Embryo model and construction method therefor

Abstract

Provided is an embryo model, which is obtained by means of self-assembly of induced pluripotent stem cells and induced hypoblast stem cells, wherein both the induced pluripotent stem cells and the induced hypoblast stem cells are obtained by means of somatic cell reprogramming. The self-assembly of the stem cells of two different lineages generated by means of somatic cell reprogramming forms an embryo model simulating human peri-gastrulation. The embryo model better reproduces the interaction and communication mechanism among cells of multiple lineages during human embryonic development by means of combining cells of different lineages. With the embryo model, by means of a gene editing tool, lineage tracing and gene regulatory network research can be performed on cells of different lineages, thereby enabling an in-depth understanding of the molecular regulatory mechanisms of early human embryonic development.

Description

An embryo model and a method for constructing the same

[0001] Cross-reference to Related Applications

[0002] This application claims priority to the Chinese patent application No. 202411386178.4, filed on September 30, 2024, and entitled “An embryo model and a method for constructing the same”, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to an embryo model and a method for constructing the same, and belongs to the technical field of stem cells. BACKGROUND

[0004] Human early embryonic development begins with the fertilization of an egg, during which a zygote is formed. The zygote then enters the cleavage stage to form a 2-cell, 4-cell, 8-cell, and then a morula, and further develops into a blastocyst. After the blastocyst implants in the uterus, it enters the gastrulation stage to form a gastrula, neurula, and finally enters the early organogenesis stage. According to the morphological and structural characteristics of human early embryos, the development process of the first 60 days of embryonic period can be roughly divided into 23 different stages, which are called Carnegie stages (CS). After human embryos implant, the primitive epiblast cells (Epi) acquire polarity and build the primitive amniotic cavity, eventually forming the amnion and implanted epiblast cells. At the same time, the hypoblast (Hypo) differentiates into the splanchnic mesoderm and yolk sac mesoderm, forming the primitive yolk sac and secondary yolk sac structures. The epiblast cells and splanchnic mesoderm together generate the blastoderm 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 tracking post-implantation embryos) and ethical limitations (14-day rule), researchers have lacked systematic and in-depth understanding of the development process of human embryos in the two weeks after implantation.

[0005] In recent years, the rapid development of human pluripotent stem cell (hPSC) research has provided another new method for exploring the development process of human embryos. At present, the embryo model constructed by spontaneous assembly of single or multiple types of human stem cells in vitro is generally referred to as a "embryoid", which is similar to a real embryo in cell number and composition and three-dimensional structure, and is a simplified model that simulates the explicit characteristics of a natural embryo. Human embryo samples are often very scarce, and compared with real human embryos, stem cell-based embryo models have the characteristics of scalability, ease of genetic manipulation, and fine control of variables. Therefore, the emerging field of embryoid models provides a new strategy for exploring the genetic and molecular mechanisms of human early development, and provides new research ideas for studying the differentiation trajectory of different cell lineages during early human embryonic development, as well as the clinical drug screening and regenerative medicine of major diseases such as early embryonic development defects. However, although the existing in vitro embryo models have made significant progress in simulating human embryonic development, they still have several technical defects and limitations.

[0006] For example, existing in vitro models, including human peri-gastrulation embryo models such as peri-gastruloids (see Liu, L. et al. Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids. Cell 186, 3776-3792 e3716 (2023).) and peri-gastrulation trilaminar embryonic disc (PTED) embryoids (see 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).), etc. mostly rely on the spontaneous assembly of single human embryonic stem cells (hESCs), so it is difficult to effectively simulate the complex interactions between different lineage cells in the early embryo. And because the cell lineages involved in the existing models are single, it is not possible to effectively track the lineages using gene editing tools such as CRISPR to analyze the differentiation trajectory and gene regulation mechanisms of different lineage cells during human embryonic development. In addition, most existing models based on human embryonic stem cells (hESCs) may raise ethical concerns due to the use of human embryonic cells. SUMMARY

[0007] To address the aforementioned deficiencies, this application provides an embryo model, wherein the embryo model is composed of... Induced pluripotent stem cells (i.e., stem cells) It is obtained by the spontaneous assembly of iPSCs and induced hypoblast stem cells (iHypoblast SCs); Both induced pluripotent stem cells and induced hypoblast stem cells (iHypoblast SCs) are derived from somatic cell reprogramming.

[0008] In one embodiment of this application, the method for preparing the embryo model includes: ... Induced pluripotent stem cells (i.e., stem cells) After iPSCs and induced hypodermal stem cells (iHypoblast SCs) were dispersed, they were co-seeded into culture containers and cultured sequentially with embryo assembly basal medium supplemented with leukemia suppressor factor (LIF) and CEPT (Chroman 1, Emricasan, Polyamines, Trans-ISRIB), embryo assembly basal medium supplemented with leukemia suppressor factor (LIF), and embryo assembly basal medium supplemented with bone morphogenetic protein 4 (BMP4) to obtain embryo models.

