Embryo model and construction method thereof

Abstract

The invention relates to an embryo model and a construction method thereof, and belongs to the technical field of stem cells. The invention provides an embryo model. The embryo model is obtained by spontaneously assembling # imgabs0 # induced pluripotent stem cells and induced hypoderm stem cells, the # imgabs1 # induced pluripotent stem cells and the induced hypoderm stem cells are both obtained by reprogramming somatic cells. According to the invention, stem cells of two different pedigree generated by somatic cell reprogramming are utilized to spontaneously assemble and construct an embryo model for simulating a human perioriginal period. The embryo model better reproduces the interaction and communication mechanism among the multi-lineage cells in the human embryo development process by combining the cells of different lineages; under the embryo model, pedigree tracking and gene regulation network research can be performed on cells of different pedigree through a gene editing tool, so that a molecular regulation mechanism of human early embryonic development is deeply understood.

Description

Technical Field

[0001] The present invention relates to an embryo model and a method for constructing the same, belonging to the technical field of stem cells. Background Art

[0002] The development of the human early embryo begins with the fertilization of an egg, during which a zygote is formed. The zygote then enters the cleavage stage and sequentially forms 2-cell, 4-cell, 8-cell embryos, which develop into a morula, and then further develop into a blastocyst. After the blastocyst implants in the uterus, gastrulation occurs, sequentially forming a gastrula, a neurula, and finally entering the early organogenesis stage. According to the morphological and structural characteristics of the human early embryo, the developmental process in the first 60 days of the embryonic period can be roughly divided into 23 different stages, known as Carnegie Stages (CS). After human embryo implantation, the primitive epiblast cells (Epi) acquire polarity and construct the primitive amniotic cavity, eventually forming the amnion and the post-implantation epiblast cells. At the same time, the hypoblast (Hypo) differentiates into the visceral endoderm and the yolk sac endoderm, forming the primitive yolk sac and the secondary yolk sac structures. The epiblast cells and the visceral endoderm together generate a bilaminar disc structure, and then the embryo enters the gastrulation stage, which is considered a milestone in mammalian embryonic development. However, due to technical limitations (difficulty in tracing post-implantation embryos) and ethical constraints (the 14-day rule), researchers have lacked a systematic and in-depth understanding of the developmental process of the human embryo in the two weeks after implantation.

[0003] In recent years, with the rapid development of human pluripotent stem cell (hPSC) research, a new method has been provided for exploring the developmental process of human embryos. Currently, embryo models spontaneously assembled in vitro using single or multiple types of human stem cells are generally called "embryoid models". They are similar to real embryos in terms of cell number, composition, and three-dimensional structure, and are a simplified model that mimics the clear characteristics of natural embryos. Human embryo samples are often very scarce. Compared with real human embryos, stem cell-based embryo models have the characteristics of scalability, easy genetic manipulation, and fine control of variables. Therefore, this emerging field of embryoid models provides a new strategy for exploring the genetic and molecular mechanisms of human early development, and new research ideas for studying the specialization trajectories of different cell lineages during human early embryo development, as well as clinical drug screening and regenerative medicine for major diseases such as early embryo developmental defects. However, although existing in vitro embryo models have made significant progress in simulating human embryo development, they still have several technical defects and limitations.

[0004] For example, existing in vitro models, including human peri-gastrulation embryo models such as peri-gastruloids (see the literature "Liu, L. et al. Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids. Cell 186, 3776-3792e3716 (2023).") and peri-gastrulation trilaminar embryonic disc (PTED) embryo models (see the literature "Sun, S. et al. A transgene-free, human peri-gastrulation embryo model with trilaminar embryonic disc-, amnion- and yolk sac-like structures. bioRxiv, 2024.2008.2005.606556 (2024)."), mostly rely on the spontaneous assembly of a single type of human embryonic stem cells (hESCs). Therefore, it is difficult to effectively simulate the complex interactions between different lineage cells in the early embryo. Moreover, due to the single cell lineage involved in the existing models, it is impossible to use gene editing tools such as CRISPR for effective lineage tracing to analyze the differentiation trajectories and gene regulatory mechanisms of different lineage cells during human embryonic development. In addition, most existing models based on human embryonic stem cells (hESCs) may raise ethical controversies due to the use of human embryonic cells. Summary of the Invention

[0005] To address the above deficiencies, the present invention provides an embryo model, which is obtained by the spontaneous assembly of induced pluripotent stem cells ( iPSCs) and induced hypoblast stem cells (iHypoblast SCs); both the induced pluripotent stem cells and induced hypoblast stem cells (iHypoblast SCs) are obtained by reprogramming somatic cells.

[0006] In one embodiment of the present invention, the method for preparing the embryo model includes: placing induced pluripotent stem cells ( After induced pluripotent stem cells (iPSCs) and induced hypoblast stem cells (iHypoblast SCs) are dispersed, they are co-inoculated into a culture container and cultured successively with an embryonic assembly basal medium supplemented additionally with leukemia inhibitory factor (LIF) and CEPT (Chroman 1, Emricasan, Polyamines, Trans-ISRIB), an embryonic assembly basal medium supplemented additionally with leukemia inhibitory factor (LIF), and an embryonic assembly basal medium supplemented additionally with bone morphogenetic protein 4 (BMP4) to obtain an embryonic model.

[0007] In an embodiment of the present invention, the components of the embryonic assembly basal medium include GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement, and bovine serum albumin (BSA).

[0008] In an embodiment of the present invention, by volume percentage, the concentration of GlutaMAX in the embryonic assembly basal medium is 0.5-1%; by volume percentage, the concentration of non-essential amino acids in the embryonic assembly basal medium is 0.5-1%; by volume percentage, the concentration of penicillin-streptomycin in the embryonic assembly basal medium is 0.5-1%; by volume percentage, the concentration of sodium pyruvate in the embryonic assembly basal medium is 0.5-1%; the concentration of 2-mercaptoethanol in the embryonic assembly basal medium is 0.1-0.5 mM; by volume percentage, the concentration of N2 supplement in the embryonic assembly basal medium is 0.5-1%; by volume percentage, the concentration of B27 supplement in the embryonic assembly basal medium is 1-2%; by volume percentage, the concentration of bovine serum albumin in the embryonic assembly basal medium is 0.1-0.5%; the concentration of leukemia inhibitory factor in the embryonic assembly basal medium is 10-50 ng / mL; the concentration of bone morphogenetic protein 4 in the embryonic assembly basal medium is 50-200 ng / mL; by volume percentage, the concentration of CEPT in the embryonic assembly basal medium is 0.1-0.5%.

[0009] In an embodiment of the present invention, the components of the embryonic assembly basal medium further include a matrix; the matrix includes Neurobasal medium and DMEM / F12 medium; in the matrix, the volume ratio of Neurobasal medium to DMEM / F12 medium is 0.5-1:0.5-1.

[0010] In one embodiment of the present invention, the method for preparing induced hypoblast stem cells (iHypoblast SCs) includes: first transfecting somatic cells with Yamanaka factors (OSKM), and then inducing and culturing the transfected somatic cells with an induced hypoblast stem cell medium to obtain induced hypoblast stem cells (iHypoblast SCs).

[0011] The characteristics of the induced hypoblast stem cells (iHypoblast SCs) include:

[0012] First, being in an undifferentiated, bipotent state and having the ability to differentiate into cells exhibiting one or more characteristics that belong to endoderm / yolk sac endoderm (VE / YE)-like cells and extraembryonic mesoderm (ExEM)-like cells;

[0013] Second, a colony appearance with a flat epithelial shape;

[0014] Third, the expression of one or more biochemical markers of hypoblast stem cells (e.g., PDGFRA, SOX17, GATA6, GATA4, FOXA2, FN1, COL4A1, and Laminin), which can be determined by immunohistochemistry and / or PCR detection;

[0015] Fourth, single-cell omics similar to hypoblast cells derived from human embryos;

[0016] Fifth, the ability to integrate into the extraembryonic endoderm lineage in human-mouse chimeras.

[0017] In one embodiment of the present invention, the components of the induced hypoblast stem cell medium include platelet-derived growth factor AA (PDGFAA), leukemia inhibitory factor (LIF), fibroblast growth factor 4 (FGF4), GSK-3α / β inhibitor CHIR99021, TGF-β type I receptor inhibitor A83-01, and bone morphogenetic protein 4 (BMP4).

[0018] In an embodiment of the present invention, the concentration of platelet-derived growth factor AA in the induced endoderm stem cell medium is 10 - 50 ng / mL; the concentration of leukemia inhibitory factor in the induced endoderm stem cell medium is 10 - 50 ng / mL; the concentration of fibroblast growth factor 4 in the induced endoderm stem cell medium is 25 - 125 ng / mL; the concentration of GSK-3α / β inhibitor CHIR99021 in the induced endoderm stem cell medium is 3 - 15 μM; the concentration of TGF-β type I receptor inhibitor A83-01 in the induced endoderm stem cell medium is 1 - 5 μM; the concentration of bone morphogenetic protein 4 in the induced endoderm stem cell medium is 10 - 50 ng / mL.

[0019] In an embodiment of the present invention, the components of the induced endoderm stem cell medium further include heparin, N2 supplement, B27 supplement, GlutaMAX, non-essential amino acids, 2-mercaptoethanol and / or penicillin-streptomycin.

