A reagent combination or kit for constructing a synthetic embryo and use thereof

Abstract

This invention belongs to the field of biotechnology and discloses a reagent combination or kit for constructing embryo-like cells and its applications. This reagent combination or kit 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, and has high efficiency in constructing embryo-like cells.

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 1ESCs can self-assemble into organized structures, such as blastooids, gastrulooids, and in vitro embryos, after induction in vitro [2-4]. Although this embryonic development system has opened up new avenues for early embryonic development and in vitro reproduction of developmental processes, it also faces many limitations. For example, blastooids 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 asymmetry and body axis formation [2], but cannot fully simulate the signals and morphogenesis during body axis formation. The main reason is that gastrulooids lack the primitive endoderm lineage, while signals from extraembryonic tissues are crucial for the development of the epiblast (EPI) and the establishment of the anterior-posterior axis [2]. In short, both blastocysts and gastrula have significant defects and cannot form embryos with a complete lineage.

[0005] Recently, in vitro embryo-like systems have broken through the limitations of previous methods and opened a new chapter in simulating post-implantation embryo development. The Zernicka-Goetz team constructed pre- and post-implantation embryo-like structures, namely ETS synthetic embryos / embryoids (ETS-Embryoids), by co-culturing ESCs with EPI differentiation potential, TSCs derived from TE, and extraembryonic endoderm cells (XEN) derived from PE[6]. During the gastrulation stage of ETS-like embryos, the symmetry of the embryo is disrupted because there are not enough PE cells to develop into the distal visceral endoderm (DVE) and anterior visceral endoderm (AVE). Jacob Hanna and Zernicka-Goetz's team used cells that overexpressed the key PE regulator Gata4 (iXEN) in ESCs (Dox-induced cells) to replace XENs and cells that expressed the key TSC gene Cdx2 (iTSC) to replace TSCs, forming ETiX / EiTiX embryos, achieving a major breakthrough [3,4]. The embryos formed by the ETiX / EiTiX system have a good ability to simulate post-implantation development, experiencing the peri-implantation period and gastrulation period, and also have extraembryonic tissues [7]. These reports indicate that the gastrulation and organogenesis processes in mammals can be reproduced by cross-linking in culture dishes, making it possible to reproduce the embryonic development process. Jose Silva's team found that by activating the STAT3 cell pathway through transgenes and regulating the STAT3 signaling pathway, the developmental potential of embryonic stem cells (ESCs) can be stimulated, and a new type of cell, morula-like cells (MLCs), can be obtained efficiently [8]. MLC has the ability to develop all lineages within an embryo and extraembryonic tissues, and can effectively form blastocysts and embryo-like structures with a certain developmental capacity. However, existing in vitro embryo-like systems have the following limitations: (1) They require mixing cells of different lineages, including ESC, TSC, and XEN or their substitutes, which is extremely inefficient; (2) TSC and XEN substitutes rely on transgenic activation of extraembryonic lineages, which is still inefficient; (3) The most advanced mouse embryo-like structures currently available cannot reproduce complete gastrulation, especially the specialized structures of the endoderm, mesoderm, and ectoderm, and lack the gastrulation morphology comparable to natural embryos; (4) Although they possess multiple cell lineages, the proportion and maturity of cells with different fates are not balanced; (5) The heterogeneity of recombinant cells results in an embryo formation rate of less than 0.5%. Figure 1 ).

[0006] Therefore, constructing a cell with developmental pluripotency based on small molecule induction, non-transgenic, single-cell origin, and using it to construct an in vitro embryo-like system that is more efficient and closer to the natural embryonic developmental pathway, will help overcome the limitations of previous methods, advance the development of this field, and reduce the difficulty of subsequent biomedical research applications. Summary of the Invention

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

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

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

[0010] The fourth aspect of this invention aims to provide a method for inducing stem cells to produce blastocyst-like cells.

[0011] The fifth aspect of this invention is to provide a method for constructing an embryo-like structure.

[0012] The sixth aspect of this invention is to provide a blastocyst-like lineage precursor cell.

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

[0014] An eighth aspect of the present invention aims to provide an embryo-like substance.

[0015] The object of the ninth aspect of the present invention is to provide the application of the blastocyst-like lineage precursor cells of the sixth aspect, the blastocyst-like cells of the seventh aspect, and the embryo-like cells of the eighth aspect.

[0016] The tenth aspect of this invention is to provide a product.

[0017] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0018] A first aspect of the present invention provides a reagent combination or kit comprising a first culture medium;

[0019] The first culture medium is a basal medium containing GSK-3 inhibitors, STAT3 activators, retinoic acid nuclear receptor (RAR) agonists, and TGF-β receptor kinase inhibitors.

[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 basal culture medium.

[0021] Preferably, the first culture medium is used to induce stem cells to produce blastocyst-like lineage precursor cells.

[0022] Preferably, the reagent combination or kit further comprises a second culture medium;

[0023] The second culture medium is a basal medium containing GSK-3 inhibitors, retinoic acid nuclear receptor (RAR) agonists, and TGF-β receptor kinase inhibitors.

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

[0025] Preferably, the second culture medium is used to induce stem cells to produce blastocyst-like lineage precursor cells.

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

[0027] Preferably, the STAT3 activator in the first culture medium contains at least one of leukemia inhibitory factor (LIF) and interleukin-6 (IL6); more preferably, it is leukemia inhibitory factor (LIF); and even more preferably, it is mouse leukemia inhibitory factor.

[0028] Preferably, the retinoic acid nuclear receptor (RAR) agonists in the first and second culture media are 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; more preferably, the retinoic acid nuclear receptor (RAR) agonist in the first and second culture media is AM580.

[0029] Preferably, the TGF-β receptor kinase inhibitors in the first and second culture media 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; more preferably, the TGF-β receptor kinase inhibitor in the first and second culture media is E616452.

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

[0031] Preferably, the concentration of the STAT3 activator in the first culture medium is 10-100 ng / mL; more preferably 15-25 ng / mL; and even more preferably 18-22 ng / mL.

[0032] Preferably, the concentration of the retinoic acid nuclear receptor (RAR) agonist in the first culture medium is 0.01–0.1 μM; more preferably 0.02–0.08 μM; and even more preferably 0.04–0.06 μM.

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

[0034] Preferably, the concentration of the GSK-3 inhibitor in the second culture medium is 1–15 μM; further, 2.1–9.9 μM; even further, 5.4–9.9 μM; and 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 culture medium is 0.01–0.1 μM; more preferably 0.02–0.08 μM; and even more preferably 0.04–0.06 μM.

[0036] Preferably, the concentration of the TGF-β receptor kinase inhibitor in the second culture medium is 1–20 μM; further, 1–15 μM; even further, 5–15 μM; still further, 5–12 μM; and even further, 9–11 μM.

[0037] Preferably, the reagent combination or kit comprises a first culture medium, or a first culture medium and a second culture medium, and the reagent combination or kit is used to induce stem cells to produce blastocyst-like lineage precursor cells.

[0038] Preferably, when the reagent combination or kit is used to induce stem cells to produce blastocyst-like lineage precursor cells, the reagent combination or kit further includes instruction manual 1, which describes a method for inducing stem cells to produce blastocyst-like lineage precursor cells.

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

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

[0041] 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.

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

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

[0044] 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; further, it is BMP4; and even further, it is hBMP4.

[0045] Preferably, the TGF-β activator in the third culture medium contains at least one of TGF-β and activator A; more preferably, it is activator A.

[0046] 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 paclobutrazol, CCT031374, iCRT-3, iCRT-5, iCRT-14, CPG049090, and NC043; more preferably, XAV939.

[0047] Preferably, the Lats kinase inhibitor in the third culture medium comprises at least one of TRULI, GA-017, and TDI-011536; more preferably, TRULI.

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

[0049] 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-phosphide, L-ascorbic acid 3-phosphide, and L-ascorbic acid 2-sulfate; further comprising L-ascorbic acid-2-phosphate.

[0050] Preferably, the concentration of the fibroblast growth factor in the third culture medium is 10–50 ng / mL; more preferably 15–30 ng / mL; and even more preferably 22–28 ng / mL.

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

[0052] Preferably, the concentration of the TGF-β activator in the third culture medium is 10–40 ng / mL; more preferably 15–30 ng / mL; and even more preferably 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; more preferably 2–5 μM; and even more preferably 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; more preferably 1.5–3 μM; and even more preferably 1.8–2.2 μM.

[0055] Preferably, the concentration of the anticoagulant in the third culture medium is 0.5–2.0 μg / mL; more preferably 0.8–1.5 μg / mL; and even more preferably 0.9–1.1 μg / mL.

[0056] Preferably, the concentration of ascorbic acid or its derivative in the third culture medium is 50-500 μM; more preferably 100-250 μM; and even more preferably 180-220 μM.

[0057] Preferably, the concentration of the insulin-transferrin-selenium additive in the third culture medium is 0.5–2×; more preferably 0.6–1.5×; and even more preferably 0.9–1.1×.

[0058] Preferably, the reagent combination or kit comprises: a first culture medium and a third culture medium; or

[0059] First culture medium, second culture medium, and third culture medium;

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

[0061] Preferably, when the reagent combination or kit is used to induce stem cells to produce blastocyst-like cells, the reagent combination or kit further includes instruction manual 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 invention.

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

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

[0065] Preferably, the fourth culture medium is used to induce blastocyst-like cells to produce embryo-like cells.

[0066] Preferably, the reagent combination or kit comprises: a first culture medium, a third culture medium, and a fourth culture medium; or

[0067] First culture medium, second culture medium, third culture medium, and fourth culture medium;

[0068] The reagent combination or kit is used to induce stem cells to produce embryo-like structures.

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

[0070] Preferably, the method for inducing stem cells to produce embryo-like cells is the method of the fifth aspect of the present invention (the method for inducing blastocyst-like cells to produce embryo-like cells in U2 does not include the steps of culturing and / or maturing the embryo-like cells).

[0071] Preferably, the reagent combination or kit further comprises a fifth culture medium and a sixth culture medium;

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

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

[0074] Preferably, the fifth and sixth culture media are used for the culture and / or maturation of embryo-like organisms.

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

[0076] Preferably, the antibiotic in the fifth culture medium comprises at least one of amphotericin B, nystatin, gentamicin, tetracycline, erythromycin, penicillin, and streptomycin; more preferably, penicillin and streptomycin.