[0009] In one embodiment of this application, the embryo assembly basal culture medium comprises GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement, and bovine serum albumin (BSA).

[0010] In an embodiment of the present application, the concentration of GlutaMAX in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of non-essential amino acids in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of penicillin-streptomycin in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of sodium pyruvate in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of 2-mercaptoethanol in the embryonic assembly base medium is 0.1-0.5 mM; the concentration of N2 supplement in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of B27 supplement in the embryonic assembly base medium is 1-2% by volume percentage; the concentration of bovine serum albumin in the embryonic assembly base medium is 0.1-0.5% by volume percentage; the concentration of leukemia inhibitory factor in the embryonic assembly base medium is 10-50 ng / mL; the concentration of bone morphogenetic protein 4 in the embryonic assembly base medium is 50-200 ng / mL; and the concentration of CEPT in the embryonic assembly base medium is 0.1-0.5% by volume percentage.

[0011] In an embodiment of the present application, the components of the embryonic assembly base medium further comprise a matrix; the matrix comprises Neurobasal medium and DMEM / F12 medium; and the volume ratio of Neurobasal medium to DMEM / F12 medium in the matrix is 0.5-1:0.5-1.

[0012] In an embodiment of the present application, the method for preparing the induced hypoblast stem cells (iHypoblast SCs) comprises: first, transfecting somatic cells using Yamanaka factors (OSKM); and then, inducing and culturing the transfected somatic cells using an induced hypoblast stem cell medium to obtain induced hypoblast stem cells (iHypoblast SCs).

[0013] The characteristics of the induced hypoblast stem cells (iHypoblast SCs) include:

[0014] First, in an undifferentiated, bipotential state, and having the ability to differentiate into cells exhibiting one or more characteristics of endoderm / yolk sac endoderm (VE / YE) like cells and extraembryonic mesoderm (ExEM) like cells;

[0015] Second, a colony appearance of flat epithelial shape;

[0016] Third, expression of one or more biochemical markers of hypoblast stem cells (e.g., PDGFRA, SOX17, GATA6, GATA4, FOXA2, FN1, COL4A1, and Laminin) can be determined by immunohistochemistry and / or PCR detection;

[0017] Fourth, single cell omics similar to human embryonically derived hypoblast cells;

[0018] Fifth, the ability to integrate in the extraembryonic endoderm lineage in human-mouse chimeras.

[0019] In an embodiment of the present application, the components of the induced hypoblast stem cell medium comprise platelet-derived growth factor AA (PDGFAA), leukemia inhibitory factor (LIF), fibroblast growth factor 4 (FGF4), GSK-3a / b inhibitor CHIR99021, TGF-beta type I receptor inhibitor A83-01, and bone morphogenetic protein 4 (BMP4).

[0020] In an embodiment of the present application, the concentration of the platelet-derived growth factor AA in the induced hypoblast stem cell medium is 10-50 ng / mL; the concentration of the leukemia inhibitory factor in the induced hypoblast stem cell medium is 10-50 ng / mL; the concentration of the fibroblast growth factor 4 in the induced hypoblast stem cell medium is 25-125 ng / mL; the concentration of the GSK-3a / b inhibitor CHIR99021 in the induced hypoblast stem cell medium is 3-15 mM; the concentration of the TGF-beta type I receptor inhibitor A83-01 in the induced hypoblast stem cell medium is 1-5 mM; and the concentration of the bone morphogenetic protein 4 in the induced hypoblast stem cell medium is 10-50 ng / mL.

[0021] In an embodiment of the present application, the components of the induced hypoblast stem cell medium further comprise heparin, N2 supplement, B27 supplement, GlutaMAX, non-essential amino acids, 2-mercaptoethanol, and / or penicillin-streptomycin.

[0022] In an embodiment of the present application, the concentration of the N2 supplement in the induced hypoblast stem cell culture medium is 0.5-1% by volume percentage; the concentration of the B27 supplement in the induced hypoblast stem cell culture medium is 1-2% by volume percentage; the concentration of the GlutaMAX in the induced hypoblast stem cell culture medium is 0.5-1% by volume percentage; the concentration of the non-essential amino acids in the induced hypoblast stem cell culture medium is 0.5-1% by volume percentage; the concentration of the penicillin-streptomycin in the induced hypoblast stem cell culture medium is 0.5-1% by volume percentage; the concentration of the heparin in the induced hypoblast stem cell culture medium is 1-5 μg / mL; and the concentration of the 2-mercaptoethanol in the induced hypoblast stem cell culture medium is 0.1-0.5 mM.

[0023] In an embodiment of the present application, the components of the induced hypoblast stem cell culture medium further comprise a matrix; the matrix comprises Neurobasal medium and DMEM / F12 medium; and the volume ratio of Neurobasal medium to DMEM / F12 medium in the matrix is 0.5-1:0.5-1.