[0020] In an embodiment of the present invention, by volume percentage, the concentration of N2 supplement in the induced endoderm stem cell medium is 0.5 - 1%; by volume percentage, the concentration of B27 supplement in the induced endoderm stem cell medium is 1 - 2%; by volume percentage, the concentration of GlutaMAX in the induced endoderm stem cell medium is 0.5 - 1%; by volume percentage, the concentration of non-essential amino acids in the induced endoderm stem cell medium is 0.5 - 1%; by volume percentage, the concentration of penicillin-streptomycin in the induced endoderm stem cell medium is 0.5 - 1%; the concentration of heparin in the induced endoderm stem cell medium is 1 - 5 μg / mL; the concentration of 2-mercaptoethanol in the induced endoderm stem cell medium is 0.1 - 0.5 mM.

[0021] In an embodiment of the present invention, the components of the induced endoderm stem cell medium further include a matrix; the matrix includes Neurobasal medium and DMEM / F12 medium; in the matrix, the volume ratio of Neurobasal medium to DMEM / F12 medium is 0.5 - 1:0.5 - 1.

[0022] In an embodiment of the present invention, the matrix is composed of Neurobasal medium and DMEM / F12 medium; in the matrix, the volume ratio of Neurobasal medium to DMEM / F12 medium is 1:1.

[0023] In one embodiment of the present invention, the method for preparing induced hypoblast stem cells (iHypoblast SCs) includes: after inoculating somatic cells into a somatic cell culture medium, transfecting the somatic cells with Yamanaka factors (OSKM) under the mediation of a transfection vector; after the transfection is completed, dispersing the transfected somatic cells and inoculating them into a cell culture plate coated with feeder cells, and culturing them successively with a somatic cell culture medium and an induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632; after the culture is completed, selecting PDGFRA-positive hypoblast-like cells and re-inoculating them into a cell culture plate coated with feeder cells, and culturing the cells in the cell culture plate successively with an induced hypoblast stem cell culture medium supplemented with ROCK-I / II inhibitor Y27632 and an induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632; after the culture is completed, selecting hypoblast-like colonies in the cell culture plate and transferring them to a cell culture plate coated with feeder cells, and culturing them successively with an induced hypoblast stem cell culture medium supplemented with ROCK-I / II inhibitor Y27632 and an induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632 until the hypoblast-like cells in the cell culture plate reach 80-90% confluence; after the culture is completed, dispersing the hypoblast-like cells and inoculating them into a cell culture plate coated with feeder cells, and culturing them successively with an induced hypoblast stem cell culture medium supplemented with ROCK-I / II inhibitor Y27632 and an induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632 to obtain induced hypoblast stem cells (iHypoblast SCs).

[0024] In one embodiment of the present invention, the concentration of the ROCK-I / II inhibitor Y27632 in the induced hypoblast stem cell culture medium is 5-10 μM.

[0025] In one embodiment of the present invention, the somatic cells include human fibroblasts (e.g., adult dermal fibroblasts and neonatal dermal fibroblasts), amniotic mesenchymal stem cells, cardiac fibroblasts, CD34+ blood cells, mammary epithelial cells, nasal epithelial cells, peripheral blood monocytes, skeletal muscle myoblasts, T cells, umbilical vein epithelial cells, and / or urethral epithelial cells.

[0026] In one embodiment of the present invention, the somatic cell culture medium includes fibroblast culture medium and / or 106 medium.

[0027] In one embodiment of the present invention, the feeder cells include inactivated mouse embryonic fibroblasts and / or inactivated human fibroblasts.

[0028] In one embodiment of the present invention, the Yamanaka factors (OSKM) for reprogramming somatic cells (e.g., human fibroblasts) into a dedifferentiated or pluripotent state include OCT3 / 4, c-MYC, KLF4, SOX2, L-MYC, NANOG, LIN28, and / or chemical reprogramming.

[0029] In one embodiment of the present invention, the transfection vector includes virus, liposome, mRNA, and / or exosome.

[0030] In one embodiment of the present invention, the virus includes Sendai virus, adenovirus, and / or lentivirus.

[0031] The present invention also provides a method for constructing the above-mentioned embryonic model, the method comprising: After dispersing induced pluripotent stem cells ( iPSCs) and induced hypoblast stem cells (iHypoblast SCs), co-inoculating them into a culture container, and culturing them successively with an embryonic assembly basal medium supplemented additionally with leukemia inhibitory factor (LIF) and CEPT (Chroman 1, Emricasan, Polyamines, Trans-ISRIB), an embryonic assembly basal medium supplemented additionally with leukemia inhibitory factor (LIF), and an embryonic assembly basal medium supplemented additionally with bone morphogenetic protein 4 (BMP4) to obtain an embryonic model.

[0032] In one embodiment of the present invention, the components of the embryonic assembly basal medium include GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement, and bovine serum albumin (BSA).

[0033] In one embodiment of the present invention, calculated by volume percentage, the concentration of GlutaMAX in the basic medium for embryo assembly is 0.5-1%; calculated by volume percentage, the concentration of non-essential amino acids in the basic medium for embryo assembly is 0.5-1%; calculated by volume percentage, the concentration of penicillin-streptomycin in the basic medium for embryo assembly is 0.5-1%; calculated by volume percentage, the concentration of sodium pyruvate in the basic medium for embryo assembly is 0.5-1%; the concentration of 2-mercaptoethanol in the basic medium for embryo assembly is 0.1-0.5 mM; calculated by volume percentage, the concentration of N2 supplement in the basic medium for embryo assembly is 0.5-1%; calculated by volume percentage, the concentration of B27 supplement in the basic medium for embryo assembly is 1-2%; calculated by volume percentage, the concentration of bovine serum albumin in the basic medium for embryo assembly is 0.1-0.5%; the concentration of leukemia inhibitory factor in the basic medium for embryo assembly is 10-50 ng / mL; the concentration of bone morphogenetic protein 4 in the basic medium for embryo assembly is 50-200 ng / mL; calculated by volume percentage, the concentration of CEPT in the basic medium for embryo assembly is 0.1-0.5%.

[0034] In one embodiment of the present invention, the components of the basic medium for embryo assembly further include a matrix; the matrix includes Neurobasal medium and DMEM / F12 medium; in the matrix, the volume ratio of Neurobasal medium to DMEM / F12 medium is 0.5-1:0.5-1.

[0035] In one embodiment of the present invention, the method for preparing induced hypoblast stem cells (iHypoblast SCs) includes: first transfecting somatic cells with Yamanaka factors (OSKM), and then inducing and culturing the transfected somatic cells with an induced hypoblast stem cell medium to obtain induced hypoblast stem cells (iHypoblast SCs).

[0036] In one embodiment of the present invention, the components of the induced hypoblast stem cell medium include platelet-derived growth factor AA (PDGFAA), leukemia inhibitory factor (LIF), fibroblast growth factor 4 (FGF4), GSK-3α / β inhibitor CHIR99021, TGF-β type I receptor inhibitor A83-01, and bone morphogenetic protein 4 (BMP4).

[0037] In an embodiment of the present invention, the concentration of platelet-derived growth factor AA in the induced hypoblast stem cell medium is 10-50 ng / mL; the concentration of leukemia inhibitory factor in the induced hypoblast stem cell medium is 10-50 ng / mL; the concentration of fibroblast growth factor 4 in the induced hypoblast stem cell medium is 25-125 ng / mL; the concentration of GSK-3α / β inhibitor CHIR99021 in the induced hypoblast stem cell medium is 3-15 μM; the concentration of TGF-β type I receptor inhibitor A83-01 in the induced hypoblast stem cell medium is 1-5 μM; the concentration of bone morphogenetic protein 4 in the induced hypoblast stem cell medium is 10-50 ng / mL.

[0038] In an embodiment of the present invention, the components of the induced hypoblast stem cell medium further include heparin, N2 supplement, B27 supplement, GlutaMAX, non-essential amino acids, 2-mercaptoethanol and / or penicillin-streptomycin.

[0039] In an embodiment of the present invention, by volume percentage, the concentration of the N2 supplement in the induced hypoblast stem cell medium is 0.5-1%; by volume percentage, the concentration of the B27 supplement in the induced hypoblast stem cell medium is 1-2%; by volume percentage, the concentration of GlutaMAX in the induced hypoblast stem cell medium is 0.5-1%; by volume percentage, the concentration of non-essential amino acids in the induced hypoblast stem cell medium is 0.5-1%; by volume percentage, the concentration of penicillin-streptomycin in the induced hypoblast stem cell medium is 0.5-1%; the concentration of heparin in the induced hypoblast stem cell medium is 1-5 μg / mL; the concentration of 2-mercaptoethanol in the induced hypoblast stem cell medium is 0.1-0.5 mM.

[0040] In an embodiment of the present invention, the components of the induced hypoblast stem cell medium further include a matrix; the matrix includes Neurobasal medium and DMEM / F12 medium; in the matrix, the volume ratio of Neurobasal medium to DMEM / F12 medium is 0.5-1:0.5-1.