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

[0078] Preferably, the concentration of glutamine in the fifth culture medium is 0.5–2×; more preferably 0.7–1.5×; and even more preferably 0.9–1.1×.

[0079] Preferably, the concentration of insulin-transferrin-seleno-aminoethanol in the fifth culture medium is 0.5-2×; more preferably 0.7-1.5×; and even more preferably 0.9-1.1×.

[0080] Preferably, the concentration of the thyroid hormone receptor agonist in the fifth culture medium is 50–200 nM; more preferably 60–150 nM; and even more preferably 90–110 nM.

[0081] Preferably, the concentration of estradiol in the fifth culture medium is 5–11 nM; more preferably 6–10 nM; and even more preferably 7.2–8.8 nM.

[0082] Preferably, the concentration of progesterone in the fifth culture medium is 100–300 ng / mL; more preferably 150–270 ng / mL; and even more preferably 180–220 ng / mL.

[0083] Preferably, the concentration of acetylcysteine ​​in the fifth culture medium is 5–45 μM; more preferably 10–35 μM; and even more preferably 22.5–27.5 μM.

[0084] Preferably, the concentration of glucose in the fifth culture medium is 0.5–2 mg / mL; more preferably 0.7–1.5 mg / mL; and even more preferably 0.9–1.1 mg / mL.

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

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

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

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

[0089] Preferably, the buffer salt in the sixth culture medium contains at least one of phosphate, Tris, and HEPES; more preferably, HEPES.

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

[0091] Preferably, the concentration of the serum in the sixth culture medium is 35-95% by volume; more preferably 45-89%; and even more preferably 73-84%.

[0092] Preferably, the concentration of the rat serum in the sixth culture medium is 25-60% by volume; more preferably 30-55%; and even more preferably 45-52%.

[0093] Preferably, the concentration of the human AB serum in the sixth culture medium is 10-35% by volume; more preferably 15-34%; and even more preferably 28-32%.

[0094] Preferably, the concentration of glutamine in the sixth culture medium is 0.5–2x; more preferably 0.7–1.5x; and even more preferably 0.9–1.1x.

[0095] Preferably, the concentration of pyruvate or its salt in the sixth culture medium is 0.5-2x; more preferably 0.7-1.5x; and even more preferably 0.9-1.1x.

[0096] Preferably, the concentration of the buffer salt in the sixth culture medium is 6–20 mM; more preferably 8–16 mM; and even more preferably 10–12 mM.

[0097] Preferably, the concentration of glucose in the sixth culture medium is 1–6 mg / mL; more preferably 2–5 mg / mL; and even more preferably 3.6–4.4 mg / mL.

[0098] Preferably, the concentration of the antibiotic in the sixth culture medium is 0.1-2% by volume; more preferably 0.5-1.7%; and even more preferably 0.9-1.1%.

[0099] Preferably, the reagent combination or kit comprises: a first culture medium, a third culture medium, a fourth culture medium, a fifth culture medium, and a sixth culture medium; or

[0100] First culture medium, second culture medium, third culture medium, fourth culture medium, fifth culture medium and sixth culture medium;

[0101] The reagent combination or kit is used to induce stem cells to produce embryo-like structures and to culture and / or mature these embryo-like structures.

[0102] Preferably, when the reagent combination or kit is used to induce stem cells to produce embryo-like structures and to culture and / or mature the embryo-like structures, the reagent combination or kit further includes instruction manual 4, which describes a method for inducing stem cells to produce embryo-like structures and to culture and / or mature the embryo-like structures.

[0103] Preferably, the method for inducing stem cells to generate embryo-like cells and the method for culturing and / or maturing embryo-like cells are methods of the fifth aspect of the present invention (the method for inducing blastocyst-like cells to generate embryo-like cells in U2 further includes the steps of culturing and / or maturing embryo-like cells).

[0104] Preferably, the reagent combination or kit contains any one or more of 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 (for example, the reagent combination or kit may contain a third culture medium; or the reagent combination or kit may contain a fourth culture medium and a fifth culture medium; or the reagent combination or kit may contain a fourth culture medium, a fifth culture medium, and a sixth culture medium); the purpose of the reagent combination or kit corresponds to the culture medium.

[0105] Preferably, the basal media of the first, second, third, fourth, fifth, and sixth media are each independently selected from at least one of IMDM (Iscove's Modified Dulbecco's Medium), Neurobasal medium, Eagle's Basal Medium (BME), MEM medium, DMEM medium, Ham's F-12 medium, RPMI 1640 medium, Advanced RPMI 1640 medium, Advanced DF-12 (Advanced DMEM / F-12) medium, and DMEM / F12 medium; more preferably, the basal media of the first, second, third, fourth, fifth, and sixth media are each independently selected from one of Neurobasal medium, DMEM medium, Advanced DF-12 (Advanced DMEM / F-12) medium, and DMEM / F12 medium.

[0106] Preferably, the basal medium for the first culture medium and / or the second culture medium is DMEM / F12 medium and Neurobasal medium.

[0107] Preferably, the volume ratio of DMEM / F12 medium and Neurobasal medium in the basal medium of the first culture medium and / or the second culture medium is independently selected from 1:(0.8 to 1.2); more preferably from 1:(0.9 to 1.1).

[0108] Preferably, the basal medium of the first culture medium and / or the second culture medium is a basal 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 basal medium of the first culture medium and / or the second culture medium is 0.5 to 1.5×; more preferably 0.9 to 1.1×.

[0110] Preferably, the concentration of the non-essential amino acids in the basal medium of the first culture medium and / or the second culture medium is 0.5 to 1.5 × 10⁻⁶; more preferably, it is 0.9 to 1.1 × 10⁻⁶.

[0111] Preferably, the concentration of B27 in the basal medium of the first culture medium and / or the second culture medium is 0.5 to 1.5×; more preferably 0.9 to 1.1×.

[0112] Preferably, the concentration of N2 in the basal medium of the first culture medium and / or the second culture medium is 0.5 to 1.5×; more preferably 0.9 to 1.1×.

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

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

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

[0116] Preferably, the concentration of the serum in the basal medium of the third culture medium is 10-30% by volume; more preferably 15-25%; and even more preferably 18-22%.

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

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

[0120] Preferably, the concentration of 2-mercaptoethanol in the basal medium of the third culture medium is 0.05–0.15 mM; more preferably 0.08–0.12 mM; and even more preferably 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; more preferably 0.5-1.7%; and even more preferably 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 a low-sugar, pyruvate-containing, glutamine-free, and phenol red-free DMEM medium).

[0124] Preferably, the stem cells are stem cells with multi-directional differentiation potential.

[0125] 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; more preferably, the stem cells with multipotent differentiation potential include embryonic stem cells.

[0126] Preferably, the stem cells are derived from mammals (e.g., primates (e.g., humans, chimpanzees, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits)).

[0127] Preferably, the stem cells are derived from rodents; further, from mice; and even further, from mice.

[0128] Preferably, the stem cells are derived from primates; more preferably, they are derived from humans.

[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 commercially available 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 and are within 14 days of fertilization.

[0132] A second aspect of the present invention provides the use of the reagent combination or kit of the first aspect of the present invention in any one of (1) to (10);

[0133] (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 (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, intestinal precursor cells, liver precursor cells, kidney precursor cells, etc.); (10) Preparation of products that induce stem cells to produce 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, intestinal precursor cells, liver precursor cells, kidney precursor cells, etc.).

[0134] Preferably, the stem cell is the stem cell described in the first aspect of the present invention.

[0135] A third aspect of the present invention provides a method for inducing stem cells to produce blastocyst-like lineage precursor cells, comprising the steps of using a reagent combination or kit of the first aspect of the present invention.

[0136] Preferably, the method for inducing stem cells to generate blastocyst-like lineage precursor cells includes S1 or S2:

[0137] S1: Culture stem cells using the first culture medium in the reagent combination or kit of the first aspect of the present invention;

[0138] S2: The stem cells are cultured for the first time using the second culture medium in the reagent combination or kit of the first aspect of the present invention, and then cultured for the second time using the first culture medium in the reagent combination or kit of the first aspect of the present invention.

[0139] Preferably, the culture time in S1 is 36–108 h; further, 42–78 h; and even further, 54–66 h.

[0140] Preferably, the first culture time in S2 is 12-36 hours; further, it is 18-30 hours; and even further, it is 22-26 hours.

[0141] Preferably, the second culture time in S2 is 24-72 hours; further, 24-48 hours; and even further, 32-40 hours.

[0142] Preferably, the above-mentioned culture (including the culture in S1, and the first culture and the second culture in S2) is a wall-adherent culture; more preferably, the above-mentioned culture is carried out in a culture device treated with gelatin.

[0143] Preferably, the conditions for the above-mentioned cultivation (including the cultivation in S1, and the first and second cultivations in S2) are 33-40°C and 3-7% CO2; more preferably 36-38°C and 4-6% CO2.

[0144] Preferably, the stem cell is the stem cell described in the first aspect of the present invention.

[0145] A fourth aspect of the present invention provides a method for inducing stem cells to produce blastocyst-like cells, comprising the steps of using a reagent combination or kit from the first aspect of the present invention.

[0146] Preferably, the method for inducing stem cells to generate blastocyst-like cells includes the following steps:

[0147] T1: Inducing stem cells to generate blastocyst-like lineage precursor cells: The method for inducing stem cells to generate blastocyst-like lineage precursor cells is the method for inducing stem cells to generate blastocyst-like lineage precursor cells according to the third aspect of the present invention;

[0148] T2: Induces 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 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 of the first aspect of the present invention.

[0150] Preferably, the third culture time is 24-48 hours; further, 28-44 hours; and even further, 32-40 hours.

[0151] Preferably, the third culture is a suspension culture; more preferably, the third culture is carried out using Aggrewell culture plates; even more preferably, the third culture is carried out using Aggrewell culture plates treated with anti-adhesion rinsing solution.

[0152] Preferably, the conditions for the third culture are 33–40°C and 3–7% CO2; more preferably, 36–38°C and 4–6% CO2.

[0153] Preferably, the stem cell is the stem cell described in the first aspect of the present invention.

[0154] A fifth aspect of the present invention provides a method for inducing stem cells to produce embryo-like structures, comprising the steps of using a reagent combination or kit from the first aspect of the present invention.