[0024] In an embodiment of the present application, the matrix consists of Neurobasal medium and DMEM / F12 medium; and the volume ratio of Neurobasal medium to DMEM / F12 medium in the matrix is 1:1.

[0025] In an embodiment of the present application, the method for preparing the induced hypoblast stem cells (iHypoblast SCs) comprises: after the somatic cells are inoculated into the somatic cell culture medium, the somatic cells are transfected using the Yamanaka factors (OSKM) under the mediation of the transfection vector; after the transfection is completed, the transfected somatic cells are dispersed and inoculated into a cell culture plate with feeder layer cells, and the cells in the cell culture plate are cultured using the somatic cell culture medium and the induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632 in sequence; after the culture is completed, the PDGFRA positive hypoblast-like cells are selected and re-inoculated into a cell culture plate with feeder layer cells, and the cells in the cell culture plate are cultured using the induced hypoblast stem cell culture medium with additional ROCK-I / II inhibitor Y27632 and the induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632 in sequence; after the culture is completed, the hypoblast-like colonies in the cell culture plate are selected and transferred to a cell culture plate with feeder layer cells, and the cells in the cell culture plate are cultured using the induced hypoblast stem cell culture medium with additional ROCK-I / II inhibitor Y27632 and the induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632 in sequence until the hypoblast-like cells in the cell culture plate reach 80-90% confluence; after the culture is completed, the hypoblast-like cells are dispersed and inoculated into a cell culture plate with feeder layer cells, and the cells in the cell culture plate are cultured using the induced hypoblast stem cell culture medium with additional ROCK-I / II inhibitor Y27632 and the induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632 in sequence to obtain the induced hypoblast stem cells (iHypoblast SCs).

[0026] In an embodiment of the present application, the concentration of the ROCK-I / II inhibitor Y27632 in the induced hypoblast stem cell culture medium is 5-10 μM.

[0027] In an embodiment of the present application, the somatic cells comprise human fibroblasts (for example, 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.

[0028] In an embodiment of the present application, the somatic cell culture medium comprises fibroblast culture medium and / or 106 culture medium.

[0029] In an embodiment of the present application, the feeder layer cells comprise inactivated mouse embryonic fibroblasts and / or inactivated human fibroblasts.

[0030] In one embodiment of this application, the Yamanaka factors (OSKMs) used to reprogram 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.

[0031] In one embodiment of this application, the transfection vector includes a virus, liposomes, mRNA, and / or exosomes.

[0032] In one embodiment of this application, the virus includes Sendai virus, adenovirus, and / or lentivirus.

[0033] This application also provides a method for constructing the above-mentioned embryo model, the method comprising: […]. Induced pluripotent stem cells (i.e., stem cells) After iPSCs and induced hypodermal stem cells (iHypoblast SCs) were dispersed, they were co-seeded into culture containers and cultured sequentially with embryo assembly basal medium supplemented with leukemia suppressor factor (LIF) and CEPT (Chroman 1, Emricasan, Polyamines, Trans-ISRIB), embryo assembly basal medium supplemented with leukemia suppressor factor (LIF), and embryo assembly basal medium supplemented with bone morphogenetic protein 4 (BMP4) to obtain embryo models.

[0034] In one embodiment of this application, the embryo assembly basal culture medium comprises GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement, and bovine serum albumin (BSA).

[0035] In an embodiment of the present application, the concentration of GlutaMAX in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of non-essential amino acids in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of penicillin-streptomycin in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of sodium pyruvate in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of 2-mercaptoethanol in the embryonic assembly base medium is 0.1-0.5 mM; the concentration of N2 supplement in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of B27 supplement in the embryonic assembly base medium is 1-2% by volume percentage; the concentration of bovine serum albumin in the embryonic assembly base medium is 0.1-0.5% by volume percentage; the concentration of leukemia inhibitory factor in the embryonic assembly base medium is 10-50 ng / mL; the concentration of bone morphogenetic protein 4 in the embryonic assembly base medium is 50-200 ng / mL; and the concentration of CEPT in the embryonic assembly base medium is 0.1-0.5% by volume percentage.

[0036] In an embodiment of the present application, the components of the embryonic assembly base medium further comprise a matrix; the matrix comprises Neurobasal medium and DMEM / F12 medium; and the volume ratio of Neurobasal medium to DMEM / F12 medium in the matrix is 0.5-1:0.5-1.

[0037] In an embodiment of the present application, the method for preparing the induced hypoblast stem cells (iHypoblast SCs) comprises: first, transfecting somatic cells using Yamanaka factors (OSKM); and then, inducing and culturing the transfected somatic cells using an induced hypoblast stem cell medium to obtain induced hypoblast stem cells (iHypoblast SCs).

