epiblast-like cells, primitive endoderm-like cells, and trophoectoderm-like cells. Figure 7). It can be seen that the cells obtained by further induction with the third medium have similar properties to the three lineage cells of the natural embryo E4.5 blastocyst, and the obtained blastocyst-like cells have the characteristics of cells of the three lineages of epiblast, primitive endoderm and trophoblast ectoderm (analyzed at the level of single cell transcriptome, protein and RNA). The results of the blastocyst-like lineage precursor cells obtained in step (1) and the blastocyst-like cells obtained in step (2) in Examples 19 and 20 are similar to those in Examples 12-17 and 18-1 to 18-7.

[0383] The blastocyst-like precursor cells of Q4 (60h) (Example 15) and the blastocyst-like cells of Q4-ciBP (36h) (Example 18-1) were subjected to single cell level transcriptome analysis comparison, 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 ectoderm marker genes (Cited, Rhox6, Rhox9, Id2, Krt8, Krt18) were detected. After matured by the ciBP third medium, the blastocyst-like cells of Q4-ciBP (36h) significantly expressed higher levels of marker genes than the blastocyst-like precursor cells of Q4 (60h); it can be seen that ciBP has the function of maturing and specializing the blastocyst-like precursor cells Figure 8 A). Immunofluorescence staining also showed the expression of blastocyst-like lineage-specific proteins (KRT18 / PDGFRa / OCT4) Figure 8 B). In addition, the trophoblast cells in the blastocyst stage have heterogeneity, and there are polar TE cells and mural TE cells; in the trophoblast-like cells in the present application, the markers of the two groups of cells (CDX2 and KRT18) were also found Figure 8 B and C). The heat map analysis of the three cell groups of the blastocyst-like precursor cells induced by small molecules for 96 hours and the E4.5 natural embryo showed that they have very similar molecular characteristics Figure 8 D). It can be seen that the blastocyst-like lineage cells have similar cell characteristics to the E4.5 blastocyst lineage cells at the levels of single cell transcriptome, protein and RNA.

[0384] 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 (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) were performed in sequence). 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 Figure 9 in vitro reconstitution embryo system (A). 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 on the 2.5th day, a layer of cells formed on the outside of the embryo, and cells derived from the primitive endoderm (GATA6 marker) wrapped the TE (CDX2 marker) and EPI cells (OCT4 marker) of the ci-Embryoids; on the 3.5th day, the ci-Embryoids aggregate morphology was similar to that of natural embryos at E5.5-6.5, showing a clear cup structure; on the 4.5th day, the embryo continued to develop, forming an expanded amniotic cavity, and successfully forming an asymmetric structure, and primitive streak tissue appeared (primitive streak marker T expressed on one side, AVE marker DKK1) Figure 9 in vitro reconstitution embryo system (A). In the 4.5th day ci-Embryoids, all embryo characteristics were observed, which were similar to the primitive gut movement period of natural embryos Figure 9 in vitro reconstitution embryo system (B), which was much higher than the efficiency of less than 0.5% in previous work [4], reaching an efficiency of 31.6% Figure 9(C). Immunohistochemistry was performed on representative embryos, and the results showed that the embryos possessed characteristics of the gastrula stage, including the placental cone (EPC), extra-embryonic ectoderm (EXE), chorion (Ch), amnion (Am), primitive streak (DE), epiblast, extraembryonic endoderm, extraembryonic ectoderm, mesoderm, primitive streak (PS), and visceral endoderm. Figure 9 (D); it not only possesses the original streak and epithelial-mesenchymal transition (T / E-Cad / N-Cad), forming endoderm, ectoderm, and mesoderm (FOXA2 / GATA4 / OCT4); but also has placental cones and extraembryonic ectoderm and mesoderm capable of implantation (AP2γ / OCT4 / Sox2). This in vitro synthetic embryo system is feasible. Figure 9 (EH). It can be seen that ci-BPCs, induced to produce ci-Embryoids through the fourth, fifth, and sixth culture media, and the simultaneous culturing and / or maturation of these ci-Embryoids, efficiently and with high fidelity simulated the development of the embryo around and after implantation. Figure 9 (A), and constructed a complete embryonic model of the late gastrulation stage (A). Figure 9 (A and B): Not only do they possess the unique characteristics of gastrula, such as the development of endoderm, mesoderm, and ectoderm, the formation of primitive streaks, and the epithelioid-mesenchymal transition, but they also simultaneously possess the characteristics of trophectoderm development into extraembryonic ectoderm and placental cones (…). Figure 10 (DH).