[0155] Preferably, the method for inducing stem cells to generate embryo-like structures includes 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 according to the fourth aspect of the present invention;

[0157] U2: Induces blastocyst-like cells to produce embryo-like cells.

[0158] Preferably, 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 of the first aspect of the present invention.

[0159] Preferably, the fourth culture time is 24-48 hours; more preferably 28-44 hours; and even more preferably 32-40 hours.

[0160] Preferably, the fourth culture is a suspension culture; more preferably, the fourth culture is carried out using Aggrewell culture plates; even more preferably, the fourth culture is carried out using Aggrewell culture plates treated with anti-adhesion rinsing solution.

[0161] Preferably, the conditions for the fourth culture are 33–40°C and 3–7% CO2; more preferably, 36–38°C and 4–6% CO2.

[0162] 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: the embryo-like cells obtained after the fourth culture are cultured for a fifth and a sixth time sequentially using the fifth culture medium in the reagent combination or kit of the first aspect of the present invention, and then the embryo-like cells are cultured for a seventh time using the sixth culture medium in the reagent combination or kit of the first aspect of the present invention.

[0163] Preferably, the fifth culture time is 12-36 hours; further, 20-28 hours; and even further, 22-26 hours.

[0164] Preferably, the sixth culture time is 16-32 hours; further, 20-28 hours; and even further, 22-26 hours.

[0165] Preferably, the seventh culture time is 16-32 hours; further, 20-28 hours; and even further, 22-26 hours.

[0166] Preferably, the fifth culture is a suspension culture; more preferably, the fifth culture is performed using Aggrewell culture plates; even more preferably, the fifth culture is performed using Aggrewell culture plates treated with anti-adhesion rinsing solution.

[0167] Preferably, the sixth culture is a dynamic culture; more preferably, the sixth culture is carried out using a suspension culture device at 60-150 rpm; even more preferably, the sixth culture is carried out using a suspension culture device at 70-90 rpm.

[0168] Preferably, the seventh culture is a dynamic culture; more preferably, the seventh culture is carried out in a bioreactor at 30-100 rpm; even more preferably, the seventh culture is carried out in a bioreactor at 40-60 rpm.

[0169] Preferably, the conditions for the fifth, sixth, and / or seventh culture are 33–40°C and 3–7% CO2; more preferably, 36–38°C and 4–6% CO2.

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

[0171] A sixth aspect of the present invention provides a blastocyst-like lineage precursor cell obtained by the method of a third aspect of the present invention.

[0172] Preferably, the content of GATA6-positive cells in the blastocyst-like lineage precursor cells is 21.9%–36.3%; more preferably 24.6%–34.5%; even more preferably 26.1%–34.5%; and still more preferably 28.4%–31.9%.

[0173] Preferably, the content of CDX2 positive cells in the blastocyst-like lineage precursor cells is 1.28% to 61.2%; more preferably, 6.45% to 43.5%; even more preferably, 15.6% to 29.2%; and still more preferably, 26.8% to 29.2%.

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

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

[0176] A seventh aspect of the present invention provides a blastocyst-like cell obtained by the method of a fourth aspect of the present invention.

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

[0178] Preferably, the content of trophectoderm-like cell population in the blastocyst-like cells is 7.26–41.99%; more preferably 16.87–25.00%; and even more preferably 20.87–23.48%.

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

[0180] Preferably, the blastocyst-like cells do not contain reproductive material.

[0181] An eighth aspect of the present invention provides an embryo-like form obtained by the method of the fifth aspect of the present invention.

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

[0183] A ninth aspect of the invention provides the use of blastocyst-like lineage precursor cells of the sixth aspect, blastocyst-like cells of the seventh aspect, and / or embryo-like cells of the eighth aspect in any one of a1) to a8):

[0184] a1) Preparation of embryo models;

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

[0186] a3) Screening for key genes causing developmental defects in embryos (preferably early embryos);

[0187] a4) Investigate the effects of knockout or overexpression libraries on embryonic (preferably early embryo) development;

[0188] a5) Testing 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 2This section demonstrates the analysis of blastocyst-like lineage precursor cells induced from embryonic stem cells using the kits from Examples 1-1-1-4 and 2-7 in Examples 11-1 to 11-4 and 2-7, as shown in Examples 11-1 to 11-4 and 2-7. In these examples, A represents the small molecules involved in the kits from Examples 1-1 to 11-4 and 2-7, along with related abbreviations; C (Chir99021) is a glycogen synthase kinase 3 inhibitor (GSK3i); and L (mLif) is a murine leukemia inhibitor. Factor (mLIF) 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 used in Examples 11-1 to 11-4 to induce blastocyst-like lineage precursor cells (referred to as Q0-a, Q0-b, Q0-c, Q0-d) from embryonic stem cells; C is the immunoflow cytometry results of Q0-a, Q0-b, Q0-c, and Q0-d (GATA6 represents the primitive endoderm). The text appears to be a series of seemingly unrelated phrases and sentences, making it impossible to translate coherently. It includes fragments about: "GATA6 is a marker protein for primitive endoderm (PrE), and CDX2 is a marker protein for trophectoderm (TE)"; "D is the strategy for inducing blastocyst-like lineage precursor cells (Q1, Q2, Q3, Q4, Q5, Q6) from embryonic stem cells using the kits from Examples 2-7 in Examples 12-17"; and "E is the immunoflow cytometry results of blastocyst-like lineage precursor cells (Q1, Q2, Q3, Q4, Q5, Q6) ."

[0200] Figure 3 Immunofluorescence results of blastocyst-like lineage precursor cells (referred to as Q1, Q2, Q3, Q4, Q5, and Q6) induced by embryonic stem cells using the kits in Examples 2-7 are shown in Examples 12-17. (Red represents CDX2, the marker protein for TE; purple represents GATA6, the marker protein for PrE; green represents OCT4, the marker protein for the inner cell mass and epiblast; dark blue represents the nuclear dye DAPI; scale bar is 50 μm).

[0201] Figure 4The following examples 14 and 15 demonstrate the expression of marker genes at different time points in blastocyst-like lineage precursor cells (Q3 and Q4) induced from embryonic stem cells using the kits from examples 4 and 5 (with Actin as the internal reference gene, and the relative expression levels of each gene calculated using the ΔΔCt method): A represents the expression of marker genes at different time points in blastocyst-like lineage precursor cells induced from embryonic stem cells in example 14; B represents the expression of marker genes at different time points in blastocyst-like lineage precursor cells induced from embryonic stem cells in example 15.

[0202] Figure 5 The single-cell transcriptome analysis results of blastocyst-like lineage precursor cells obtained by inducing embryonic stem cells using the kits from Examples 2 to 7 in Examples 12 to 17 are shown. Specifically, A is a non-linear dimensionality reduction algorithm UMAP analysis diagram performed on blastocyst-like lineage precursor cells generated in Example 12 and mouse early embryo reference data; B is a non-linear dimensionality reduction algorithm UMAP analysis diagram performed on blastocyst-like lineage precursor cells generated in Example 13 and mouse early embryo reference data; C is a non-linear dimensionality reduction algorithm UMAP analysis diagram performed on a mixture of blastocyst-like lineage precursor cells generated in Examples 14 and 15 and mouse early embryo reference data; D is a non-linear dimensionality reduction algorithm UMAP analysis diagram performed on blastocyst-like lineage precursor cells generated in Example 16 and mouse early embryo reference data; and E is a non-linear dimensionality reduction algorithm UMAP analysis diagram performed on blastocyst-like lineage precursor cells generated in Example 17 and mouse early embryo reference data.

[0203] Figure 6 The single-cell transcriptome analysis results of the blastocyst-like cells obtained in Examples 18-1 to 18-7 are shown below: A is a nonlinear dimensionality reduction algorithm UMAP analysis diagram of the blastocyst-like cells obtained in Example 18-2 and the mouse early embryo reference data; B is a nonlinear dimensionality reduction algorithm UMAP analysis diagram of the blastocyst-like cells obtained in Example 18-3 and the mouse early embryo reference data; C is a nonlinear dimensionality reduction algorithm UMAP analysis diagram of the blastocyst-like cells obtained in Example 18-4 and the mouse early embryo reference data; D is a nonlinear dimensionality reduction algorithm UMAP analysis diagram of the blastocyst-like cells obtained in Example 18-1 and the mouse early embryo reference data; E is a nonlinear dimensionality reduction algorithm UMAP analysis diagram of the blastocyst-like cells obtained in Example 18-5 and the mouse early embryo reference data; F is a nonlinear dimensionality reduction algorithm UMAP analysis diagram of the blastocyst-like cells obtained in Example 18-6 and the mouse early embryo reference data; G is a nonlinear dimensionality reduction algorithm UMAP analysis diagram of the blastocyst-like cells obtained in Example 18-7 and the mouse early embryo reference data.

[0204] Figure 7The results of single-cell transcriptome analysis of blastocyst-like cells obtained in Examples 18-1 to 18-7 are shown: A is a nonlinear dimensionality reduction algorithm UMAP analysis diagram of blastocyst-like cells obtained in Examples 18-1 to 18-7 and mouse early embryo reference data, as well as the single-cell transcriptome expression level of marker genes (GATA6 is the marker gene of primitive endoderm (PrE), and CDX2 is the marker gene of trophectoderm (TE)); B is the cell type ratio of blastocyst-like cells obtained in Examples 18-1 to 18-7.

[0205] Figure 8 The expression of lineage marker genes in the blastocyst-like cells obtained in Example 18-1 and the results of immunofluorescence staining are shown: A shows the single-cell transcriptome expression of lineage marker genes in the three cell populations (primitive endoderm-like cells, trophectoderm-like cells, and epiblast-like cells) of the blastocyst-like cells obtained in Example 18-1; B shows the immunofluorescence staining results of the blastocyst-like cells obtained in Example 18-1 (KRT18 / PDGFRA / OCT4); C shows the immunofluorescence staining results of the blastocyst-like cells obtained in Example 18-1 (KRT18 / CDX2 / OCT4), with two replicates; D shows the thermogram analysis of the three cell populations of blastocyst-like cells obtained in Example 18-1 and the E4.5 natural embryo.