[0038] In an embodiment of the present application, the components of the induced hypoblast stem cell medium comprise 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).

[0039] In an embodiment of the present application, the concentration of the platelet-derived growth factor AA in the induced hypoblast stem cell culture medium is 10-50 ng / mL; the concentration of the leukemia inhibitory factor in the induced hypoblast stem cell culture medium is 10-50 ng / mL; the concentration of the fibroblast growth factor 4 in the induced hypoblast stem cell culture medium is 25-125 ng / mL; the concentration of the GSK-3a / b inhibitor CHIR99021 in the induced hypoblast stem cell culture medium is 3-15 mM; the concentration of the TGF-β type I receptor inhibitor A83-01 in the induced hypoblast stem cell culture medium is 1-5 mM; and the concentration of the bone morphogenetic protein 4 in the induced hypoblast stem cell culture medium is 10-50 ng / mL.

[0040] In an embodiment of the present application, the components of the induced hypoblast stem cell culture medium further comprise heparin, N2 supplement, B27 supplement, GlutaMAX, non-essential amino acids, 2-mercaptoethanol, and / or penicillin-streptomycin.

[0041] In an embodiment of the present application, the concentration of the N2 supplement in the induced hypoblast stem cell culture medium is 0.5-1% by volume; the concentration of the B27 supplement in the induced hypoblast stem cell culture medium is 1-2% by volume; the concentration of the GlutaMAX in the induced hypoblast stem cell culture medium is 0.5-1% by volume; the concentration of the non-essential amino acids in the induced hypoblast stem cell culture medium is 0.5-1% by volume; the concentration of the penicillin-streptomycin in the induced hypoblast stem cell culture medium is 0.5-1% by volume; the concentration of the heparin in the induced hypoblast stem cell culture medium is 1-5 pg / mL; and the concentration of the 2-mercaptoethanol in the induced hypoblast stem cell culture medium is 0.1-0.5 mM.

[0042] In an embodiment of the present application, the components of the induced hypoblast stem cell culture medium further comprise a matrix; the matrix comprises Neurobasal medium and DMEM / F12 medium; and the volume ratio of Neurobasal medium to DMEM / F12 medium in the matrix is 0.5-1:0.5-1.

[0043] In an embodiment of the present application, the matrix consists of Neurobasal medium and DMEM / F12 medium; and the volume ratio of Neurobasal medium to DMEM / F12 medium in the matrix is 1:1.

[0044] In an embodiment of the present application, the method for preparing the induced hypoblast stem cells (iHypoblast SCs) comprises: after the somatic cells are inoculated into the somatic cell culture medium, the somatic cells are transfected using the Yamanaka factors (OSKM) under the mediation of the transfection vector; after the transfection is completed, the transfected somatic cells are dispersed and inoculated into a cell culture plate with feeder layer cells, and the cells in the cell culture plate are cultured using the somatic cell culture medium and the induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632 in sequence; after the culture is completed, the PDGFRA positive hypoblast-like cells are selected and re-inoculated into a cell culture plate with feeder layer cells, and the cells in the cell culture plate are cultured using the induced hypoblast stem cell culture medium with additional ROCK-I / II inhibitor Y27632 and the induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632 in sequence; after the culture is completed, the hypoblast-like colonies in the cell culture plate are selected and transferred to a cell culture plate with feeder layer cells, and the cells in the cell culture plate are cultured using the induced hypoblast stem cell culture medium with additional ROCK-I / II inhibitor Y27632 and the induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632 in sequence until the hypoblast-like cells in the cell culture plate reach 80-90% confluence; after the culture is completed, the hypoblast-like cells are dispersed and inoculated into a cell culture plate with feeder layer cells, and the cells in the cell culture plate are cultured using the induced hypoblast stem cell culture medium with additional ROCK-I / II inhibitor Y27632 and the induced hypoblast stem cell culture medium without additional ROCK-I / II inhibitor Y27632 in sequence to obtain the induced hypoblast stem cells (iHypoblast SCs).

[0045] In an embodiment of the present application, the concentration of the ROCK-I / II inhibitor Y27632 in the induced hypoblast stem cell culture medium is 5-10 μM.

[0046] In an embodiment of the present application, the somatic cells comprise human fibroblasts (for example, 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.

[0047] In an embodiment of the present application, the somatic cell culture medium comprises fibroblast culture medium and / or 106 culture medium.

[0048] In an embodiment of the present application, the feeder layer cells comprise inactivated mouse embryonic fibroblasts and / or inactivated human fibroblasts.

[0049] In an embodiment of the application, 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.

[0050] In an embodiment of the application, the transfection vehicle includes a virus, a liposome, an mRNA, and / or an exosome.

[0051] In an embodiment of the application, the virus includes a Sendai virus, an adenovirus, and / or a lentivirus.