[0385] Single-cell sequencing further confirmed that the ci-Embryoids constructed from the ciBPC obtained in this invention (Example 18-1) are highly similar to natural embryos at E7.5 in vivo, possessing different lineages developed at this stage: ectoderm, intestine, extraembryonic mesoderm, epidermal ectoderm, caudal ectoderm, primary mesoderm, amniotic villus mesoderm, embryonic endoderm, mixed mesoderm, blood and endothelial progenitor cells, protostrum, extraembryonic endoderm, apical wall endoderm, extraembryonic ectoderm, protostrum and adjacent ectoderm, caudal neuroectoderm, protostrum, visceral mesoderm, paraaxial mesoderm A / B, notochord, qualitative endoderm, etc. Figures 10-12 Furthermore, the ci-Embryoids constructed by ciBPC have the correct expression patterns. Figures 11-12Not only do the mesoderm (Mesp1, Snail, T, Mixl1), endoderm (Dkk1 / Cer1 / Foxa2), and ectoderm (Noto / Nkx1-2 / Pax6 / Hoxa1) develop correctly, but they also simultaneously develop into extraembryonic ectoderm and placental cones (Tfap2c, Sox2, Id2, etc.). Figure 13 The results of Day 4.5 ci-Embryoids obtained in step (3) of Examples 18-2 to 18-7, 19, and 20 are similar to those of Example 18-1.

[0386] The kits and / or induction methods in this invention are universally applicable. Using embryonic stem cells from other sources (stem cell line 1: C57BL / 6; stem cell line 2: ICR), blastocyst-like precursor cells are obtained through stem cell culture induction in Q3 and Q4 (i.e., the method is the same as in Examples 14 and 15, the only difference being the source of the stem cells). These cells also exhibit good levels of activation of the trophoblastic ectoderm marker protein CDX2 and the primitive endoderm marker protein GATA6. Figure 13 (A and B). Furthermore, after being cultured with ciBP to specialize into blastocyst-like cells, they underwent 3.5 days of embryonic-like culture (i.e., the method was the same as in Examples 18-1 and 18-4, the only difference being the source of stem cells). The embryonic-like cells showed a gradual development from 1.5 days to 2.5 days to 3.5 days, simulating important events such as implantation, ovum column formation, and early formation of the primitive streak. ​ (C and D). As can be seen, different stem cell lines all exhibit good induction ability and the ability to form embryo-like structures, demonstrating the universality of this invention.

[0387] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0388] References

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Claims

1. A reagent combination or kit comprising a first culture medium; The first culture medium is a basal culture medium containing GSK-3 inhibitors, STAT3 activators, retinoic acid nuclear receptor agonists, and TGF-β receptor kinase inhibitors.

2. The reagent combination or kit according to claim 1, characterized in that: The reagent combination or kit also includes a second culture medium; The second culture medium is a basal medium containing GSK-3 inhibitors, retinoic acid nuclear receptor agonists, and TGF-β receptor kinase inhibitors; Preferably, the GSK-3 inhibitors in the first and second culture media are each independently selected from at least one of GSK-3α inhibitors and GSK-3β inhibitors; more preferably, the GSK-3 inhibitors in the first and second culture media are 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, and 1-azacamparone. Preferably, the STAT3 activator in the first culture medium contains at least one of leukemia inhibitory factor and interleukin-6; Preferably, the retinoic acid nuclear receptor agonist in the first culture medium and the second culture medium is 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 inhibitors in the first culture medium and the second culture medium are 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, and BIO-013077-01.