[0206] Figure 9The results of inducing embryonic-like structures from embryonic stem cells using the kit from Example 8-1 in Example 18-1 are shown below: A is a schematic diagram of the process of inducing embryonic-like structures from embryonic stem cells (scale bar: 50 μm); B is a representative sample of Day 4.5 embryonic-like structures (ci-Embryoids) induced from embryonic stem cells using the kit from Example 8-1 in Example 18-1 (scale bar: 100 μm); C is a table comparing the formation efficiency of embryonic-like structures generated in Example 18-1 with that of previously reported EiTiX embryonic-like structures at different stages; D is an immunohistochemical staining image of a representative sample of Day 4.5 embryonic-like structures (ci-Embryoids) induced from embryonic stem cells using the kit from Example 8-1 in Example 18-1 (scale bar: 100 μm); E is an immunohistochemical staining image of a representative sample of Day 4.5 embryonic-like structures (ci-Embryoids) induced from embryonic stem cells using the kit from Example 8-1 in Example 18-1. Immunofluorescence staining results (AP2γ / SOX2 / OCT4); F is the immunofluorescence staining result of a representative sample of Day 4.5 embryonic ci-Embryoids induced by embryonic stem cells using the kit of Example 8-1 in Example 18-1 (SOX2 / T / Bry) (scale bar is 100μm); G is the immunofluorescence staining result of a representative sample of Day 4.5 embryonic ci-Embryoids induced by embryonic stem cells using the kit of Example 8-1 in Example 18-1 (N-cad / T, N-cad / E-cad, T) (scale bar: top image is 40μm, bottom image is 10μm); H is the immunofluorescence staining result of a representative sample of Day 4.5 embryonic ci-Embryoids induced by embryonic stem cells using the kit of Example 8-1 in Example 18-1 (FOXA2 / GATA4) (scale bar is 100μm).

[0207] Figure 10 The nonlinear dimensionality reduction algorithm UMAP analysis plot (generated from 10×genomic scRNA-seq) is shown for single-cell transcriptome samples of Day 4.5 embryonic-like cells induced by embryonic stem cells using the kit of Example 8-1 in Example 18-1 and natural mouse embryonic E7.5 samples.

[0208] Figure 11 This study demonstrates the single-cell transcriptome expression levels of marker genes in E7.5 samples from natural mouse embryos.

[0209] Figure 12 This demonstrates the single-cell transcriptome expression levels of embryonic marker genes generated on Day 4.5 by embryonic stem cells induced by the kit in Example 8-1 using Example 18-1.

[0210] Figure 13 This invention demonstrates the universality of its kit and / or induction method (using stem cell lines from two sources other than OG2 mouse embryonic stem cells: C57BL / 6 mouse inner cell mass and ICR mouse background stem cells, respectively). Immunofluorescence staining images of cells induced using Q3 and Q4 small molecule induction methods and their constructed embryo-like structures are shown: A shows the immunofluorescence image of blastocyst-like lineage precursor cells generated from other cell line 1 (C57BL / 6 mouse source) after induction (the method is the same as in Examples 14 and 15, the only difference being the source of the stem cells). The images show lineage marker proteins GATA6, CDX2, and SOX2 (50 μm) for primitive endoderm-like cell populations, trophoblast-like cell populations, and epiblast-like cell populations, respectively. B shows the immunofluorescence image of other cell line 2 (ICR mouse source). Immunofluorescence images of blastocyst-like lineage precursor cells induced by cell lineage (method same as in Examples 14 and 15, the only difference being the source of stem cells) (scale bar 50 μm); C is the immunofluorescence image of embryo-like cells constructed by induction from other cell line 1 (C57BL / 6 mouse source) (i.e., method same as in Examples 18-1 and 18-4, the only difference being the source of stem cells) at different days (CDX2 / GATA4 / OCT4; AP2γ / SOX2) (scale bar 50 μm); D is the immunofluorescence image of embryo-like cells constructed by induction from other cell line 2 (ICR mouse source) (i.e., method same as in Examples 18-1 and 18-4, the only difference being the source of stem cells) at different days (CDX2 / GATA4 / OCT4; AP2γ / SOX2) (scale bar 50 μm). Detailed Implementation

[0211] The present invention will be further described in detail below through specific embodiments.

[0212] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0213] Definitions:

[0214] In this invention, "blastocyst-like lineage precursor cells" refers to a cell population that, after being induced by a specific combination of small molecules through culture, expresses the key transcription factors GATA6, CDX2, and OCT4 of the three lineages (primordial endoderm-like cells, trophectoderm-like cells, and epiblast-like cells) during the blastocyst stage. Although the molecular level has not yet fully reached the molecular level of the blastocyst stage, the cell population has already acquired the ability to mature into blastocyst-like cells of the three lineages.

[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] The OG2 mouse embryonic stem cell line was derived from the inner cell mass of embryos 3.5 days after mating 129 female mice with OG2 male mice (B6; CBA-Tg(Pou5f1-EGFP)2Mnn / J). Other stem cell lines 1 were derived from the inner cell mass of embryos 3.5 days after self-crossing of C57BL / 6 mice; and stem cell line 2 was derived from the inner cell mass of embryos 3.5 days after self-crossing of ICR mice. The methods for preparing stem cell lines are described in [9]. All mice were purchased from The Jackson Laboratory.

[0224] Mouse embryonic stem cells were seeded in cell culture plates treated with 1% gelatin (STEMCELL, 07903) and cultured in 2iLIF medium. 2iLIF medium was N2B27 basal medium containing 20 ng / mL mouse leukemia inhibitory factor (murine LIF, qKine, Qk018), 3 μM CHIR99021 (Selleck, S2924), and 1 μM PD0325901 (Selleck, S1036). The N2B27 basal medium was a mixed medium containing 1×B27 (Gibco, 17504044), 1×B27 (Gibco, 17502048), 1×GlutaMAX (Gibco, 35050061), 1×MEM-NEAA (Gibco, 11140050), and 0.1 mM 2-mercaptoethanol (Gibco, 21985023). This mixed medium was prepared by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a 1:1 volume ratio. The cell culture medium was updated daily and the cells were cultured at 37°C, 20% O2, and 5% CO2.

[0225] 3. Rat serum

[0226] The preparation method for rat serum is as follows: Prepare 3% isoflurane (RWD, R510-22-10) gas anesthetic and inflate it into the anesthesia equipment box at a flow rate of 3.0 L / min for 10 minutes. Transfer the prepared rats to the box for anesthesia. Gently stimulate the rat's paws with forceps; if there is no response, the anesthesia is complete. Then cover the rat's nose with an inhalation mask connected to the gas anesthetic to ensure the rat is deeply anesthetized during blood collection. Use large scissors to cut the skin and abdominal wall towards the forelimbs to further expose the posterior abdominal cavity. Use forceps to turn excess fat out of the abdominal cavity. Use forceps to symmetrically separate the visceral fat along the midline of the rat's abdomen to expose the abdominal aorta. Repeat this step to clear the fat in the longitudinal region of the abdominal aorta to clearly identify the bifurcation of the abdominal aorta. Carefully remove the fat from the abdominal aorta and bifurcation with forceps to separate the fat from the target blood vessel.

[0227] Blood was collected from the abdominal aorta of male rats using a 0.5 mm venipuncture needle (SANLI, F0326-9-1) and a coagulation-promoting tube (BD Biosciences, 367955). After each collection, the blood was inverted and mixed 6 to 7 times, then allowed to stand at room temperature for at least 30 minutes. Once clear stratification was observed, the blood was centrifuged at 2,000 × g for 15 minutes at room temperature. After centrifugation, the supernatant serum was carefully transferred to a 15 mL centrifuge tube in a laminar flow hood and inactivated at 55 °C for 45 minutes. The serum was then flash-frozen in liquid nitrogen and stored at -80 °C. Before use, the serum was thawed and filtered through a 0.45 μm (Millipore, HAWP04700) filter membrane.

[0228] 4. Immunofluorescence of cells and embryonic forms

[0229] Cells were fixed with 4% PFA (Beyotime, P0099) at room temperature for half an hour, and embryonic cells were fixed with 4% PFA at 4°C overnight. After fixation, the samples were blocked with blocking buffer (DPBS containing 5% v / v FBS, 2% v / v BSA, and 0.3% v / v Trition X-100) for 1 hour, and then the primary antibody diluted with blocking buffer was added and incubated at 4°C overnight. Primary antibody information: GATA6 (CellSignaling Technology, 5851); CDX2 (Biogenex, MU392A-UC); OCT4 (Cell Signaling Technology, 83932); T (Abcam, ab209665); SOX2 (Invitrogen eBioscience, 14-9811-82); AP2γ (Santa Cruz, sc-8977); PDGRFA (Invitrogen eBioscience, PA5-16571); KRT18 (Sigma-Aldrich, SAB4501665); DKK1 (Santa Cruz, sc-374574); GATA4 (Invitrogene Bioscience, 14-9980-82); N-Cadherin (Invitrogen eBioscience, 33-3900); E-Cadherin (Invitrogen eBioscience, 14-3249-82); FOXA2 (Cell Signaling Technology, 8186S).

[0230] On the second day, the sample was washed three times with PBST (DPBS containing 0.05% v / v Tween-20), and then incubated with Alexa Fluor secondary antibody diluted with blocking buffer at room temperature for 1 hour. The corresponding secondary antibodies were purchased from Thermo Fisher Scientific, catalog numbers: 21202, 31573, 31570, 31572, 31571, 21472, and 21447. Finally, DAPI (Sigma, D9542) was added for nuclear staining, and the sample was washed three times with PBST before imaging.

[0231] For embryos aged 3.5 days and older, clearing was performed after staining. The clearing procedure involved gradient treatment with 25%, 50%, 75%, 95%, and 100% thioethanol (v / v, diluted in DPBS; Sigma, 166782).

[0232] Finally, the samples were observed and photographed using a confocal microscope FV3000 (Olympus), and the images were analyzed using imageJ software (NIH, USA).