[0052] The present application also provides an embryonic assembly medium, wherein the components of the embryonic 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);

[0053] The components of the embryonic 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);

[0054] Alternatively, the components of the embryonic 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).

[0055] In an embodiment of the present application, the concentration of GlutaMAX in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of non-essential amino acids in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of penicillin-streptomycin in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of sodium pyruvate in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of 2-mercaptoethanol in the embryonic assembly base medium is 0.1-0.5 mM; the concentration of N2 supplement in the embryonic assembly base medium is 0.5-1% by volume percentage; the concentration of B27 supplement in the embryonic assembly base medium is 1-2% by volume percentage; the concentration of bovine serum albumin in the embryonic assembly base medium is 0.1-0.5% by volume percentage; the concentration of leukemia inhibitory factor in the embryonic assembly base medium is 10-50 ng / mL; the concentration of bone morphogenetic protein 4 in the embryonic assembly base medium is 50-200 ng / mL; and the concentration of CEPT in the embryonic assembly base medium is 0.1-0.5% by volume percentage.

[0056] In an embodiment of the present application, the components of the embryonic assembly base medium further comprise a matrix; the matrix comprises Neurobasal medium and DMEM / F12 medium; and the volume ratio of Neurobasal medium to DMEM / F12 medium in the matrix is 0.5-1:0.5-1.

[0057] The present application also provides a method for screening drugs for preventing and / or treating diseases, which comprises screening drugs for preventing and / or treating diseases using the embryonic model described above; the diseases include genetic diseases and / or developmental disorders.

[0058] In an embodiment of the present application, the genetic diseases include GATA6 mutation-induced genetic pancreatic agenesis, HNF1B-related renal dysplasia, and / or situs inversus; and the developmental disorders include endoderm developmental disorders, fore-aft axis developmental disorders, and / or early embryonic developmental arrest.

[0059] In an embodiment of the present application, the method comprises first modeling the embryonic model to obtain an in-vitro model of diseases, and then screening drugs for preventing and / or treating diseases using the in-vitro model of diseases.

[0060] In an embodiment of the present application, the modeling comprises inducing diseases by drugs and / or inducing diseases by gene editing.

[0061] In an embodiment of the present application, the modeling comprises: applying SB431542, A83-01 and Activin A drugs to the embryonic model to obtain an in vitro model of Nodal / Activin signaling pathway and periblastula stage embryonic development; or applying CHIR99021, IWP-2 and XAV939 drugs to the embryonic model to obtain an in vitro model of Wnt / β-Catenin signaling pathway and periblastula stage embryonic development; or applying FGF2 and SU5402 drugs to the embryonic model to obtain an in vitro model of FGF signaling pathway and periblastula stage embryonic development.

[0062] The present application also provides a method for evaluating drug toxicity, which comprises: applying a drug to the above-mentioned embryonic model, and evaluating the toxicity of the drug by observing the changes of the indicators of the embryonic model.

[0063] In an embodiment of the present application, the toxicity of the drug comprises potential embryonic / fetal toxicity, neurotoxicity, immunotoxicity, genotoxicity, endocrine interference, embryonic lethality and / or teratogenicity.

[0064] The present application also provides the use of the above-mentioned embryonic model or the above-mentioned method or the above-mentioned embryonic assembly basal medium in screening drugs for preventing and / or treating diseases, or in evaluating drug toxicity, wherein the diseases comprise genetic diseases and / or developmental disorders.

[0065] In an embodiment of the present application, the genetic diseases comprise genetic pancreatic agenesis caused by GATA6 mutation, HNF1B-related kidney dysplasia and / or situs inversus; and the developmental disorders comprise endoderm developmental disorder, anterior-posterior axis developmental disorder and / or early embryonic developmental arrest.

[0066] In an embodiment of the present application, the toxicity of the drug comprises potential embryonic / fetal toxicity, neurotoxicity, immunotoxicity, genotoxicity, endocrine interference, embryonic lethality and / or teratogenicity.

[0067] The present application also provides a cell therapy drug for preventing and / or treating diseases, wherein the components of the cell therapy drug comprise the above-mentioned embryonic model, or the cell therapy drug is prepared from the above-mentioned embryonic model.

[0068] In an embodiment of the present application, the preparation method of the cell therapy drug comprises: extracting stem cells and / or precursor cells of different lineages at a specific developmental stage using the above-mentioned embryonic model, and further differentiating them in vitro to generate specific functional cell types, to obtain the cell therapy drug.

[0069] In an embodiment of the present application, the precursor cells comprise hemogenic endothelial progenitor cell-like cells.

[0070] In one embodiment of this application, the prevention and / or treatment of diseases includes hematopoietic function repair, angiogenesis, and treatment of related diseases.

[0071] This application also provides the use of the above-mentioned embryonic model in the preparation of cell therapy drugs for the prevention and / or treatment of diseases.