3. The reagent combination or kit according to any one of claims 1 to 2, characterized in that: The reagent combination or kit also includes a third culture medium; The third culture medium is a basal culture medium containing fibroblast growth factor, BMP4 signaling pathway activator, TGF-β activator, WNT signaling pathway inhibitor, Lats kinase inhibitor, anticoagulant, ascorbic acid or its derivatives, and insulin-transferrin-selenium additive. Preferably, the fibroblast growth factor (FGF) is at least one of FGF1 to FGF23; Preferably, the BMP4 signaling pathway activator in the third culture medium comprises at least one of BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoliquiritin, geraniol, apigenin, and chickpea sprout extract. Preferably, the TGF-β activator in the third culture medium contains at least one of TGF-β and activator A; Preferably, the WNT signaling pathway inhibitor in the third culture 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, pyrantel pamoate, CCT031374, iCRT-3, iCRT-5, iCRT-14, CPG049090, and NC043; Preferably, the Lats kinase inhibitor in the third culture medium comprises at least one of TRULI, GA-017, and TDI-011536; Preferably, the anticoagulant in the third culture medium contains at least one of heparin and EDTA salt; Preferably, the ascorbic acid or its derivative in the third culture medium comprises at least one of ascorbic acid, calcium ascorbate, magnesium ascorbate, zinc ascorbate, potassium ascorbate, sodium ascorbate, dehydroascorbic acid, L-threonic acid, L-xylitolic acid, L-lythreonic acid, L-ascorbic acid monostearate, L-ascorbic acid dipalmitoate, L-ascorbic acid 6-hexadecanoic acid compound, L-ascorbic acid 2-phosphoside, L-ascorbic acid 3-phosphoside, and L-ascorbic acid 2-sulfate.

4. The reagent combination or kit according to any one of claims 1 to 3, characterized in that: The reagent combination or kit also includes a fourth culture medium; The fourth culture medium is the base culture medium of the third culture medium; Preferably, the reagent combination or kit further comprises a fifth culture medium and a sixth culture medium; The fifth culture medium is a basal culture medium containing serum, glutamine, insulin-transferrin-selenoethanol, thyroid hormone receptor agonist, estradiol, progesterone, acetylcysteine, glucose and antibiotics. The sixth culture medium is a basal culture medium containing serum, glutamine, pyruvate or its salts, buffer salts, glucose and antibiotics; Preferably, the thyroid hormone receptor agonist in the fifth culture medium comprises at least one of thyroxine or a salt thereof, triiodothyronine or a salt thereof.

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

6. The use of the reagent combination or kit according to any one of claims 1 to 5 in any one of (1) to (10); (1) Preparation of blastocyst-like lineage precursor cells; (2) Preparation of blastocyst-like cells; (3) Preparation of embryo-like cells; (4) Preparation of products that induce stem cells to produce blastocyst-like lineage precursor cells; (5) Preparation of products that induce stem cells to produce blastocyst-like cells; (6) Preparation of products that induce stem cells to produce embryo-like cells; (7) Preparation of embryo-like cells in the gastrulation stage; (8) Preparation of products that induce stem cells to produce embryo-like cells in the gastrulation stage; (9) Preparation of tissue and / or organ precursor cells; (10) Preparation of products that induce stem cells to produce tissue and / or organ precursor cells; Preferably, the stem cells are stem cells with multi-directional differentiation potential; Preferably, the stem cells with multipotent differentiation potential include at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells.

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

8. The method according to claim 7, characterized in that: The method for inducing stem cells to generate blastocyst-like lineage precursor cells includes S1 or S2: S1: Culture stem cells using the first culture medium in the reagent combination or kit according to any one of claims 1 to 5; S2: The stem cells are cultured for the first time using the second culture medium in the reagent combination or kit according to any one of claims 2 to 5, and then cultured for the second time using the first culture medium in the reagent combination or kit according to any one of claims 1 to 5. Preferably, the culture time in S1 is 36–108 h; Preferably, the first culture time in S2 is 12-36 hours; Preferably, the second culture time in S2 is 24–72 hours; Preferably, the culture in S1, the first culture in S2, and / or the second culture in S2 are adherent cultures.