[0233] 5. Cell flow cytometry analysis

[0234] Cells were digested with Accutase (Invitrogen, 00455556) and resuspended in a suitable culture medium. The cell immunocytometry procedure was as follows: first, cells were blocked with a blocking buffer (containing 5% v / v FBS, 5% v / v NGS, and 2% v / v BSA in a Ca2+-free medium). 2+ and Mg 2+ Block with DPBS at room temperature for 20 minutes. Then add primary antibody (GATA6 (Cell Signaling Technology, 5851); CDX2 (Biogenex, MU392A-UC)) diluted in blocking buffer and incubate at room temperature for 1 hour. Then block with DPBS (Ca-free) 2+ and Mg 2+ After washing three times, add the secondary antibody diluted with blocking buffer (Thermo Fisher Scientific, 31571, 31572) and incubate at room temperature for 30 minutes. Then, use DPBS (Ca-free) to wash the solution. 2+ and Mg 2+ After washing three times, the cells were filtered through a 40μm filter (BD Biosciences, 352340), and flow cytometry analysis was performed using an LSR Tortessa X-20 (BD Biosciences). The results were then analyzed using FlowJo software. FACS cell sorting was performed using the BD FACSAriaⅢ instrument (BD Biosciences) to sort the target cell populations for subsequent experiments.

[0235] 6. RNA extraction and qPCR detection

[0236] Total RNA was extracted using the Ultrapure RNA Kit (CWBIO, CW0581M) and analyzed according to RevertAid. TM The FirstStrand cDNA Synthesis Kit (Thermo, K16225) instructions recommend using 1 μg of total RNA for reverse DNA synthesis. For qRT-PCR detection, use a TB [presumably a specific brand or kit]. Premix Ex Taq TM The Takara (RR820A) kit, with primers shown in Table 1, was used for detection via QuantStudio 3 Real-Time PCR System (Applied Biosystems) after spotting. The relative fold change was calculated using the ΔΔCt method, with Actin as the internal reference gene.

[0237] Table 1 qPCR primers

[0238]

[0239]

[0240] 7. Single-cell suspension preparation and sequencing

[0241] In the examples, the induced cells were digested with Accutase. Day 4.5 ci-embryoid cells induced in the examples were digested with 100 μL of 10 U / mL papain (Worthington Biochemicals, LS003126) at 37°C for 15 minutes. The dissociated cells were then digested with pre-cooled DPBS (Ca-free). 2+ and Mg 2+ After washing twice, the cells were filtered through a 40 μm filter (BD Biosciences, 352340) to obtain a single-cell suspension, which was then loaded into a 10×Genomics Chromium system at a concentration of 16,000 single cells per sample using a Single Cell 3'ReagentKits V3.1. The library was sequenced in PE 100 mode on an MGISEQ 2000 (MGI Tech) system.

[0242] 8. Preprocessing of single-cell RNA sequencing data

[0243] A reference genome was constructed following the guidelines provided on the 10xGenomics official website, and all single-cell RNA sequencing data were analyzed accordingly (https: / / support.10xgenomics.com / single-cell-gene-expression / software / pipelines / latest / using / tutorial_mr). The pre-constructed genome sequences and gene annotation files used were downloaded from the GRCm39 104 version of the Ensembl database. First, the raw data were quality controlled using fastp (v.0.21.0, https: / / github.com / OpenGene / fastp) with default parameters. Then, STAR (v.2.7.9a) was used, following the standard STARsolo analysis workflow (https: / / github.com / alexdobin / STAR / blob / master / docs / STARsolo.md) for alignment, annotation, PCR repeat removal, and gene expression quantification of the quality-controlled data.

[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 fertilized egg to E7.5, which includes all known cell lineages) were downloaded and preprocessed using the same methods as the data generated in this study, or adjusted and postprocessed as instructed by the authors of the source articles.

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

[0247] Linear dimensionality reduction and principal component analysis (PCA) were performed. Further analysis was conducted using Seurat software (v.4.3.0) in R 4.2.2. Based on preliminary assessments of each cell quality control indicator, cells with fewer than 4000 detected genes (Smart-seq2 data), 2000 or more (10× genome data), or greater than or equal to 5% of mitochondrial gene percentages were removed, and only genes detected in at least 3 cells were retained. The filtered sequencing data were 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 with default parameters based on the selected highly variable genes. The PCA coordinates were then used as input for a 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 Day 4.5 ci-embryoid dataset obtained in this embodiment with the public natural embryo dataset, the standard canonical correlation analysis (CCA) method was used. Figure 10 This process followed the standard procedures provided on the Seurat website (https: / / satijalab.org / seurat / articles / integration_introduction.html). This was done to integrate public data from multiple sources across different cell lineages and developmental stages. Figure 5 , 6The inventors used the fastMNN method, treating each dataset from different experiments as a batch, with each batch containing at least one cell population shared with other cells. The data was log-normalized using the `computeSumFactors` function from the `scran` package (v.1.20.1), and then scaled normalized across batches using the `multiBatchNorm` function from the `batchchelor` package (v.1.8.1). The log-normalized and batch-effect-corrected datasets were then integrated using the fastMNN method, implemented via `SeuratWrappers` (v.0.3.0, https: / / github.com / satijalab / seurat-wrappers). The low-dimensional coordinates of the MNN 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 constructed a shared nearest neighbor (SNN) graph using the FindNeighbors function, then partitioned it using the Louvain algorithm implemented with the FindClusters function, with the resolution range selected based on 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 generate blastocyst-like lineage precursor cells, comprising: a first culture medium; the first culture medium is N2B27 basal medium (CL6A) containing 1 μM CHIR99021 (Selleck, S2924), 10 ng / mL mouse leukemia inhibitory factor (murine LIF, qKine, Qk018), 1 μM E616452 (Selleck, S7223) and 0.01 μM AM580 (Selleck, S2933);

[0254] The N2B27 basal medium was 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). The mixed medium was prepared 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 produce 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 N2B27 basal medium (CL6A) containing 3 μM CHIR99021 (Selleck, S2924), 20 ng / mL mouse leukemia inhibitory factor (murine LIF, qKine, Qk018), 10 μM ME616452 (Selleck, S7223) and 0.05 μM AM580 (Selleck, S2933);

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

[0258] Examples 1-3: A kit for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells

[0259] A kit for inducing stem cells to generate blastocyst-like lineage precursor cells, comprising: a first culture medium; the first culture medium is N2B27 basal medium (CL6A) containing 6 μM CHIR99021 (Selleck, S2924), 20 ng / mL mouse leukemia inhibitory factor (murine LIF, qKine, Qk018), 10 μM E616452 (Selleck, S7223) and 0.05 μM AM580 (Selleck, S2933);

[0260] The N2B27 basal medium was 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). The mixed medium was prepared by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0261] Examples 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 generate blastocyst-like lineage precursor cells, comprising: a first culture medium; the first culture medium is N2B27 basal medium (CL6A) containing 15 μM CHIR99021 (Selleck, S2924), 100 ng / mL mouse leukemia inhibitory factor (murine LIF, qKine, Qk018), 15 μM E616452 (Selleck, S7223) and 0.1 μM AM580 (Selleck, S2933);

[0263] The N2B27 basal medium was 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). The mixed medium was prepared by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0264] Example 2: A kit for inducing mouse embryonic stem cells to produce 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 was N2B27 basal medium (CL6A) containing 1 μM CHIR99021, 10 ng / mL mouse leukemia inhibitory factor, 1 μME616452 and 0.01 μMAM580;

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

[0268] The N2B27 basal 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). The mixed medium is prepared by mixing DMEM / F12 (Gibco, C11330500CP) and Neurobasal (Gibco, 21103049) at a volume ratio of 1:1.

[0269] Example 3: A kit for inducing mouse embryonic stem cells to produce 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 was N2B27 basal medium (CL6A) containing 3 μM CHIR99021, 20 ng / mL mouse leukemia inhibitory factor, 5 μME616452 and 0.02 μMAM580;

[0272] The second culture medium was N2B27 basal medium (C6A) containing 3 μM CHIR99021, 5 μM E616452 and 0.02 μM AM580;

[0273] The N2B27 basal 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). The mixed medium is prepared 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 produce blastocyst-like lineage precursor cells

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

[0276] The first culture medium was N2B27 basal medium (CL6A) containing 6 μM CHIR99021, 20 ng / mL mouse leukemia inhibitory factor, 10 μME616452 and 0.05 μMAM580;

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

[0278] The N2B27 basal 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). The mixed medium is prepared 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 produce blastocyst-like lineage precursor cells

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

[0281] The first culture medium was N2B27 basal medium (CL6A) containing 3 μM CHIR99021, 20 ng / mL mouse leukemia inhibitory factor, 10 μME616452 and 0.05 μMAM580;

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

[0283] The N2B27 basal 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). The mixed medium is prepared 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 produce 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 was N2B27 basal medium (CL6A) containing 9 μM CHIR99021, 50 ng / mL mouse leukemia inhibitory factor, 12 μME616452 and 0.08 μMAM580;

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

[0288] The N2B27 basal 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). The mixed medium is prepared 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 produce blastocyst-like lineage precursor cells

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

[0291] The first culture medium was N2B27 basal medium (CL6A) containing 15 μM CHIR99021, 100 ng / mL mouse leukemia inhibitory factor, 15 μME616452 and 0.1 μMAM580;

[0292] The second culture medium was N2B27 basal medium (C6A) containing 15 μM CHIR99021, 15 μM E616452 and 0.1 μM AM580;

[0293] The N2B27 basal 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). The mixed medium is prepared 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 embryo-like structures

[0295] 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;

[0296] The first culture medium and the second culture medium are the first culture medium and the second culture medium in Example 5, which are used to induce mouse embryonic stem cells to produce blastocyst-like lineage precursor cells.

[0297] The third culture medium was FC basal medium (ciBP medium) containing 25 ng / mL rhFGF4 (R&D, 235F4), 10 ng / mL hBMP4 (Miltenyi, 130111167), 20 ng / mL activin A (Activin A, PeproTech, 12014E), 3 μM XAV939 (Sigma, X3004), 2 μM TRULI (Selleck, E1061), 1 μg / mL heparin (Heparin, Sigma, H3149), 200 μM L-ascorbic acid 2-phosphate (Sigma, A8960) and 1× insulin-transferrin-selenium supplement (ITS-G, Gibco, 41400045), which was used to induce blastocyst-like lineage precursor cells to produce blastocyst-like cells;

[0298] The fourth culture medium is FC basal medium;

[0299] The fourth culture medium is used to induce blastocyst-like cells to produce embryo-like cells;

[0300] The fifth culture medium contained 30% (v / v) FBS (Carpicorn Scientific, FBS52A), 1x GlutaMax (Gibco, 35050038), 1× insulin-transferrin-selenoethanol (ITS-X, Gibco, 51500056), 100 nMT3 (3,3',5-triiodo-L-thyronine sodium salt, Sigma, T6397), 8 nM estradiol (β-estradiol, Sigma, E8875), 200 ng / mL progesterone (progesterone, Sigma, P0130), 25 μM acetylcysteine ​​(N-acetyl-L-cysteine, Sigma, C7880), and 1 mg / mL Advanced DMEM / F12 medium (Gibco, 12634028) of D-glucose (D-glucose, sigma, G8644) and 1% (v / v) penicillin / streptomycin (Gibco, 15140122) [4,13] (IVC1 medium);

[0301] The sixth culture medium was DMEM 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). This medium was low in glucose, contained pyruvate, and was glutamine-free and phenol-free. red), Gibco, 11880) [4,13] (IVC2 medium);

[0302] The fifth and sixth culture media are used for the culture and / or maturation of embryo-like organisms;

[0303] 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].