[0072] In one embodiment of this application, the method for preparing the cell therapy drug includes: extracting stem cells and / or precursor cells of different lineages at a specific developmental stage using the above-mentioned embryonic model, and further differentiating them in vitro to generate cell types with specific functions, thereby obtaining the cell therapy drug.

[0073] In one embodiment of this application, the precursor cells comprise hematopoietic endothelial progenitor cells.

[0074] In one embodiment of this application, the prevention and / or treatment of diseases includes hematopoietic function repair, angiogenesis, and treatment of related diseases.

[0075] This application also provides an organ graft prepared from the above-described embryo model.

[0076] In one embodiment of this application, the method for preparing the organ transplant includes: using the above-mentioned embryo model for extended culture to allow it to reach the organogenesis stage in vitro, thereby obtaining an organ transplant.

[0077] This application also provides the application of the above-mentioned embryonic model in the preparation of organ grafts.

[0078] In one embodiment of this application, the method for preparing the organ transplant includes: using the above-mentioned embryo model for extended culture to allow it to reach the organogenesis stage in vitro, thereby obtaining an organ transplant.

[0079] The technical solution of this application has the following advantages:

[0080] This application provides an embryo model, which is composed of... Induced pluripotent stem cells (i.e., stem cells) It is obtained by the spontaneous assembly of iPSCs and induced hypoblast stem cells (iHypoblast SCs); Induced pluripotent stem cells (i.e., stem cells) Both induced pluripotent stem cells (iPSCs) and induced hypoblast stem cells (iHypoblast SCs) are derived from somatic cell reprogramming. This application utilizes two different lineages of stem cells generated through somatic cell reprogramming. Induced pluripotent stem cells and induced hypoblast stem cells) spontaneously assemble to construct an embryonic model that simulates the human peri-gastrula stage, called induced embryoids (iEmbryoids). This embryonic model has the following advantages:

[0081] First, this embryonic model better recapitulates the interactions and communication mechanisms between cells of different lineages during human embryonic development by combining cells of different lineages, effectively restoring the developmental characteristics of human peri-gastrula stage embryos. Second, under this embryonic model, different lineages of cells can be tracked and the gene regulation network can be studied using CRISPR and other gene editing tools, thereby providing a deeper understanding of the molecular regulation 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 instead of traditional human embryonic stem cells (hESCs), which can to some extent avoid ethical issues and provide a more feasible solution for extensive basic research and clinical applications. In addition, this embryonic model can be used to extract stem cells and precursor cells of different lineages (such as hemogenic endothelial progenitor cells, etc.) at specific developmental stages, and further differentiate them in vitro to generate specific functional cell types, which can be used for specific cell therapy (including hematopoietic function repair, vascular regeneration, and treatment of related diseases). Finally, in the future, this embryonic model can be cultured for a longer period of time to reach the organogenesis stage in vitro, and 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 shortage of organ donors by cultivating functional tissues and organs in vitro, and by reprogramming patient's own cells and introducing them into the embryonic model, not only can generate organs that match the patient's genes, but also can greatly reduce the risk of immune rejection. This technology is expected to provide a revolutionary new solution for organ transplantation, using cutting-edge regenerative medicine technology to promote the development of personalized medicine, thereby providing an effective solution to 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.

[0082] Further, the preparation method of the embryonic model comprises: Induced pluripotent stem cells (iPSCs) iPSCs) and induced hypoblast stem cells (iHypoblast SCs) are dispersed and co-inoculated into a culture vessel, and are cultured in turn using embryonic assembly basal medium additionally supplemented with leukemia inhibitory factor (LIF) and CEPT (Chroman 1, Emricasan, Polyamines, Trans-ISRIB), embryonic assembly basal medium additionally supplemented with leukemia inhibitory factor (LIF), and embryonic assembly basal medium additionally supplemented with bone morphogenetic protein 4 (BMP4) to obtain an embryoid 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). The use of the embryonic assembly basal medium additionally supplemented with leukemia inhibitory factor + CEPT, leukemia inhibitory factor, or bone morphogenetic protein 4 for culturing Induced pluripotent stem cells iPSCs) and induced hypoblast stem cells (iHypoblast SCs) can precisely regulate important signaling pathways (BMP and JAK / STAT signaling) of cells of two lineages, and thus successfully achieve spontaneous assembly and stable maintenance of cells of two lineages. The components of the embryonic assembly basal medium additionally supplemented with leukemia inhibitory factor + CEPT, leukemia inhibitory factor, or bone morphogenetic protein 4 can ensure that cells of two lineages maintain specific lineage characteristics during differentiation, and ensure that cells of two lineages maintain stable self-renewal ability in long-term culture. Research has confirmed that the use of the embryonic assembly basal medium additionally supplemented with leukemia inhibitory factor + CEPT, leukemia inhibitory factor, or bone morphogenetic protein 4 significantly improves the spontaneous assembly efficiency of iPSCs and induced hypoblast stem cells (iHypoblast SCs), and enhances the operability and experimental reproducibility of iEmbryoids. Induced pluripotent stem cells iPSCs) and induced hypoblast stem cells (iHypoblast SCs), and enhances the operability and experimental reproducibility of iEmbryoids. BRIEF DESCRIPTION OF DRAWINGS