[0041] In an embodiment of the present invention, the matrix consists of Neurobasal medium and DMEM / F12 medium; in the matrix, the volume ratio of Neurobasal medium to DMEM / F12 medium is 1:1.

[0042] In one embodiment of the present invention, the method for preparing induced hypoblast stem cells (iHypoblast SCs) comprises: inoculating somatic cells into a somatic cell culture medium, and then transfecting the somatic cells with Yamanaka factors (OSKM) under the mediation of a transfection vector; after the transfection is completed, dispersing the transfected somatic cells and inoculating them into a cell culture plate pre-coated with feeder cells, and culturing them successively with a somatic cell culture medium and an induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632; after the culture is completed, selecting PDGFRA-positive hypoblast-like cells and re-inoculating them into a cell culture plate pre-coated with feeder cells, and culturing the cells in the cell culture plate successively with an induced hypoblast stem cell culture medium supplemented with ROCK-I / II inhibitor Y27632 and an induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632; after the culture is completed, selecting hypoblast-like colonies in the cell culture plate and transferring them to a cell culture plate pre-coated with feeder cells, and culturing them successively with an induced hypoblast stem cell culture medium supplemented with ROCK-I / II inhibitor Y27632 and an induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632 until the hypoblast-like cells in the cell culture plate reach 80-90% confluence; after the culture is completed, dispersing the hypoblast-like cells and inoculating them into a cell culture plate pre-coated with feeder cells, and culturing them successively with an induced hypoblast stem cell culture medium supplemented with ROCK-I / II inhibitor Y27632 and an induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632 to obtain induced hypoblast stem cells (iHypoblast SCs).

[0043] In one embodiment of the present invention, the concentration of the ROCK-I / II inhibitor Y27632 in the induced hypoblast stem cell culture medium is 5-10 μM.

[0044] In one embodiment of the present invention, the somatic cells include human fibroblasts (e.g., adult dermal fibroblasts and neonatal dermal fibroblasts), amniotic mesenchymal stem cells, cardiac fibroblasts, CD34+ blood cells, mammary epithelial cells, nasal epithelial cells, peripheral blood mononuclear cells, skeletal muscle myoblasts, T cells, umbilical vein epithelial cells, and / or urethral epithelial cells.

[0045] In one embodiment of the present invention, the somatic cell culture medium includes fibroblast culture medium and / or 106 medium.

[0046] In one embodiment of the present invention, the feeder cells include inactivated mouse embryonic fibroblasts and / or inactivated human fibroblasts.

[0047] In one embodiment of the present invention, the Yamanaka factors (OSKM) for reprogramming somatic cells (e.g., human fibroblasts) into a dedifferentiated or pluripotent state include OCT3 / 4, c-MYC, KLF4, SOX2, L-MYC, NANOG, LIN28, and / or chemical reprogramming.

[0048] In one embodiment of the present invention, the transfection vector includes a virus, liposome, mRNA, and / or exosome.

[0049] In one embodiment of the present invention, the virus includes Sendai virus, adenovirus, and / or lentivirus.

[0050] The present invention also provides an embryo assembly medium, and the components of the embryo assembly medium include leukemia inhibitory factor, GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement, and bovine serum albumin (BSA);

[0051] The components of the embryo assembly medium include leukemia inhibitory factor, CEPT, GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement, and bovine serum albumin (BSA);

[0052] Alternatively, the components of the embryo assembly medium include bone morphogenetic protein 4, GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement, and bovine serum albumin (BSA).

[0053] In one embodiment of the present invention, by volume percentage, the concentration of GlutaMAX in the basic embryo assembly medium is 0.5 - 1%; by volume percentage, the concentration of non-essential amino acids in the basic embryo assembly medium is 0.5 - 1%; by volume percentage, the concentration of penicillin-streptomycin in the basic embryo assembly medium is 0.5 - 1%; by volume percentage, the concentration of sodium pyruvate in the basic embryo assembly medium is 0.5 - 1%; the concentration of 2-mercaptoethanol in the basic embryo assembly medium is 0.1 - 0.5 mM; by volume percentage, the concentration of N2 supplement in the basic embryo assembly medium is 0.5 - 1%; by volume percentage, the concentration of B27 supplement in the basic embryo assembly medium is 1 - 2%; by volume percentage, the concentration of bovine serum albumin in the basic embryo assembly medium is 0.1 - 0.5%; the concentration of leukemia inhibitory factor in the basic embryo assembly medium is 10 - 50 ng / mL; the concentration of bone morphogenetic protein 4 in the basic embryo assembly medium is 50 - 200 ng / mL; by volume percentage, the concentration of CEPT in the basic embryo assembly medium is 0.1 - 0.5%.

[0054] In one embodiment of the present invention, the components of the basic embryo assembly medium further include a matrix; the matrix includes Neurobasal medium and DMEM / F12 medium; in the matrix, the volume ratio of Neurobasal medium to DMEM / F12 medium is 0.5 - 1:0.5 - 1.

[0055] The present invention also provides a method for screening drugs for preventing and / or treating diseases, the method comprising: screening drugs for preventing and / or treating diseases using the above-mentioned embryo model; the diseases include genetic diseases and / or developmental disorders.

[0056] In one embodiment of the present invention, the genetic diseases include hereditary pancreatic hypoplasia caused by GATA6 mutation, HNF1B-related renal dysplasia, and / or situs inversus; the developmental disorders include endoderm developmental disorders, anteroposterior axis developmental disorders, and / or early embryo developmental arrest.

[0057] In one embodiment of the present invention, the method comprises: first modeling the embryo model to obtain an in vitro disease model, and then screening drugs for preventing and / or treating diseases using the in vitro disease model.

[0058] In one embodiment of the present invention, the modeling includes inducing diseases by drugs and / or inducing diseases by gene editing.

[0059] In one embodiment of the present invention, the model establishment includes: administering drugs SB431542, A83-01, and Activin A to an embryonic model to obtain an in vitro model of the Nodal / Activin signaling pathway and perigastrulation embryonic development; or, administering drugs CHIR99021, IWP-2, and XAV939 to an embryonic model to obtain an in vitro model of the Wnt / β-Catenin signaling pathway and perigastrulation embryonic development; or, administering drugs FGF2 and SU5402 to an embryonic model to obtain an in vitro model of the FGF signaling pathway and perigastrulation embryonic development.

[0060] The present invention also provides a method for evaluating drug toxicity, which includes: administering a drug to the above-mentioned embryonic model and evaluating the toxicity of the drug by observing the index changes of the embryonic model.

[0061] In one embodiment of the present invention, the toxicity of the drug includes potential embryo / fetal toxicity, neurotoxicity, immunotoxicity, genotoxicity, endocrine disruption, embryonic lethality, and / or teratogenicity.

[0062] The present invention also provides the use of the above-mentioned embryonic model, or the above-mentioned method, or the above-mentioned basic medium for embryonic assembly in screening drugs for preventing and / or treating diseases or evaluating drug toxicity, and the diseases include genetic diseases and / or developmental disorders.

[0063] In one embodiment of the present invention, the genetic diseases include hereditary pancreatic hypoplasia caused by GATA6 mutation, HNF1B-related renal dysplasia, and / or situs inversus; the developmental disorders include endodermal developmental disorders, anteroposterior axis developmental disorders, and / or early embryonic developmental arrest.

[0064] In one embodiment of the present invention, the toxicity of the drug includes potential embryo / fetal toxicity, neurotoxicity, immunotoxicity, genotoxicity, endocrine disruption, embryonic lethality, and / or teratogenicity.

[0065] The present invention also provides a cell therapy drug for preventing and / or treating diseases, and the composition of the cell therapy drug includes the above-mentioned embryonic model, or the cell therapy drug is prepared from the above-mentioned embryonic model.

[0066] In one embodiment of the present invention, the preparation method of the cell therapy drug includes: extracting different lineages of stem cells and / or progenitor cells from the above-mentioned embryonic model at a specific developmental stage and further differentiating them in vitro to generate cell types with specific functions to obtain the cell therapy drug.

[0067] In one embodiment of the present invention, the progenitor cells include hematopoietic endothelial progenitor cell-like.

[0068] In one embodiment of the present invention, the prevention and / or treatment of diseases includes hematopoietic function repair, blood vessel regeneration, and the treatment of related diseases.

[0069] The present invention also provides the use of the above-mentioned embryonic model in the preparation of a cell therapy drug for preventing and / or treating diseases.

[0070] In one embodiment of the present invention, the method for preparing the cell therapy drug includes: extracting stem cells and / or progenitor cells of different lineages from the above-mentioned embryonic model at a specific developmental stage and further differentiating them in vitro to generate cell types with specific functions, thereby obtaining the cell therapy drug.

[0071] In one embodiment of the present invention, the progenitor cells include hematopoietic endothelial progenitor cell-like.

[0072] In one embodiment of the present invention, the prevention and / or treatment of diseases includes hematopoietic function repair, blood vessel regeneration, and the treatment of related diseases.

[0073] The present invention also provides an organ graft, which is prepared from the above-mentioned embryonic model.

[0074] In one embodiment of the present invention, the method for preparing the organ graft includes: performing extended culture using the above-mentioned embryonic model to reach the organogenesis stage in vitro, thereby obtaining the organ graft.