[0304] Example 8-2: A kit for constructing embryo-like structures

[0305] A kit for constructing embryo-like structures is identical to the kit for constructing embryo-like structures in Example 8-1, except that the first and second culture media are the same as those in Example 2.

[0306] Example 8-3: A kit for constructing embryo-like structures

[0307] A kit for constructing embryo-like structures is identical to the kit for constructing embryo-like structures in Example 8-1, except that the first and second culture media are the same as those in Example 3.

[0308] Example 8-4: A kit for constructing embryo-like structures

[0309] A kit for constructing embryo-like structures is identical to the kit for constructing embryo-like structures in Example 8-1, except that the first and second culture media are the same as those in Example 4.

[0310] Example 8-5: A kit for constructing embryo-like structures

[0311] A kit for constructing embryo-like structures is identical to the kit for constructing embryo-like structures in Example 8-1, except that the first and second culture media are the same as those in Example 6.

[0312] Examples 8-6: A kit for constructing embryo-like structures

[0313] A kit for constructing embryo-like structures is identical to the kit for constructing embryo-like structures in Example 8-1, except that the first and second culture media are the same as those in Example 7.

[0314] Example 9: A kit for constructing embryo-like structures

[0315] 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;

[0316] The first culture medium and the second culture medium are the first culture medium and the second culture medium in Example 2, which are used to induce mouse embryonic stem cells to produce blastocyst-like lineage precursor cells.

[0317] The third culture medium was FC basal medium containing 10 ng / mL rhFGF4 (R&D, 235F4), 5 ng / mL hBMP4 (Miltenyi, 130111167), 10 ng / mL activin A (Activin A, PeproTech, 12014E), 1 μM XAV939 (Sigma, X3004), 1 μM TRULI (Selleck, E1061), 0.5 μg / mL heparin (Heparin, Sigma, H3149), 50 μM L-ascorbic acid 2-phosphate (Sigma, A8960), and 0.5 × insulin-transferrin-selenium supplement (ITS-G, Gibco, 41400045), which was used to induce blastocyst-like lineage precursor cells to produce blastocyst-like cells;

[0318] The fourth culture medium is FC basal medium;

[0319] The fourth culture medium is used to induce blastocyst-like cells to produce embryo-like cells;

[0320] The fifth culture medium contained 10% (v / v) FBS (Carpicorn Scientific, FBS52A), 0.5 mM GlutaMax (Gibco, 35050038), 0.5× insulin-transferrin-selenoethanol (ITS-X, Gibco, 51500056), 50 nM T3 (3,3',5-triiodo-L-thyronine sodium salt, Sigma, T6397), 5 nM estradiol (β-estradiol, Sigma, E8875), 100 ng / mL progesterone (progesterone, Sigma, P0130), 5 μM acetylcysteine ​​(N-acetyl-L-cysteine, Sigma, C7880), and 0.5 mg / mL... Advanced DMEM / F12 medium (Gibco, 12634028) (IVC1 medium) containing D-glucose (D-glucose, Sigma, G8644) and 0.1% (v / v) penicillin / streptomycin (Gibco, 15140122).

[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 culture medium was FC basal medium containing 50 ng / mL rhFGF4 (R&D, 235F4), 20 ng / mL hBMP4 (Miltenyi, 130111167), 40 ng / mL activin A (Activin A, PeproTech, 12014E), 9 μM XAV939 (Sigma, X3004), 5 μM TRULI (Selleck, E1061), 2 μg / mL heparin (Heparin, Sigma, H3149), 500 μM L-ascorbic acid 2-phosphate (Sigma, A8960) and 2× insulin-transferrin-selenium supplement (ITS-G, Gibco, 41400045), which was used to induce blastocyst-like lineage precursor cells to produce blastocyst-like cells;

[0328] The fourth culture medium is FC basal medium;

[0329] The fourth culture medium is used to induce blastocyst-like cells to produce embryo-like cells;

[0330] The fifth culture medium contained 40 (v / v) FBS (Carpicorn Scientific, FBS52A), 2×GlutaMax (Gibco, 35050038), 2×Insulin-Transferrin-Selenyl-Aminoethanol (ITS-X, Gibco, 51500056), 200 nMT3 (3,3',5-Triiodo-L-thyronine sodium salt, Sigma, T6397), 11 nM estradiol (β-estradiol, Sigma, E8875), 300 ng / mL progesterone (progesterone, Sigma, P0130), 45 μM acetylcysteine ​​(N-acetyl-L-cysteine, Sigma, C7880), and 2 mg / mL Advanced DMEM / F12 medium (Gibco, 12634028) (IVC1 medium) containing D-glucose (D-glucose, sigma, G8644) and 2% (v / v) penicillin / streptomycin (Gibco, 15140122).

[0331] The sixth culture medium was DMEM containing 60% (v / v) rat serum (made in house), 35% (v / v) human serum AB (Off the Clot, Gemini, 100-318), 2×GlutaMax (Gibco, 35050038), 2×sodium pyruvate (Gibco, 11360039), 20mM HEPES (Gibco, 15630130), 6mg / mL D-glucose (Sigma, G8644), and 2% (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);

[0332] The fifth and sixth culture media are used for the culture and / or maturation of embryo-like organisms;

[0333] 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].

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

[0335] A method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells, comprising the steps of using the kit of Example 1-1, as detailed below:

[0336] With 2×10 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 for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0347] A method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as in Example 12, except that the kit in Example 2 is replaced with the kit in Example 3, the culture time of the second culture medium is 30 h, and the culture time of the first culture medium is 48 h.

[0348] Example 14: A method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0349] A method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as in Example 12, except that the kit in Example 2 is replaced with the kit in Example 4, the culture time of the second culture medium is 24 h, and the culture time of the first culture medium is 36 h.

[0350] Example 15: A method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0351] A method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as in Example 12, except that the kit in Example 2 is replaced with the kit in Example 5, the culture time of the second culture medium is 24 h, and the culture time of the first culture medium is 36 h.

[0352] Example 16: A method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0353] A method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as in Example 12, except that the kit in Example 2 is replaced with the kit in Example 6, the culture time in the second culture medium is 18 h, and the culture time in the first culture medium is 24 h.

[0354] Example 17: A method for inducing mouse embryonic stem cells to generate blastocyst-like lineage precursor cells

[0355] A method for inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as in Example 12, except that the kit in Example 2 is replaced with the kit in Example 7, the culture time in the second culture medium is 12 h, and the culture time in the first culture medium is 24 h.

[0356] Example 18-1 A method for constructing an embryo-like structure

[0357] A method for constructing embryo-like structures, comprising the steps of using the kit from Example 8-1, is as follows:

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

[0359] (2) Induction of blastocyst-like lineage precursor cells to generate blastocyst-like cells (ciBPCs): Cells obtained in step (1) were digested with Accutase (Invitrogen, 00455556), centrifuged, resuspended in DMEM medium (Gibco, A1443001), and counted; Aggrewell 400 plates (Stemcell, 34415) treated with anti-adhesion rinsing solution (Stemcell, 7010) were prepared; cells were sputtered at 3 × 10⁻⁶ cells / cm². 4 The cells were seeded at a density of cells / well in Aggrewell 400, and 1.5 mL of the third medium from Example 8-1 was added. The cells were then incubated at 37°C and 5% CO2 for 36 h.

[0360] (3) Induction of ci-Embryoids from blastocyst-like cells (ciBPCs): Remove the third culture medium (referred to as Day 0 for embryo-like induction and culture), and gently wash twice with the fourth culture medium. Add 1 mL of the fourth culture medium from Example 8-1, and culture at 37°C and 5% CO2 for 1.5 days (36 h) (Day 1.5 is the end of the culture). Remove the fourth culture medium, add 1.5 mL of the fifth culture medium from Example 8-1, and culture at 37°C and 5% CO2 for 1 day (24 h) (Day 2.5 is the end of the culture). Gently resuspend the embryo-like cells in Aggrewell 400 and transfer them to a six-well suspension culture plate (Greiner, 657185). Add 4 mL of the fifth culture medium and culture on a shaker (Zhichu, ZCLY180N, shaker settings: 80 rpm, 37°C, 5%). Continue culturing on CO2 for another day (24h) (Day 3.5 is the end of the culture); transfer the embryo-like cells to a 5mL bioreactor (ABLEBiott, BWV-S005A) and add 3mL of the sixth culture medium from Example 8-1. Continue culturing on the substrate of the bioreactor magnetic stir system (6-ch) (Bioreactor Magnetic Stir System-6ch.Base plate, ABLE Biott, BWS-S03N0S-6B) at 37°C and 5% CO2 for another day (24h) (Day 4.5 is the end of the culture), with the system speed set to 50rpm.

[0361] Example 18-2 A method for constructing an embryo-like structure

[0362] A method for constructing embryo-like cells includes 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 of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as the method of Example 12, and the culture time of the third culture medium is 24 hours.

[0363] Example 18-3 A method for constructing embryo-like structures

[0364] A method for constructing embryo-like cells includes 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 of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as the method of Example 13, and the culture time of the third culture medium is 32h.

[0365] Example 18-4 A method for constructing embryo-like structures

[0366] A method for constructing embryo-like cells includes the steps of using the kit of Examples 8-4, which is the same as the method of Examples 18-1, except that the kit of Example 8-1 is replaced with the kit of Example 8-4, the method of inducing mouse embryonic stem cells to produce blastocyst-like lineage precursor cells is the same as the method of Example 14, and the culture time of the third culture medium is 36 hours.