[0083] FIG. 1: Spontaneous assembly of induced pluripotent stem cells (iPSCs) and induced hypoblast stem cells (iHypoblast SCs) by co-culturing homologous cells iPSCs were used to construct a human embryo model. In Figure 1, (a) a cartoon illustration shows the embryo-like assembly experimental process of human iEmbryoids; (b) bright-field plots of iEmbryoids at different days during the assembly process from day 0 to day 8; (c) a bar chart of the assembly efficiency of the D8 iEmbryoid model according to morphological criteria. Mean ± standard error, n = 473 iEmbryoids; (d) hematoxylin-eosin staining of real human embryos (Carnegie stage CS6b); (e) immunofluorescence images of iEmbryoids on day 8 (n = 3 independent experiments, scale bar: 50 μm).

[0084] Figure 2: Immunofluorescence staining of embryonic models at different time points. In Figure 3, (a) immunofluorescence staining of iEmbryoids (day 4, day 6, day 8) embryonic models at different time points (SOX2, SOX17) (n = 3 independent experiments, scale bar: 50 μm); (b) immunofluorescence staining of iEmbryoids (day 4, day 6, day 8) embryonic models at different time points (OCT3 / 4, GATA6) (n = 3 independent experiments, scale bar: 50 μm).

[0085] Figure 3: Single induced hypoblast stem cells (iHypoblast SCs) or iPSCs construct cell aggregates. In Figure 3, (a) the initial cells are only... (a) Bright-field plots of iPSCs and iEmbryoids assembled on different days (n = 3 independent experiments, scale bar: 50 μm); (b) Starting cells only (c) Immunofluorescence images of iPS and iEmbyroid on day 4, OCT3 / 4, GATA6, SOX2, and SOX17 (n = 3 independent experiments, scale bar: 50 μm); (d) Immunofluorescence images of iEmbyroid on day 4, starting cells only iHypoblast SCs, OCT3 / 4, GATA6, SOX2, and SOX17 (n = 3 independent experiments, scale bar: 50 μm); (e) Immunofluorescence images of iEmbyroid on day 4, starting cells only iHypoblast SCs, OCT3 / 4, GATA6, SOX2, and SOX17 (n = 3 independent experiments, scale bar: 50 μm).

[0086] Figure 4: Immunofluorescence staining of embryoid models. In Figure 4, (a) Representative immunofluorescence images of day 4 iEmbryoids (OCT3 / 4 and F-ACTIN) (n = 3 independent experiments, scale bar: 50 pm); (b) Representative immunofluorescence images of day 6 iEmbryoids (ISL1, SOX2 and FOXA2) (arrows and dashed areas indicate putative amnion-like cells, n = 3 independent experiments, scale bar: 50 pm); (c) Representative immunofluorescence images of day 8 iEmbryoids (NANOG, SOX17 and TFAP2C) (arrows and dashed areas indicate putative primordial germ cell (PGC)-like cells, n = 3 independent experiments, scale bar: 50 pm); (d) Representative immunofluorescence images of day 8 iEmbryoids (OCT3 / 4, T and OTX2) (arrows and dashed areas indicate putative anterior-posterior axis (A-P axis), n = 3 independent experiments, scale bar: 50 pm).

[0087] Figure 5: Immunofluorescence staining of embryoid models. In Figure 5, (a) Representative immunofluorescence images of day 4 iEmbryoids (SOX2 and F-ACTIN) (n = 3 independent experiments, scale bar: 50 pm); (b) Representative immunofluorescence images of day 6 iEmbryoids (SOX2, TFAP2A and SOX17) (arrows and dashed areas indicate putative amnion-like cells, n = 3 independent experiments, scale bar: 50 pm); (c) Representative immunofluorescence images of day 8 iEmbryoids (OCT3 / 4, SOX17 and BLIMP1) (arrows and dashed areas indicate putative primordial germ cell (PGC)-like cells, n = 3 independent experiments, scale bar: 50 pm); (d) Representative immunofluorescence images of day 8 iEmbryoids (OCT3 / 4, T and LEFTY) (arrows and dashed areas indicate putative distal endoderm (AVE)-like cells and primitive streak (PS)-like cells, n = 3 independent experiments, scale bar: 50 pm); (e) Representative immunofluorescence images of day 8 iEmbryoids (OCT3 / 4, T and CER1) (arrows and dashed areas indicate putative distal endoderm (AVE)-like cells and primitive streak (PS)-like cells, n = 3 independent experiments, scale bar: 50 pm).