[0075] The present invention also provides the use of the above-mentioned embryonic model in the preparation of an organ graft.

[0076] In one embodiment of the present invention, the method for preparing the organ graft includes: performing extended culture using the above-mentioned embryonic model to reach the organogenesis stage in vitro, thereby obtaining the organ graft.

[0077] The technical solution of the present invention has the following advantages:

[0078] The present invention provides an embryonic model, which is spontaneously assembled from induced pluripotent stem cells ( iPSCs) and induced hypoblast stem cells (iHypoblast SCs); the induced pluripotent stem cells ( induced pluripotent stem cells transformed from induced pluripotent stem cells iPSC reprogrammed from somatic cells) and induced hypoblast stem cells (iHypoblast SCs) are both reprogrammed from somatic cells. The present invention utilizes two different lineages of stem cells generated by somatic cell reprogramming ( Induced pluripotent stem cells and induced hypoblast stem cells spontaneously assembled to construct an embryonic model simulating the human perigastrulation stage, called induced embryoids (iEmbryoids). This embryonic model has the following advantages:

[0079] First, by combining cells of different lineages, this embryonic model better reproduces the interaction and communication mechanisms between multi-lineage cells during human embryonic development, effectively restoring the developmental characteristics of human perigastrulation embryos. Second, under this embryonic model, through gene editing tools such as CRISPR, lineage tracing and gene regulatory network studies can be carried out on cells of different lineages, so as to deeply understand the molecular regulatory mechanisms of human early embryonic development. Third, this embryonic model uses induced pluripotent stem cells (iPSCs) and induced hypoblast stem cells (iHypoblast SCs) generated by somatic cell reprogramming to replace traditional human embryonic stem cells (hESCs), which can avoid ethical issues to a certain extent and provide a more feasible solution for extensive basic research and clinical applications. In addition, using this embryonic model to extract stem cells and progenitor cells of different lineages (such as hematopoietic endothelial progenitor cell-like, etc.) at specific developmental stages and further differentiating them in vitro can generate cell types with specific functions, and these cells can be used for specific cell therapies (including hematopoietic function repair, blood vessel regeneration, and treatment of related diseases). Finally, in the future, by extending the culture of this embryonic model, it can reach the organogenesis stage in vitro. This technology can be used to develop personalized artificial organ substitutes, providing a new breakthrough in the field of regenerative medicine. This method is expected to solve the problem of donor shortage faced by current human organ transplantation by culturing functional tissues and organs in vitro; by reprogramming the patient's autologous cells and introducing them into this embryonic model, not only can organs matching the patient's genes be generated, but also the risk of immune rejection can be greatly reduced. This technology is expected to provide a revolutionary new solution for organ transplantation, promoting the development of personalized medicine using cutting-edge technologies in regenerative medicine, and thus providing an effective solution for the global demand for organ transplantation in the future. Therefore, this embryonic model has great application prospects in the fields of regenerative medicine, developmental biology, and drug development.

[0080] Furthermore, the preparation method of the embryonic model includes: Induced pluripotent stem cells ( After induced pluripotent stem cells (iPSCs) and induced hypoblast stem cells (iHypoblast SCs) were dispersed, they were co-seeded into a culture vessel and cultured successively with an embryonic assembly basal medium supplemented additionally with leukemia inhibitory factor (LIF) and CEPT (Chroman 1, Emricasan, Polyamines, Trans-ISRIB), an embryonic assembly basal medium supplemented additionally with leukemia inhibitory factor (LIF), and an embryonic assembly basal medium supplemented additionally with bone morphogenetic protein 4 (BMP4) to obtain an embryonic model; the components of the embryonic assembly basal medium include GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement, and bovine serum albumin (BSA). Using this embryonic assembly basal medium supplemented additionally with leukemia inhibitory factor + CEPT, leukemia inhibitory factor, or bone morphogenetic protein 4 for culture induced pluripotent stem cells( iPSCs) and induced hypoblast stem cells (iHypoblast SCs) can precisely regulate the important signaling pathways (BMP and JAK / STAT signaling) of the two lineages of cells, and then successfully achieve the spontaneous assembly and stable maintenance of the two lineages of cells. The components of this embryonic assembly basal medium supplemented additionally with leukemia inhibitory factor + CEPT, leukemia inhibitory factor, or bone morphogenetic protein 4 can ensure that the two lineages of cells maintain specific lineage characteristics during differentiation, and moreover, ensure that the two lineages of cells maintain a stable self-renewal ability during long-term culture. Research has confirmed that the use of this embryonic assembly basal medium supplemented additionally with leukemia inhibitory factor + CEPT, leukemia inhibitory factor, or bone morphogenetic protein 4 significantly improves induced pluripotent stem cells( iPSCs) and the spontaneous assembly efficiency of induced hypoblast stem cells (iHypoblast SCs), and moreover, enhances the operability and experimental reproducibility of iEmbryoids. Brief Description of the Drawings

[0081] Figure 1 : Constructing a human embryonic model by combining homologous induced hypoblast stem cells (iHypoblast SCs) and iPSCs. Figure 1In (a), the cartoon schematic diagram shows the experimental procedure for the assembly of human iEmbryoids into embryo-like structures; (b) bright-field images of iEmbryoids at different days during the assembly process from day 0 to day 8; (c) bar graph showing the assembly efficiency of the embryo-like model D8iEmbryoid according to morphological criteria. Mean ± standard error, n = 473 iEmbryoids; (d) hematoxylin-eosin staining of a real human embryo (Carnegie stage CS6b); (e) immunofluorescence image of day 8 iEmbryoids (n = 3 independent experiments, scale bar: 50 μm).

[0082] Figure 2 Immunofluorescent staining of embryo models at different time points. Figure 3 In (a), immunofluorescent staining images of embryo-like models of iEmbryoids at different time points (day 4, day 6, day 8) (SOX2, SOX17) (n = 3 independent experiments, scale bar: 50 μm); (b) immunofluorescent staining images of embryo-like models of iEmbryoids at different time points (day 4, day 6, day 8) (OCT3 / 4, GATA6) (n = 3 independent experiments, scale bar: 50 μm).

[0083] Figure 3 Single-induced hypoblast stem cells (iHypoblast SCs) or iPSCs were used to construct cell aggregates. Figure 3 In (a), the starting cells were only iPSCs, and bright-field images of iEmbryoids at different days of assembly (n = 3 independent experiments, scale bar: 50 μm); (b) the starting cells were only iPS, and immunofluorescent image of iEmbyroid on day 4, OCT3 / 4, GATA6, SOX2 and SOX17 (n = 3 independent experiments, scale bar: 50 μm); (c) the starting cells were only iHypoblast SCs, and bright-field images of iEmbryoids at different days of assembly (n = 3 independent experiments, scale bar: 50 μm); (d) the starting cells were only iHypoblast SCs, and immunofluorescent image of iEmbyroid on day 4, OCT3 / 4, GATA6, SOX2 and SOX17 (n = 3 independent experiments, scale bar: 50 μm).

[0084] Figure 4 Immunofluorescent staining of embryo models. Figure 4Among them, (a) Representative immunofluorescence images of iEmbryoids on day 4 (OCT3 / 4 and F-ACTIN) (n = 3 independent experiments, scale bar: 50 μm); (b) Representative immunofluorescence images of iEmbryoids on day 6 (ISL1, SOX2 and FOXA2) (arrows and dashed areas indicate putative amniotic-like cells, n = 3 independent experiments, scale bar: 50 μm); (c) Representative immunofluorescence images of iEmbryoids on day 8 (NANOG, SOX17 and TFAP2C) (arrows and dashed areas indicate putative primordial germ cell (PGC)-like cells, n = 3 independent experiments, scale bar: 50 μm); (d) Representative immunofluorescence images of iEmbryoids on day 8 (OCT3 / 4, T and OTX2) (arrows and dashed areas indicate the putative anteroposterior axis (A-P axis), n = 3 independent experiments, scale bar: 50 μm).

[0085] Figure 5 : Immunofluorescence staining of embryo models. Figure 5 Among them, (a) Representative immunofluorescence images of iEmbryoids on day 4 (SOX2 and F-ACTIN) (n = 3 independent experiments, scale bar: 50 μm); (b) Representative immunofluorescence images of iEmbryoids on day 6 (SOX2, TFAP2A and SOX17) (arrows and dashed areas indicate putative amniotic-like cells, n = 3 independent experiments, scale bar: 50 μm); (c) Representative immunofluorescence images of iEmbryoids on day 8 (OCT3 / 4, SOX17 and BLIMP1) (arrows and dashed areas indicate putative primordial germ cell (PGC)-like cells, n = 3 independent experiments, scale bar: 50 μm); (d) Representative immunofluorescence images of iEmbryoids on day 8 (OCT3 / 4, T and LEFTY) (arrows and dashed areas indicate putative anterior visceral endoderm (AVE)-like cells and primitive streak (PS)-like cells, n = 3 independent experiments, scale bar: 50 μm); (e) Representative immunofluorescence images of iEmbryoids on day 8 (OCT3 / 4, T and CER1) (arrows and dashed areas indicate putative anterior visceral endoderm (AVE)-like cells and primitive streak (PS)-like cells, n = 3 independent experiments, scale bar: 50 μm).