[0367] Example 18-5 A method for constructing embryo-like structures

[0368] A method for constructing embryo-like cells includes the steps of using the kits of Examples 8-4, which are the same as the method of Examples 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 produce blastocyst-like lineage precursor cells is the same as the method of Example 14, and the culture time of the third culture medium is 48 hours.

[0369] Example 18-6 A method for constructing embryo-like structures

[0370] A method for constructing embryo-like cells includes the steps of using the kits of Examples 8-5, which are the same as the method of Examples 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 produce blastocyst-like lineage precursor cells is the same as the method of Example 16, and the culture time of the third culture medium is 40 h.

[0371] Example 18-7 A method for constructing embryo-like structures

[0372] A method for constructing embryo-like cells includes the steps of using the kits of Examples 8-6, which are the same as those of Examples 18-1, except that the kit of Example 8-1 is replaced with the kit of Example 8-6, 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 in the third culture medium is 48 hours.

[0373] Example 19: A method for constructing an embryo-like structure

[0374] A method for constructing embryo-like structures includes 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 an embryo-like structure

[0376] A method for constructing embryo-like structures includes 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 is the same as in Example 11-1, except that the first culture medium in Example 1-1 is replaced with 2iLIF culture medium.

[0379] Effect Example

[0380] 1. The blastocyst-like lineage precursor cells obtained in Examples 11-1 to 11-4 and 12 to 17 (referred to sequentially as Q0-a, Q0-b, Q0-c, Q0-d, Q1, Q2, Q3, Q4, Q5, and Q6) and the mouse embryonic stem cells (ESCs) obtained in Comparative Example 1 were subjected to flow cytometry analysis. The results are as follows: Figure 2As shown: In the mouse embryonic stem cells (ESCs) obtained in Comparative Example 1: 0.52% were GATA6 positive cells, i.e., PrE lineage precursor cells; 0.088% were CDX2 positive cells, i.e., TE lineage precursor cells; and 98.9% were OCT4 positive cells, i.e., EPI-like cells. In the cells obtained in Example 11-1 (Q0-a): 35.3% were GATA6 positive cells, i.e., PrE lineage precursor cells; 5.11% were CDX2 positive cells, i.e., TE lineage precursor cells; and 68.2% were OCT4 positive cells, i.e., EPI-like cells. In the cells obtained in Example 11-2 (Q0-b): 36.3% were GATA6 positive cells, i.e., PrE lineage precursor cells. In Example 11-3 (Q0-c), 1.28% of the cells were CDX2-positive (TE lineage precursor cells), and 90.5% were OCT4-positive (EPI-like cells). In Example 11-4 (Q0-d), 22.8% of the cells were GATA6-positive (PrE lineage precursor cells), 56.3% were CDX2-positive (TE lineage precursor cells), and 61.2% were OCT4-positive (EPI-like cells). In Example 11-4 (Q0-d), 21.9% were GATA6-positive (PrE lineage precursor cells), 61.2% were CDX2-positive (TE lineage precursor cells), and 60.6% were OCT4-positive (EPI-like cells). Figure 2In Example 12 (Q1), 34.3% of the cells were GATA6 positive (PrE lineage precursor cells), 6.45% were CDX2 positive (TE lineage precursor cells), and 67.5% were OCT4 positive (EPI-like cells). In Example 13 (Q2), 34.5% of the cells were GATA6 positive (PrE lineage precursor cells), 15.6% were CDX2 positive (TE lineage precursor cells), and 87.6% were OCT4 positive (EPI-like cells). In Example 14 (Q3), 31.9% of the cells were GATA6 positive (PrE lineage precursor cells), 26.8% were CDX2 positive (TE lineage precursor cells), and 67.9% were OCT4 positive (EPI-like cells). Cells; In Example 15 (Q4), 26.1% were GATA6-positive cells (PrE lineage precursor cells), 29.2% were CDX2-positive cells (TE lineage precursor cells), and 67.1% were OCT4-positive cells (EPI-like cells); In Example 16 (Q5), 28.4% were GATA6-positive cells (PrE lineage precursor cells), 26.8% were CDX2-positive cells (TE lineage precursor cells), and 59.8% were OCT4-positive cells (EPI-like cells); In Example 17 (Q6), 24.6% were GATA6-positive cells (PrE lineage precursor cells), 43.5% were CDX2-positive cells (TE lineage precursor cells), and 64.7% were OCT4-positive cells (EPI-like cells). Figure 2 (D and E); Similarly, joint analysis of the distribution of GATA6-positive and CDX2-positive cells in the total cell population showed that Q0-a, Q0-b, Q0-c, Q0-d, Q1, Q2, Q3, Q4, Q5, and Q6 all had high GATA6 activation, while CDX2 activation was less distributed in Q0-a, Q0-b, and Q1, and excessive in Q0-c, Q0-d, and Q6. The distribution ratios of CDX2 and GATA6-positive cells were similar in Q3, Q4, and Q5. Figure 2 (C and E); It can be seen that the kits in Examples 1-1 to 1-4 and 2-7 can induce embryonic stem cells to produce PrE lineage precursor cells, TE lineage precursor cells, and EPI-like cells, thus obtaining complete blastocyst lineage precursor cells. Examples 11-1 to 11-4 can obtain all lineages of blastocyst-like lineage precursor cells; however, the proportions of the three blastocyst-like lineage cell populations are relatively unbalanced, with Q0-a and Q0-b containing fewer CDX2-positive cells, while Q0-c and Q0-d have a higher proportion of CDX2-positive cells.

[0381] 2. Immunofluorescence experiments were performed on the blastocyst-like lineage precursor cells obtained in Examples 12-17 (referred to sequentially as Q1, Q2, Q3, Q4, Q5, and Q6) and the mouse embryonic stem cells (ESCs) obtained in Comparative Example 1. The results are as follows: Figure 3 As shown: ESC cells exhibit a round morphology and express only the EPI-tagged protein OCT4; while Q1, Q2, Q3, Q4, Q5, and Q6 cells showed varying levels of the TE-tagged protein CDX2 and the PrE lineage marker protein GATA6; among them, Q3, Q4, and Q5 cells not only showed a relatively balanced GATA6 and CDX2 activation ratio in both immunoflow cytometry and immunofluorescence analysis, but also exhibited relatively healthy cell morphology. Figure 2 China E and Figure 3 As can be seen, the blastocyst-like lineage precursor cells obtained in Examples 14-16 have three balanced blastocyst-like lineage cells ( Figure 2 China E and Figure 3 RNA extraction and qPCR detection were performed on cells cultured for different times (0, 12, 24, 36, 48, 60 h) from Examples 14 and 15 (referred to as Q3 and Q4, respectively). The results showed that the expression of trophectoderm marker genes (Gata3, Hand1, Id2) gradually increased, with Cdx2 showing a peak expression at 24-36 h. Primitive endoderm marker genes (Gata6, Gata4, Sox17, Foxa2) showed a gradient increase over time. Furthermore, the primitive marker genes Nanog and Sox2 were downregulated, while the primordial genes Otx2 and Fgf5 did not increase. This indicates that the cells did not differentiate, but rather, the trophectoderm and primitive endoderm lineages were stimulated from the epiblast lineage cells. Figure 4 ).

[0382] 3. Take Examples 12-17 (corresponding to...) Figure 5 The values ​​are Q1 (108h), Q2 (78h), Q3 (60h), Q4 (60h), Q5 (42h), and Q6 (36h). Figure 5 In the text, Q3 & Q4 (60h) represent the blastocyst-like lineage precursor cells obtained from Example 14 (Q3 (60h)) and Example 15 (Q4 (60h)), respectively. Examples 18-1 to 18-7 (corresponding to...) Figure 6 , 7The blastocyst-like cells obtained from Q4-ciBP (36h), Q1-ciBP (24h), Q2-ciBP (32h), Q3-ciBP (36h), Q3-ciBP (48h), Q5-ciBP (40h), and Q6-ciBP (48h) were subjected to single-cell transcriptome analysis (single-cell suspension preparation and sequencing, single-cell RNA sequencing data preprocessing, single-cell RNA sequencing data downstream analysis, batch effect correction and integration of multi-source Smart-seq2 and 10x Genomics single-cell RNA sequencing data, visualization using graph-based clustering and Uniform Manifold Approximation and Projection (UMAP) were performed sequentially). The blastocyst-like lineage precursor cells obtained in step (1) of Example 18-1 (i.e., the blastocyst-like lineage precursor cells obtained in Example 15) and the blastocyst-like cells obtained in step (2) were subjected to RNA extraction and qPCR detection, as well as immunofluorescence experiments. The results are as follows. Figures 5-8 As shown: Blastocyst-like precursor cells obtained after induction with small molecules Q1 (108h), Q2 (78h), Q3 (60h), Q4 (60h), Q5 (42h), and Q6 (36h) can be divided into four main groups based on early embryo reference data: epiblast precursor cells, primitive endoderm precursor cells, trophectoderm precursor cells, and intermediate cells. Figure 5 Among them, epiblast precursor-like cells are located between the E3.5 inner cell mass and the E4.5 epiblast cells, exhibiting a certain degree of reprogramming, and belong to epiblast-like precursor cells; primitive endoderm precursor-like cells are located between the E4.5 primitive endoderm cells and the E3.5 inner cell mass, possessing the potential to develop into PrE, and are precursors of this cell type; trophectoderm precursor-like cells are located between the epiblast and trophectoderm cells, and are trophectoderm precursor cells; while intermediate cells possess characteristics of precursors from two or three lineages, hence they are intermediate cells. Figure 5 After culturing blastocyst-like lineage precursor cells with ciBP (Q4-ciBP (36h), Q1-ciBP (24h), Q2-ciBP (32h), Q3-ciBP (36h), Q3-ciBP (48h), Q5-ciBP (40h), and Q6-ciBP (48h), three lineage-specific cell types were obtained: epiblast-like cells, primitive endoderm-like cells, and trophectoderm-like cells; these three cell groups clustered with E4.5 EPI, E4.5 PrE, and E4.5 TE, respectively. Figure 6 Gene dimensionality reduction analysis showed that after ciBP specialization, blastocyst-like precursor cells exhibited significant clustering and yielded three different proportions of blastocyst lineage cells: epiblast-like cells, primitive endoderm-like cells, and trophectoderm-like cells. Figure 7It can be seen that the cells obtained after further induction in the third culture medium have properties similar to the three lineages of cells from the natural embryo E4.5 blastocyst. The obtained blastocyst-like cells have the cell characteristics of three lineages: epiblast, primitive endoderm, and trophectoderm (analyzed at the single-cell transcriptome, protein, and RNA levels). The results of the blastocyst-like lineage precursor cells obtained in step (1) and the blastocyst-like cells obtained in step (2) of Examples 19 and 20 are similar to those of Examples 12-17 and 18-1 to 18-7.