9. A method for inducing stem cells to produce blastocyst-like cells, comprising the step of using the reagent combination or kit according to any one of claims 1 to 5; Preferably, the method for inducing stem cells to generate blastocyst-like cells includes the following steps: 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 described in claim 7 or 8; T2: Induces blastocyst-like lineage precursor cells to produce blastocyst-like cells.

10. The method according to claim 9, characterized in that: The method for inducing blastocyst-like lineage precursor cells to produce blastocyst-like cells as described in T2 includes the following steps: culturing the blastocyst-like lineage precursor cells for the third time using the third culture medium in the reagent combination or kit according to any one of claims 3 to 5. Preferably, the third culture time is 24-48 hours; Preferably, the third culture is a suspension culture.

11. A method for inducing stem cells to produce embryo-like structures, comprising the step of using the reagent combination or kit described in any one of claims 1 to 5; Preferably, the method for inducing stem cells to generate embryo-like structures includes the following steps: U1: Inducing stem cells to produce blastocyst-like cells: The method for inducing stem cells to produce blastocyst-like cells is the method described in claim 9 or 10; U2: Induces blastocyst-like cells to produce embryo-like cells.

12. The method according to claim 11, characterized in that: The method for inducing blastocyst-like cells to produce embryo-like cells as described in U2 includes the following steps: culturing the blastocyst-like cells for the fourth time using the fourth culture medium in the reagent combination or kit according to any one of claims 4 to 5; Preferably, the fourth culture time is 24–48 hours; Preferably, the fourth culture is a suspension culture; Preferably, the method for inducing blastocyst-like cells to produce embryo-like cells as described in U2 further includes the steps of culturing and / or maturing the embryo-like cells: sequentially performing a fifth and a sixth culture on the embryo-like cells obtained after the fourth culture using the fifth culture medium in the reagent combination or kit according to any one of claims 4 to 5, and then performing a seventh culture on the embryo-like cells using the sixth culture medium in the reagent combination or kit according to any one of claims 4 to 5. Preferably, the fifth culture time is 12-36 hours; Preferably, the sixth culture time is 16-32 hours; Preferably, the seventh culture time is 16-32 hours; Preferably, the fifth culture is a suspension culture; Preferably, the sixth culture is a dynamic culture; Preferably, the seventh culture is a dynamic culture. 13.d1)~d3) Any one of the following biological materials: d1) A biomaterial, said biomaterial being a blastocyst-like lineage precursor cell, said blastocyst-like lineage precursor cell being obtained by the method described in claim 7 or 8; d2) A biomaterial, wherein the biomaterial is a blastocyst-like cell, the blastocyst-like cell being obtained by the method of claim 9 or 10; d3) A biomaterial, wherein the biomaterial is an embryo-like cell, the embryo-like cell being 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% to 36.3%; Preferably, the content of CDX2 positive cells in the blastocyst-like lineage precursor cells is 1.28% to 61.2%; Preferably, the content of OCT4-positive cells in the blastocyst-like lineage precursor cells is 59.8% to 90.5%; Preferably, the content of the primitive endoderm-like cell population in the blastocyst-like cells is 15.8% to 37.49%; Preferably, the content of trophectoderm-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 embryo-like structure has the ability to reproduce post-implantation embryonic development and / or gastrulation.

14. The use of at least one of the biomaterials described in claims d1) to d3) of claim 13 in any one of a1) to a8): a1) Preparation of embryo models; a2) Embryo research; a3) Screening for key genes causing embryonic developmental defects; a4) Investigate the effects of knockout or overexpression libraries on embryonic development; a5) Testing drug safety; a6) Preparation of gastrulation phase cells; a7) Prepare tissue and / or organ precursor cells; a8) Prepare a product, said product being used in any one of a1) to a7).

15. A product comprising at least one of the biomaterials described in claims d1) to d3) of claim 13.

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