[0086] Figure 6 : Immunofluorescence staining of embryo models. Figure 6Among them, (a) Representative immunofluorescence images of day 8 iEmbryoids (OCT3 / 4, GATA6, and VIM) (arrows and dashed areas indicate putative ectoderm mesoderm (ExEM)-like cells, n = 3 independent experiments, scale bar: 50 μm); (b) Representative immunofluorescence images of day 8 iEmbryoids (OCT3 / 4, SOX17, and BST2) (arrows and dashed areas indicate putative ExEM-like cells, n = 3 independent experiments, scale bar: 50 μm); (c) Representative immunofluorescence images of day 8 iEmbryoids (OCT3 / 4, GATA4, and FOXF1) (arrows and dashed areas indicate putative ExEM-like cells, n = 3 independent experiments, scale bar: 50 μm); (d) Representative immunofluorescence images of day 8 iEmbryoids (CD34, ERG, and GATA6) (arrows and dashed areas indicate putative hematopoietic endothelial progenitor (HEP)-like cells, n = 3 independent experiments, scale bar: 50 μm).

[0087] Figure 7 : Single-cell sequencing analysis of embryo models. Figure 7 Among them, (a) Bright-field images of day 8 iEmbryoids for single-cell RNA sequencing (sc-RNAseq); (b) UMAP plots representing individual cell clusters derived from day 8 iEmbryoids (colors correspond to cell type annotations); (c) UMAP shows cell populations from different lineages in day 8 iEmbryoids (colors correspond to cell type annotations; where Epi, ectoderm; Am, amnion; PGC, primordial germ cell; PS, primitive streak; Earlymeso, early mesoderm; Meso, mesoderm; ExEM, extraembryonic mesoderm; Intermediate, intermediate cells; VE / YS, visceral endoderm / yolk sac; SYS, secondary yolk sac; HEP, hematopoietic endothelial progenitor cells); (d) Dot plots reveal the expression of key markers in 11 cell type clusters in day 8 iEmbryoids.

[0088] Figure 8 : Single-cell sequencing analysis of embryo models. Figure 8Among them, (a) UMAP analysis of the integrated data of iEmbryoids, in vitro cultured embryos and CS7 human gastrula on day 8 (the colors on the left correspond to the cell type annotations in their respective datasets; among them, EPI, ectoderm; Am, amnion; PGC, primordial germ cell; PS, primitive streak; Earlymeso, early mesoderm; Meso, mesoderm; ExEM, extraembryonic mesoderm; VE / YS, visceral endoderm / yolk sac; SYS, secondary yolk sac; Blood, blood cells; the colors on the right correspond to the aggregated plots of iEmbryoids and each dataset); (b) heatmap evaluating the relative expression levels of lineage-specific markers in different datasets; (c) subpopulation analysis of the blood cell clusters in the UMAP of the integrated data.

[0089] Figure 9 : Single-cell sequencing analysis of embryo models. Figure 9 Among them, (a) expression of epiblast (Epi) markers (OCT3 / 4, SOX2, NANOG, DPPA5 and ESRG) in the UMAP of iHypoblast SCs; (b) expression of amnion markers (ISL1, TFAP2A, GARBP, HEY1 and VTCN1) in the UMAP of iHypoblast SCs; (c) expression of primitive streak markers (CDH1, WNT8A, GAL, TBXT and MSGN1) in the UMAP of iHypoblast SCs; (d) expression of visceral endoderm / yolk sac (VE / YS) markers (SOX17, RSPO3, BMP6, FOXA2 and GATA4) in the UMAP of iHypoblast SCs.

[0090] Figure 10 : Single-cell sequencing analysis of embryo models. Figure 10 Among them, (a) expression of secondary yolk sac (SYS) markers (APOA1, APOA2, APOB, AFP and TTR) in the UMAP of iHypoblast SCs; (b) expression of primordial germ cell (PGC) markers (BLIMP, CXCR4, TFAP2C, NANOS3 and PDPN) in the UMAP of iHypoblast SCs; (c) expression of early mesoderm markers (MESP1, MESP2, EOMES, MIXL1 and BMP4) in the UMAP of iHypoblast SCs; (d) expression of mesoderm markers (ACTC1, GATA6, HAND1, PDGFRA and SNAI2) in the UMAP of iHypoblast SCs.

[0091] Figure 11 : Single-cell sequencing analysis of embryo models.Figure 11 In (a), the expression of hematopoietic endothelial progenitor cell (HEP) markers (CD34, ERG, RUNX1, MEF2C, and PECAM1) in iHypoblast SCs UMAP; (b) the expression of extraembryonic mesoderm lineage (ExEM) markers

[0092] (POSTN, DCN, and HAND2) in iHypoblast SCs UMAP; (c) the expression of neuroectoderm (NE) markers (OTX2 and PAX6) in iHypoblast SCs UMAP. Specific implementation manners

[0093] The following examples are provided to better understand the present invention further. They are not limited to the described best implementation manners, and do not constitute a limitation on the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other existing technologies falls within the protection scope of the present invention.

[0094] For those not specifying specific experimental steps or conditions in the following examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments not indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0095] The materials and reagents involved in the following examples are shown in Table 1.

[0096] Table 1 Experimental materials and reagents

[0097]

[0098]

[0099]

[0100] Example 1: An embryonic model and its construction method

[0101] This example provides an embryonic model, which is obtained by induced pluripotent stem cells ( iPSCs) and induced hypoblast stem cells (iHypoblast SCs) assembling spontaneously; the induced pluripotent stem cells ( induced pluripotent stem cells transformed from induced pluripotent stem cells iPSC reprogrammed from somatic cells) and induced hypoblast stem cells (iHypoblast SCs) are both reprogrammed from somatic cells.

[0102] The preparation process of the embryo model is as follows:

[0103] 1. Construct induced hypoblast stem cells through somatic cell reprogramming

[0104] Step 1: Use the CytoTune-iPS2.0 Sendai reprogramming kit to reprogram primary human adult skin fibroblasts (including donor 1 and donor 2) according to the instructions. The reprogramming process is as follows: Seed human fibroblasts at a cell density of 5×10 4 cells in mouse embryonic fibroblast medium and transfect them under the mediation of Sendai virus with a multiplicity of infection (MOI) of 5 (KOS), 5 (c-Myc), and 6 (KLF4).

[0105] Step 2: On the 7th day after transfection, dissociate the transfected human fibroblasts in the fibroblast medium with TryPLE Select by standing at room temperature (25) for 5 minutes (TryPLE Select dosage: 0.5 mL / well) and re-seed them at an inoculation amount of 6×10 4 cells / well in a cell culture plate pre-coated with feeder cells 1 day in advance (using mouse embryonic fibroblasts as feeder cells. For the plating method of feeder cells, refer to the literature "Liu, X. et al. Reprogramming roadmap reveals route to human induced trophoblast stem cells. Nature 586, 101–107 (2020).") (in addition to feeder cells in the cell culture plate, 1 mL / well of mouse embryonic fibroblast medium is also added), and culture them in a 37°C cell culture incubator with 5% (v / v) CO 2 and 5% (v / v) O 2 .

[0106] Step 3: After 24 hours of culture, replace the medium in the cell culture plate with induced hypoblast stem cell medium and continue to culture it in a 37°C cell culture incubator with 5% (v / v) CO 2 and 5% (v / v) O 2 .

[0107] Step 4: After 20 days of culture, enrich and screen hypoblast-like stem cells with high PDGFRA expression by FACS (fluorescence-activated cell sorting).

[0108] Step 5: Select PDGFRA-positive hypoblast-like cells and seed them at 6×10 4The inoculum of cells per well was re-inoculated into a cell culture plate pre-coated with feeder cells one day in advance (in addition to the feeder cells in the cell culture plate, 1 mL / well of induced hypoblast stem cell medium supplemented with 10 μM ROCK-I / II inhibitor Y27632 was added), and cultured in a 37 °C cell culture incubator with 5% (v / v) CO 2 and 5% (v / v) O 2 .

[0109] Step Six: After culturing for 24 hours, the medium in the cell culture plate was replaced with induced hypoblast stem cell medium without additional supplementation of ROCK-I / II inhibitor Y27632, and continued to be cultured in a 37 °C cell culture incubator with 5% (v / v) CO 2 and 5% (v / v) O 2 .

[0110] Step Seven: After culturing for 5 days, the hypoblast-like colonies in the cell culture plate were selected and transferred to a cell culture plate pre-coated with feeder cells one day in advance at an inoculum of 6×10 4 cells per well (in addition to the feeder cells in the cell culture plate, 1 mL / well of induced hypoblast stem cell medium supplemented with 10 μM ROCK-I / II inhibitor Y27632 was added), and cultured in a 37 °C cell culture incubator with 5% (v / v) CO 2 and 20% (v / v) O 2 . During the culture period, the medium was changed once a day.