[0383] Single-cell transcriptome analysis was performed on blastocyst-like precursor cells from Q4 (60h) (Example 15) and Q4-ciBP (36h) (Example 18-1). Epiblast marker genes (Nanog, Sox2, Pou5f1, Igfbp2, Fgf4, Tdgf1), primitive endoderm marker genes (Gata6, Gata4, Sox17, Foxa2, Cubn, Srgn), and trophectoderm marker genes (Cited, Rhox6, Rhox9, Id2, Krt8, Krt18) were detected. After maturation in ciBP's third medium, Q4-ciBP (36h) blastocyst-like cells expressed significantly higher levels of these marker genes than Q4 (60h) blastocyst-like precursor cells. This indicates that ciBP has the function of maturing and specializing blastocyst-like precursor cells. Figure 8 (A). Immunofluorescence staining also showed expression of proteins specific to the blastocyst-like lineage (KRT18 / PDGFRa / OCT4). Figure 8 (B). Furthermore, trophoblast cells in the blastocyst stage exhibit heterogeneity, containing both polar trophoblast cells (TE) and mural trophoblast cells (TE); markers for these two cell groups (CDX2 and KRT18) were also found in the trophoblast-like cells of this invention. Figure 8 (B and C). Thermal analysis of three cell populations of blastocyst-like precursor cells induced by small molecules at 96 hours with E4.5 natural embryos showed that they had very similar molecular characteristics. Figure 8 (D). It can be seen that blastocyst-like lineage cells have similar cellular characteristics to E4.5 blastocyst lineage cells at the single-cell transcriptome, protein, and RNA levels.

[0384] 4. Take the embryo-like cells (ci-Embryoids) obtained in step (3) of Example 18-1 for Day 1.5, Day 2.5, Day 3.5 and Day 4.5 for microscopic observation, immunohistochemical staining, immunofluorescence staining, and single-cell transcriptome analysis (preparing single-cell suspension and sequencing, preprocessing single-cell RNA sequencing data, downstream analysis of single-cell RNA sequencing data, batch effect correction and integration of multi-source Smart-seq2 and 10x Genomics single-cell RNA sequencing data, and visualization using graph-based clustering and Uniform Manifold Approximation and Projection (UMAP)). It is known that blastocyst-like cells have full lineage primitive cell characteristics and developmental totipotency. In order to further verify their developmental totipotency and construct in vitro synthetic embryos, and to study their lineage differentiation and gastrulation potential, an in vitro reconstructed embryo system based on ciBPC was established. First, ciBPCs were induced at timed intervals by FC, IVC1, and IVC2, combined with a static and dynamic culture mode, to obtain ci-Embryoids ( Figure 9 (A). Observations on embryonic development are as follows: After overnight incubation, ciBPCs aggregated into clusters. On day 2.5, rosette-to-lumen structural changes began, with a cell layer forming on the outside of the embryo. Cells derived from the primitive endoderm (GATA6 marker) encapsulated embryonic TE (CDX2 marker) and EPI cells (OCT4 marker). On day 3.5, the morphology of the embryonic aggregates resembled that of natural embryos at E5.5-6.5, showing a clear cup-shaped structure. On day 4.5, the embryo continued to develop, forming an enlarged amniotic cavity and successfully forming an asymmetric structure, with the appearance of protostome tissue (protostome marker T expressed on one side, AVE marker DKK1). Figure 9 (A). In day 4.5 embryonic-like structures, all embryonic characteristics resembling those of the natural embryonic gastrulation stage were observed. Figure 9 (B), which is much higher than the efficiency of previous work [4] which was less than 0.5%, 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 9 (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. Figure 10 Furthermore, the ci-Embryoids constructed by ciBPC have the correct expression patterns. Figure 10-12Not 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 11-12 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. Figure 13 (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.

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Claims

1. A reagent combination or kit comprising a first culture medium and a second 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; The GSK-3 inhibitor in the first culture medium was CHIR-99021, with a concentration of 1–15 μM; The STAT3 activator in the first culture medium is a leukemia inhibitory factor, with a concentration of 10–100 ng / mL; The retinoic acid nuclear receptor agonist in the first culture medium was AM580, at a concentration of 0.01–0.1 μM; The TGF-β receptor kinase inhibitor in the first culture medium is E616452, with a concentration of 1–20 μM; The second culture medium is a basal medium containing GSK-3 inhibitors, retinoic acid nuclear receptor agonists, and TGF-β receptor kinase inhibitors; The GSK-3 inhibitor in the second culture medium was CHIR99021, with a concentration of 1–15 μM; The retinoic acid nuclear receptor agonist in the second culture medium was AM580, with a concentration of 0.01–0.1 μM; The TGF-β receptor kinase inhibitor in the second culture medium was E616452, with a concentration of 1–20 μM.

2. The reagent combination or kit according to claim 1, 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. The fibroblast growth factor in the third culture medium is FGF4, with a concentration of 10–50 ng / mL; The BMP4 signaling pathway activator in the third culture medium is BMP4, with a concentration of 5–20 ng / mL; The TGF-β activator in the third culture medium is activator A, with a concentration of 10–40 ng / mL; The WNT signaling pathway inhibitor in the third culture medium is XAV939, with a concentration of 1–9 μM. The Lats kinase inhibitor in the third culture medium is TRULI, with a concentration of 1–5 μM; The anticoagulant in the third culture medium contains at least one of heparin and EDTA salt; The ascorbic acid or its derivatives in the third culture medium include at least one of the following: ascorbic acid, calcium ascorbate, magnesium ascorbate, zinc ascorbate, potassium ascorbate, sodium ascorbate, dehydroascorbic acid, L-ascorbic acid monostearate, L-ascorbic acid dipalmitate, L-ascorbic acid 6-hexadecanoic acid compound, L-ascorbic acid 2-phosphide, L-ascorbic acid 3-phosphide, and L-ascorbic acid 2-sulfate. The concentration of the anticoagulant in the third culture medium is 0.5–2.0 μg / mL; The concentration of ascorbic acid or its derivative in the third culture medium is 50–500 μM; The concentration of the insulin-transferrin-selenium additive in the third culture medium is 0.5–2×.

3. The reagent combination or kit according to claim 2, 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.

4. The reagent combination or kit of claim 3, wherein: The reagent combination or kit also includes 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 thyroid hormone receptor agonist in the fifth culture medium contains at least one of thyroxine or its salt, triiodothyronine or its salt; The concentration of the serum in the fifth culture medium was 10-40% by volume. The concentration of glutamine in the fifth culture medium is 0.5–2×. The concentration of insulin-transferrin-seleno-aminoethanol in the fifth culture medium is 0.5–2×. The concentration of the thyroid hormone receptor agonist in the fifth culture medium is 50–200 nM; The concentration of estradiol in the fifth culture medium was 5–11 nM; The concentration of progesterone in the fifth culture medium is 100–300 ng / mL; The concentration of acetylcysteine ​​in the fifth culture medium is 5–45 μM; The concentration of glucose in the fifth culture medium is 0.5–2 mg / mL; The sixth culture medium is a basal culture medium containing serum, glutamine, pyruvate or its salts, buffer salts, glucose and antibiotics; The concentration of the serum in the sixth culture medium was 35-95% by volume. The concentration of glutamine in the sixth culture medium is 0.5–2×. The concentration of pyruvate or its salt in the sixth culture medium is 0.5–2×. The concentration of the buffer salt in the sixth culture medium is 6–20 mM; The concentration of glucose in the sixth culture medium is 1–6 mg / mL; The concentration of the antibiotic in the fifth and sixth culture media was 0.1% to 2% by volume.

5. The reagent combination or kit according to claim 4, characterized in that: 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 Basal 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.

6. The reagent combination or kit according to claim 5, characterized in that: The basal medium for the first culture medium and / or the second culture medium is DMEM / F12 medium and Neurobasal medium; 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; The basal medium for the third culture medium is DMEM medium; 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; The basal medium for the fifth culture medium is Advanced DMEM / F12 medium; The basal medium for the sixth culture medium is DMEM medium.

7. The use of the reagent combination or kit according to any one of claims 1 to 6 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; The stem cells are stem cells with multi-directional differentiation potential; The stem cells with multi-directional differentiation potential are embryonic stem cells.

8. A method for inducing stem cells to generate blastocyst-like lineage precursor cells, comprising the step of using the reagent combination or kit according to any one of claims 1 to 6.

9. The method according to claim 8, 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 6; 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 6, and then the stem cells are cultured for the second time using the first culture medium in the reagent combination or kit according to any one of claims 1 to 6. The incubation time described in S1 is 36–108 h; The first culture time described in S2 is 12–36 hours; The second culture time described in S2 is 24–72 hours; The culture in S1, the first culture in S2, and / or the second culture in S2 are adherent cultures.

10. 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 6; The method for inducing stem cells to produce 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 8 or 9; T2: Induces blastocyst-like lineage precursor cells to produce blastocyst-like cells.

11. The method according to claim 10, 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 6. The third culture period is 24–48 hours; The third culture was a suspension culture.

12. 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 6; 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 10 or 11; U2: Induces blastocyst-like cells to produce embryo-like cells.

13. The method according to claim 12, 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 6; The fourth culture period is 24–48 hours. The fourth culture was a suspension culture.

14. The method according to claim 13, characterized in that: 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: the embryo-like cells obtained after the fourth culture are cultured for the fifth time and the sixth time in sequence using the fifth culture medium in the reagent combination or kit according to any one of claims 5 to 6, and then the embryo-like cells are cultured for the seventh time using the sixth culture medium in the reagent combination or kit according to any one of claims 5 to 6. The fifth culture period lasts 12–36 hours; The sixth culture period lasted 16–32 hours. The seventh culture period lasted 16–32 hours. The fifth culture was a suspension culture; The sixth cultivation was a dynamic cultivation; The seventh culture was a dynamic culture.

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