[0111] Step Eight: When the hypoblast-like cells in the cell culture plate reached 80 - 90% confluence, the hypoblast-like cells in the cell culture plate were dissociated with Accutase at room temperature (25 °C) for 5 minutes (Accutase dosage: 0.5 mL / well) and transferred to a cell culture plate pre-coated with feeder cells one day in advance at an inoculum of 6×10 4 cells per well (in addition to the feeder cells in the cell culture plate, 1 mL / well of induced hypoblast stem cell medium supplemented with 10 μM ROCK-I / II inhibitor Y27632 was added), and cultured in a 37 °C cell culture incubator with 5% (v / v) CO 2 and 20% (v / v) O 2 . After culturing for 24 hours, induced hypoblast stem cells (iHypoblast SCs) were successfully constructed.

[0112] Step Nine: After the successful construction of induced hypoblast stem cells (iHypoblast SCs), the induced hypoblast stem cells (iHypoblast SCs) were passaged at a passage ratio of 1:5 every 3 days, and the passage culture process was the same as in Step Eight.

[0113] Among them, the mouse embryonic fibroblast medium (MEF medium) is DMEM medium containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) non-essential amino acids, 1% (v / v) GlutaMAX, 1% (v / v) penicillin-streptomycin, 0.1 mM 2-mercaptoethanol and 1% (v / v) sodium pyruvate.

[0114] The induced hypoblast stem cell medium is N2B27 basal medium containing 10 ng / mL human PDGFAA, 10 ng / mL human LIF, 25 ng / mL human FGF4, 1 μg / mL heparin (promoting the binding of FGF4 to its receptor and optimizing the biological function of FGF4), 3 μM CHIR99021, 1 μM A83-01 and 10 ng / mL human BMP4; the N2B27 basal medium is a mixed medium containing 1% (v / v) N2, 2% (v / v) B27, 1% (v / v) GlutaMAX, 1% (v / v) non-essential amino acids, 0.1 mM 2-mercaptoethanol and 1% (v / v) penicillin-streptomycin; the mixed medium is composed of Neurobasal medium and DMEM / F12 at a volume ratio of 1:1.

[0115] 2. Culture of induced pluripotent stem cells

[0116] Step 1: Referring to the literature "Mazid, M.A. et al. Rolling back human pluripotent stem cells to an eight-cell embryo-like stage. Nature 605, 315–324 (2022).", use E8 complete medium to culture the primed induced pluripotent stem cell line (primed iPSC) in a cell culture plate to convert the primed induced pluripotent stem cell line (primed iPSC) into induced pluripotent stem cells ( iPSCs) (the dosage of E8 complete medium is 2 mL / well).

[0117] Step 2: The induced pluripotent stem cells obtained by transformation are at 1×10 5The inoculum of cells per well was re-inoculated into a cell culture plate pre-coated with feeder cells 1 day in advance (using mouse embryonic fibroblasts as feeder cells. For the method of plating feeder cells, see the literature "Liu, X. et al. Reprogramming roadmap reveals route to human induced trophoblast stem cells. Nature 586, 101–107 (2020).") (the cell culture plate was supplemented with 4CL medium with an additional 10 μM Y27632 at 2 mL / well), and cultured in a 37 °C cell culture incubator with 5% (v / v) CO 2 、5% (v / v) O 2 2. The culture was carried out in a 37 °C cell culture incubator with 5% (v / v) CO₂ and 5% (v / v) O₂.

[0118] Among them, the E8 complete medium is the E8 basal medium containing 1% (v / v) E8 supplement and 1% (v / v) penicillin-streptomycin.

[0119] The 4CL medium is a mixed medium containing 1% (v / v) N2, 1% (v / v) B27, 1% (v / v) sodium pyruvate, 1% (v / v) non-essential amino acids, 1% (v / v) GlutaMAX, 1% (v / v) penicillin-streptomycin, 50 μg / mL L-ascorbic acid, 10 nM DZNep, 5 nM TSA, 1 μM PD0325901, 5 μM IWR-1, 20 ng / mL human leukemia inhibitory factor (LIF), 20 ng / mL activin A, and 0.2% (v / v) Matrigel matrix glue; the mixed medium consists of Neurobasal medium and DMEM / F12 at a volume ratio of 1:1.

[0120] 3. Assembly of induced embryoids (iEmbryoids)

[0121] Step 1: Take the induced pluripotent stem cells after the first passage in Part 2. When the cells reach a density of 70-80%, discard the medium in the cell culture plate. First, wash the induced pluripotent stem cells in the cell culture plate with DPBS buffer (the amount of DPBS buffer used is 1 mL / well, and wash once), and then use TryPLE Select to dissociate the induced pluripotent stem cells in the cell culture plate at room temperature (25 °C) for 5 minutes (the amount of TryPLE Select used is 1 mL / well, dissociating into single cells). induced pluripotent stem cells, and when the cells reach a density of 70-80%, discard the medium in the cell culture plate. First, wash the induced pluripotent stem cells in the cell culture plate with DPBS buffer (the amount of DPBS buffer used is 1 mL / well, and wash once), and then use TryPLE Select to dissociate the induced pluripotent stem cells in the cell culture plate at room temperature (25 °C) for 5 minutes (the amount of TryPLE Select used is 1 mL / well, dissociating into single cells). induced pluripotent stem cells (the amount of DPBS buffer used is 1 mL / well, and wash once), and then use TryPLE Select to dissociate the induced pluripotent stem cells in the cell culture plate at room temperature (25 °C) for 5 minutes (the amount of TryPLE Select used is 1 mL / well, dissociating into single cells). induced pluripotent stem cells were dissociated with TryPLE Select at room temperature (25 °C) for 5 minutes (the amount of TryPLE Select used is 1 mL / well, dissociating into single cells).

[0122] Step 2: After dissociation, centrifuge the cell culture plate to discard the supernatant, and resuspend the cell pellet in the cell culture plate with mouse embryonic fibroblast medium supplemented with 0.1% (v / v) CEPT (1 mL of medium per well) to obtain a cell suspension.

[0123] Step 3: Add 0.1% Gelatin to the cell culture plate at an amount of 1.5 mL per well, and let it stand at room temperature (25 °C) for 10 minutes to obtain a 0.1% gelatin-treated cell culture plate.

[0124] Step 4: Perform cell counting on the cell suspension, and inoculate the induced pluripotent stem cells (inoculate together with the cell suspension) on the 0.1% gelatin-treated cell culture plate at an inoculation density of 1×10 cells per well (the cell culture plate is supplemented with 1 mL of mouse embryonic fibroblast medium supplemented with 0.1% (v / v) CEPT per well), and incubate in a 37 °C cell culture incubator with 5% (v / v) CO 6 2 and 20% (v / v) O 2 2 for 15 minutes to remove the feeder layer cells remaining in the cell culture plate 2 during stem cell maintenance culture.

[0125] Step 5: After incubation, centrifuge the cell culture plate to discard the supernatant, and resuspend the cell pellet in the cell culture plate with embryonic assembly basal medium supplemented with 10 ng / mL human LIF and 0.1% (v / v) CEPT (1 mL of medium per well) to obtain suspension A.

[0126] Step 6: Take the induced hypoblast stem cells (iHypoblast SCs) from the first passage in Part 1, and when the cells reach a density of 70 - 80%, discard the medium in the cell culture plate. First, wash the induced hypoblast stem cells in the cell culture plate with DPBS buffer (1 mL of DPBS buffer per well, wash once), and then dissociate the induced hypoblast stem cells in the cell culture plate with Accutase at room temperature (25 °C) for 5 minutes (1 mL of Accutase per well, dissociate into single cells).

[0127] Step 7: After dissociation, centrifuge the cell culture plate to discard the supernatant, and resuspend the cell pellet in the cell culture plate with mouse embryonic fibroblast medium supplemented with 0.1% (v / v) CEPT (1 mL of medium per well) to obtain a cell suspension.

[0128] ​Step 8: Add 0.1% Gelatin to the cell culture plate at an addition amount of 1.5 mL / well, and let it stand at room temperature (25) °C for 10 minutes to obtain a cell culture plate treated with 0.1% gelatin.

[0129] Step 9: Perform cell counting on the cell resuspension, and re-inoculate the induced hypoblast stem cells in the cell resuspension (inoculate together with the cell and the resuspension) on the 0.1% gelatin-treated cell culture plate at an inoculation amount of 1×10 6 cells / well (the cell culture plate contains mouse embryonic fibroblast medium supplemented with 0.1% (v / v) CEPT at 1 mL / well), and incubate it in a 37 °C cell culture incubator with 5% (v / v) CO 2 and 20% (v / v) O 2 for 15 minutes to remove the feeder cells in the residual of the cell culture plate in the maintenance culture of the induced hypoblast stem cells.

[0130] Step 10: After the culture is completed, discard the medium in the cell culture plate. First, wash the induced hypoblast stem cells in the cell culture plate with DPBS buffer (the amount of DPBS buffer is 1 mL / well, wash once), and then let the induced hypoblast stem cells in the cell culture plate stand at room temperature (25 °C) for 5 minutes with Accutase for dissociation (the amount of Accutase is 1 mL / well, dissociate into single cells).

[0131] Step 11: After the incubation is completed, centrifuge the cell culture plate to discard the supernatant, and resuspend the cell pellet in the cell culture plate with the embryonic assembly basal medium supplemented with 10 ng / mL human LIF and 0.1% (v / v) CEPT (the amount of the medium is 1 mL / well) to obtain resuspension B.

[0132] Step 12: Perform cell counting on resuspension A and resuspension B, and re-inoculate both the induced pluripotent stem cells in resuspension A (inoculate together with the resuspension) and the induced hypoblast stem cells in resuspension B on each micropore of the AggreWell 400 embryoid body culture plate at an inoculation amount of 50 cells / well (each micropore contains the embryonic assembly basal medium supplemented with 10 ng / mL human LIF and 0.1% (v / v) CEPT at 1 mL / well). First, centrifuge at 100 rcf for 3 minutes to make the cells evenly fall into the micropores, and then culture it in a 37 °C cell culture incubator with 5% (v / v) CO and 20% (v / v) O 2 and 20% (v / v) O 2 for culture. During the culture period, change the medium once a day. When changing the medium, only 10 ng / mL human LIF needs to be additionally supplemented in the embryonic assembly basal medium starting from the second day of culture, and 0.1% (v / v) CEPT does not need to be additionally supplemented.

[0133] Step 13: After culturing for 4 days, replace the medium in the AggreWell 400 embryoid culture plate with the embryonic assembly basal medium supplemented with 200 ng / mL human BMP4, and continue culturing in a 37 °C cell culture incubator with 5% (v / v) CO 2 and 20% (v / v) O 2 . During the culturing period, change the medium once a day. After culturing for 8 days, the embryonic model is successfully constructed and is called induced embryoids (iEmbryoids).

[0134] Among them, the embryonic assembly basal medium is a mixed medium containing 1% (v / v) GlutaMAX, 1% (v / v) non-essential amino acids, 1% (v / v) penicillin-streptomycin, 1% (v / v) sodium pyruvate, 0.1 mM 2-mercaptoethanol, 1% (v / v) N2, 1% (v / v) B27, and 0.3% (v / v) BSA; the mixed medium is composed of Neurobasal medium and DMEM / F12 at a volume ratio of 1:1.

[0135] Experimental Example 1: Exploration of the construction conditions of induced embryoids (iEmbryoids) and verification of the performance of induced embryoids

[0136] This experimental example provides an experiment for exploring the construction conditions of induced embryoids (iEmbryoids) and verifying the performance of induced embryoids. The experimental process is as follows:

[0137] 1. Spontaneous assembly of iPSCs and iHypoblast SCs to form induced embryoids

[0138] Understanding the cellular and molecular processes of human post-implantation development is one of the fundamental issues in developmental and stem cell biology. During the post-implantation stage of human embryogenesis, the epiblast (Epi) acquires apical-basal polarity to construct the proamniotic cavity and ultimately forms the amnion and post-implantation epiblast. Meanwhile, the hypoblast (Hypo) differentiates into the visceral endoderm (VE) and yolk sac (YS) endoderm, forming the primitive yolk sac and the secondary yolk sac cavity. Epi and VE generate a bilayer disc structure, and then the embryo enters the gastrulation stage ( Figure 1 d), which is considered a milestone in mammalian embryonic development. Therefore, this study attempts to develop a robust model by combining iPSCs and iHypoblast SCs to simulate human perigastrulation embryonic development.

[0139] Leukemia inhibitory factor (LIF) plays an important role in maintaining mammalian embryonic stem cells and is included in iPSCs( in the medium of 4CL) and iHypoblast SCs (Hypo medium 26#). Therefore, in this study, LIF was first introduced into the basic aggregation medium to promote the survival and aggregation of iPSCs and iHypoblast SCs from day 0 to day 4 ( Figure 1 a). It has been shown that bone morphogenetic protein (BMP) signaling can initiate gastrulation, including downstream WNT and NODAL signaling, mimicking embryonic development during the human perigastrulation period. Therefore, in this study, exogenous BMP4 was introduced into the basic aggregation medium from day 4 to day 8 to promote the formation of a perigastrulation embryonic model ( Figure 1 a, Figure 1 b, Figure 1 e).

[0140] During early human embryonic development, the inner cell mass separates into ectodermal and hypoblast lineages on days 5 to 7 of embryonic development. During the post-implantation stage, the ectoderm forms a ring layer expressing OCT3 / 4 and SOX2, while the hypoblast lies beneath the ectoderm and expresses GATA6 and SOX17. Consistent with this, in this study, it was observed that when iPSCs and iHypoblastSCs were introduced into a 3D AggreWell 400 plate on day 0, Epi-like cells and hypoblast-like cells separated into two distinct regions on day 4. The aggregates on day 4 clearly expressed Epi and hypoblast lineage markers such as OCT3 / 4, SOX2, as well as GATA6, SOX17, and at the same time showed signs of early cavity formation ( Figure 2 a~ Figure 2 b, Figure 4 a, Figure 5 a).

[0141] Next, this study attempted to extend the developmental potential of day 4 aggregates by activating BMP4 signaling. Time-lapse imaging from day 4 to day 8 showed that the cavities of ectoderm-like cells and hypoblast-like cells gradually expanded, forming an amnion-like cavity with low OCT3 / 4+SOX2 and a yolk sac-like cavity with GATA6+SOX17+ on day 6, i.e., a bilayer disc structure, and further expanding on day 8 ( Figure 2 a~ Figure 2 b). In addition, this study formed aggregates of each cell line separately to understand the contribution of iPSCs and iHypoblast SCs to the aggregates. In the individual aggregates of iPSCs or iHypoblast SCs, no obvious amnion-like and YS-like cavities were observed on day 4, and these aggregates gradually collapsed on day 8, indicating Cell interactions between iPSCs and iHypoblast SCs are crucial for inducing morphogenetic changes.( Figure 3 a– Figure 3 d).

[0142] Considering that the aggregates on day 8 were all derived from human skin fibroblasts, this study named this induced stem cell-based embryonic model "iEmbryoids". The efficiency of forming well-structured day 8 iEmbryoids was estimated to be 17.09%( Figure 1 c). Overall, these findings indicate that iEmbryoids are consistent with the characteristics of human post-implantation embryonic development.

[0143] 2. Induced embryoids recapitulate key developmental landmarks of the human perigastrulation period

[0144] Next, in this study, through immunofluorescence staining analysis, we examined whether iEmbryoids could mimic the key developmental landmarks of pre-implantation and perigastrulation embryos. The amnion plays a signaling hub role in regulating the ectoderm (Epi) and initiating gastrulation. On day 6, compared with the other side, the expression of SOX2 was reduced in ISL1+TFAP2A+ cells, and their amnion-like identity was confirmed by morphology and gene expression( Figure 4 b). During primate development, primordial germ cells (PGCs) first appear in the amnion and then migrate to the visceral endoderm (VE). On day 8, co-immunofluorescence staining of PGC markers (OCT4+SOX17+BLIMP1+ and NANOG+SOX17+TFAP2C+) indicated the presence of PGC-like cells in some iEmbryoids( Figure 4 c, Figure 5 c), which is consistent with the position and expression pattern in the human embryonic model.

[0145] Subsequently, this study verified the initiation process of gastrulation through the formation of the anteroposterior axis (A-P axis). The early establishment of the A-P axis is a common feature of mammals, which occurs when a subset of cells in the ectoderm begins to express the T gene in the posterior region, while their antagonists CER1 and LEFTY are expressed in the anterior region of the embryonic disc. On day 8, T+ ELCs were located posterior to the ectoderm-like cells of iEmbryoids( Figure 5 d– Figure 5 e). Meanwhile, the anterior VE-like cells of CER1 and LEFTY were located below the anterior side of the ectoderm-like cells of iEmbryoids, forming an anterior signal center for the ectoderm pattern, which together with the posterior T+ ectoderm-like cells constituted the putative A-P axis and symmetry breaking( Figure 5 d– Figure 5e). Further immunofluorescence staining analysis confirmed the positional information of the anterior VE marker OTX2 on the adjacent side of the ectoderm-like cells ( Figure 4 d). Notably, a group of cells expressed GATA6 and GATA4 but not SOX17 ( Figure 6 a– Figure 6 c), surrounding the YS-like cells, which was consistent with the ExEM pattern in marmoset embryos. Meanwhile, these cells also expressed multiple ExEM markers, including BST2, FOXF1, and VIM, further validating the identity of ExEM-like cells in day 8 iEmbryoids ( Figure 6 a– Figure 6 c). In addition, this study also observed some flattened epithelial cells located on the YS-like structure, co-expressing CD34 and ERG ( Figure 6 d), indicating that these cells represented the fate of hematopoietic endothelial progenitors (HEPs).

[0146] Overall, day 8 iEmbryoids recapitulated key milestone events of human perigastrulation embryonic development, including amnion and yolk sac cavity formation, PGC specification, gastrulation initiation, A-P axis formation, ExEM expansion, and the emergence of HEPs, and were suitable as a model for understanding primitive streak formation and the process of human gastrulation embryonic development.

[0147] 3. Single-cell transcriptomics study of induced embryoids

[0148] To further understand the cellular composition and transcriptional landscape of iEmbryoids, rather than just the expression of a few key genes, this study performed Chromium 10X single-cell RNA sequencing (scRNA-seq) analysis on approximately 200 iEmbryoids ( Figure 7 a). UMAP analysis revealed a total of 11 distinct cell clusters ( Figure 7 b). This study annotated these cell clusters based on the expression patterns of lineage-specific markers, enabling this study to classify all identified cell clusters ( Figure 7 c– Figure 7 d). This annotation identified a wide range of embryonic and extraembryonic cell types present during human perigastrulation embryonic development ( Figure 7 c– Figure 7 d).

[0149] Specifically, post-implantation Epi-like cells expressing core pluripotency markers, such as POU5F1 (also known as OCT3 / 4), SOX2, NANOG, TERF1, and ESRG, were observed in the Epi cluster ( Figure 9a). Additionally, this study observed a cell cluster expressing ISL1, TFAP2A, GARBP, HEY1, and VTCN1, which was defined as the amnion cluster ( Figure 9 b). Consistent with the immunofluorescence staining results of day 8 iEmbryoids, this study detected the presence of PGC-like cells characterized by the expression of BLIMP, CXCR4, TFAP2C, NANOS3, and PDPN ( Figure 10 b). This study also identified two hypoblast lineage clusters, including the VE / YS and SYS clusters. The VE / YS cluster expressed hypoblast markers SOX17, RSPO3, BMP6, FOXA2, and GATA4 ( Figure 9 d), while cells within the SYS cluster showed high expression of the endoderm marker AFP, the nutrient transport marker TTR, and various apolipoprotein markers (APOA1, APOA2, APOB), supporting its classification as the SYS-like region ( Figure 10 a).

[0150] Next, this study identified the primitive streak marker TBXT (also known as Brachyury) that binds to CDH1, WNT8A, GAL, and MSGN1 ( Figure 9 c). In the dorsal cells of the primitive streak, the expression of early mesoderm markers (such as MESP1, MESP2, EOMES, MIXL1, and BMP4) increased and was annotated as early mesoderm ( Figure 10 c). In contrast, the mesoderm cluster showed a broad spectrum of heterogeneous marker genes, similar to the CS7 gastrula, expressing genes of multiple mesoderm types, including ACTC1, GATA6, HAND1, PDGFRA, and SNAI2 ( Figure 10 d). The co-expression of HAND1 and GATA6 marks the anterior heart field lateral plate mesoderm. This study observed this co-expression pattern in multiple clusters, including the early mesoderm, mesoderm, and ExEM clusters as well as the amnion cluster ( Figure 11 b), indicating that these embryonic mesoderm clusters do not represent specific mesoderm subtypes but transitional mesoderm states. In mice, POSTN is a marker of ExEM, marking not only the amnion but also the yolk sac. This study detected multiple clusters expressing the ExEM markers POSTN, DCN, and HAND2, which were defined as ExEM clusters ( Figure 11 b). There have been multiple reports suggesting that the origin of ExEM is the hypoblast, while the data of this study indicate that ExEM-like cells are closer to the mesoderm lineage. This finding may support the hypothesis that ExEM originates from primitive streak cells in iEmbryoids. Additionally, this study verified the expression of the human hematopoietic progenitor cell markers CD34 and ERG by immunofluorescence staining ( Figure 6d). Consistent with the immunofluorescence staining results of day 8 iEmbryoids, the HEP clusters expressed hematopoietic and endothelial markers CD34, ERG, RUNX1, MEF2C, and PECAM1, located near the mesodermal clusters ( Figure 11 a).

[0151] To further evaluate the transcriptional similarity between iEmbryoids and human embryos, this study integrated the scRNA-seq data of iEmbryoids with three previously reported scRNA-seq datasets, which were from human CS6 in vitro cultured embryos and genuine CS7 gastrulas. UMAP analysis revealed a high degree of consistency between iEmbryoid cells and human embryo cells ( Figure 8 a). This study observed significant similarities between multiple cell lineages in iEmbryoids and CS7 gastrulas, while the similarities between iEmbryoids and pre-gastrula CS6 embryos cultured in vitro were lower, indicating that iEmbryoids are similar to CS6b human embryos ( Figure 8 a). In addition, the ExEM and PGC annotations of CS7 gastrulas and the heatmap analysis of ExEM-like cells and PGC-like cells in vitro showed similar expression patterns to the iEmbryoid cluster counterparts ( Figure 8 b). Notably, the subset analysis of the integrated blood cells revealed five different subsets, labeled as hematopoietic endothelium, blood progenitor cells, erythromyeloid progenitor cells, and myeloid progenitor cells ( Figure 8 c). In summary, the aforementioned single-cell RNA sequencing (scRNA-seq) analysis supported that iEmbryoids recapitulate the major cell types of CS6b human embryos.

[0152] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. An embryo model, characterized in that: The embryo model is Induced pluripotent stem cells and induced hypoblast stem cells are spontaneously assembled; Both induced pluripotent stem cells and induced hypoblast stem cells are derived from somatic cell reprogramming.

2. The embryo model according to claim 1, characterized in that: The method for preparing the embryo model comprises: After the induced pluripotent stem cells and induced hypoblast stem cells are dispersed, they are co-inoculated into a culture container and cultured in sequence using embryo assembly basal medium additionally supplemented with leukemia inhibitory factor and CEPT, embryo assembly basal medium additionally supplemented with leukemia inhibitory factor, and embryo assembly basal medium additionally supplemented with bone morphogenetic protein 4 to obtain an embryo model.

3. The embryo model according to claim 2, characterized in that: The components of the embryo assembly basal medium include GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement and bovine serum albumin.

4. The embryo model according to claim 3, characterized in that: In terms of volume percentage, the concentration of GlutaMAX in the embryo assembly basal medium is 0.5-1%; The concentration of the non-essential amino acids in the embryo assembly basal medium is 0.5-1% by volume; In terms of volume percentage, the concentration of penicillin-streptomycin in the embryo assembly basal medium is 0.5-1%; in terms of volume percentage, the concentration of sodium pyruvate in the embryo assembly basal medium is 0.5-1%; in terms of volume percentage, the concentration of 2-mercaptoethanol in the embryo assembly basal medium is 0.1-0.5 mM; in terms of volume percentage, the concentration of N2 supplement in the embryo assembly basal medium is 0.5-1%; In terms of volume percentage, the concentration of the B27 supplement in the embryo assembly basal medium is 1-2%; in terms of volume percentage, the concentration of the bovine serum albumin in the embryo assembly basal medium is 0.1-0.5%; the concentration of the leukemia inhibitory factor in the embryo assembly basal medium is 10-50 ng / mL; the concentration of the bone morphogenetic protein 4 in the embryo assembly basal medium is 50-200 ng / mL; and in terms of volume percentage, the concentration of the CEPT in the embryo assembly basal medium is 0.1-0.5%.

5. The embryo model according to claim 3 or 4, characterized in that: The components of the embryo assembly basal culture medium also include a matrix; the matrix includes Neurobasal culture medium and DMEM / F12 culture medium; in the matrix, the volume ratio of Neurobasal culture medium to DMEM / F12 culture medium is 0.5-1:0.5-1.

6. The embryo model according to any one of claims 1 to 5, characterized in that: The method for preparing induced hypoblast stem cells comprises: firstly transfecting somatic cells with Yamanaka factors, and then inducing and culturing the transfected somatic cells with induced hypoblast stem cell culture medium to obtain induced hypoblast stem cells.

7. A method for constructing the embryo model according to any one of claims 1 to 6, characterized in that: The method comprises: After the induced pluripotent stem cells and induced hypoblast stem cells are dispersed, they are co-inoculated into a culture container and cultured in sequence using embryo assembly basal medium additionally supplemented with leukemia inhibitory factor and CEPT, embryo assembly basal medium additionally supplemented with leukemia inhibitory factor, and embryo assembly basal medium additionally supplemented with bone morphogenetic protein 4 to obtain an embryo model.

8. An embryo assembly culture medium, characterized in that The components of the embryo assembly medium include leukemia inhibitory factor, GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement and bovine serum albumin; The components of the embryo assembly medium include leukemia inhibitory factor, CEPT, GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement and bovine serum albumin; Alternatively, the components of the embryo assembly medium include bone morphogenetic protein 4, GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement and bovine serum albumin.

9. A method for screening drugs for preventing and / or treating diseases, characterized in that: The method comprises: using the embryo model described in any one of claims 1 to 6 to screen drugs for preventing and / or treating diseases; the diseases include genetic diseases and / or developmental disorders.

10. A method for evaluating drug toxicity, characterized in that: The method comprises: administering a drug to the embryo model according to any one of claims 1 to 6, and evaluating the toxicity of the drug by observing changes in indicators of the embryo model.

11. Use of the embryo model according to any one of claims 1 to 6, the method according to claim 7, or the embryo assembly basal medium according to claim 8 in screening drugs for preventing and / or treating diseases or evaluating drug toxicity, characterized in that: The diseases include genetic diseases and / or developmental disorders.

12. A cell therapy drug for preventing and / or treating a disease, characterized in that: The cell therapy drug comprises the embryo model according to any one of claims 1 to 6, or the cell therapy drug is prepared from the embryo model according to any one of claims 1 to 6.

13. Use of the embryo model according to any one of claims 1 to 6 in the preparation of cell therapy drugs for preventing and / or treating diseases.

14. An organ transplant, characterized in that: The organ transplant is prepared from the embryo model according to any one of claims 1 to 6.

15. Use of the embryo model according to any one of claims 1 to 6 in preparing organ transplants.

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