[0088] Figure 6: Immunofluorescence staining of embryoid models. In Figure 6, (a) Representative immunofluorescence images of day 8 iEmbryoids (OCT3 / 4, GATA6 and VIM) (arrows and dotted areas indicate putative ExEM-like cells, n = 3 independent experiments, scale bar: 50 pm); (b) Representative immunofluorescence images of day 8 iEmbryoids (OCT3 / 4, SOX17 and BST2) (arrows and dotted areas indicate putative ExEM-like cells, n = 3 independent experiments, scale bar: 50 pm); (c) Representative immunofluorescence images of day 8 iEmbryoids (OCT3 / 4, GATA4 and FOXF1) (arrows and dotted areas indicate putative ExEM-like cells, n = 3 independent experiments, scale bar: 50 pm); (d) Representative immunofluorescence images of day 8 iEmbryoids (CD34, ERG and GATA6) (arrows and dotted areas indicate putative HEP-like cells, n = 3 independent experiments, scale bar: 50 pm).

[0089] Figure 7: Single-cell sequencing analysis of embryoid models. In Figure 7, (a) Brightfield images of day 8 iEmbryoids for single-cell RNA sequencing (sc-RNA seq); (b) UMAP plot representing individual cell clusters derived from day 8 iEmbryoids (colors correspond to the annotation of cell types); (c) UMAP displaying cell clusters from different lineages in day 8 iEmbryoids (colors correspond to the annotation of cell types; where, Epi, epiblast; Am, amnion; PGC, primordial germ cell; PS, primitive streak; Early meso, early mesoderm; Meso, mesoderm; ExEM, extraembryonic mesoderm; Intermediate, intermediate cell; VE / YS, visceral endoderm / yolk sac; SYS, secondary yolk sac; HEP, hemogenic endothelial progenitor); (d) Dot plot revealing the expression of key markers in 11 cell type clusters in day 8 iEmbryoids.

[0090] FIG. 8: Embryoid single-cell sequencing analysis. In FIG. 8, (a) UMAP analysis of the integrated data of day 8 iEmbryoids, in vitro cultured embryos and CS7 human primitive gut embryo (left color corresponds to cell type annotation in each dataset; where, EPI, ectoderm; Am, amnion; PGC, primordial germ cell; PS, primitive streak; Early meso, early mesoderm; Meso, mesoderm; ExEM, extraembryonic mesoderm; VE / YS, visceral endoderm / yolk sac; SYS, secondary yolk sac; Blood, blood cells; right color corresponds to iEmbryoids and aggregated plot of each dataset); (b) Heatmap to evaluate the relative expression level of each lineage cell-specific marker in different datasets; (c) Subgroup analysis of blood-like cell cluster in UMAP of integrated data.

[0091] FIG. 9: Embryoid single-cell sequencing analysis. In FIG. 9, (a) Expression of ectoderm (Epi) markers (OCT3 / 4, SOX2, NANOG, DPPA5 and ESRG) in iHypoblast SCs UMAP; (b) Expression of amnion markers (ISL1, TFAP2A, GARBP, HEY1 and VTCN1) in iHypoblast SCs UMAP; (c) Expression of primitive streak markers (CDH1, WNT8A, GAL, TBXT and MSGN1) in iHypoblast SCs UMAP; (d) Expression of visceral endoderm / yolk sac (VE / YS) markers (SOX17, RSPO3, BMP6, FOXA2 and GATA4) in iHypoblast SCs UMAP.

[0092] FIG. 10: Embryoid single-cell sequencing analysis. In FIG. 10, (a) Expression of secondary yolk sac (SYS) markers (APOA1, APOA2, APOB, AFP and TTR) in iHypoblast SCs UMAP; (b) Expression of primordial germ cell (PGC) markers (BLIMP, CXCR4, TFAP2C, NANOS3 and PDPN) in iHypoblast SCs UMAP; (c) Expression of early mesoderm markers (MESP1, MESP2, EOMES, MIXL1 and BMP4) in iHypoblast SCs UMAP; (d) Expression of mesoderm markers (ACTC1, GATA6, HAND1, PDGFRA and SNAI2) in iHypoblast SCs UMAP.

[0093] Figure 11: Embryoid model single cell sequencing analysis. In Figure 11, (a) expression of hemogenic endothelial progenitor cell (HEP) markers (CD34, ERG, RUNX1, MEF2C and PECAM1) in iHypoblast SCs UMAP; (b) expression of extraembryonic mesoderm lineage (ExEM) markers (POSTN, DCN and HAND2) in iHypoblast SCs UMAP; (c) expression of neural ectoderm (NE) markers (OTX2 and PAX6) in iHypoblast SCs UMAP.

[0094] Figure 12: Human embryoid model constructed by combining induced hypoblast stem cells (iHypoblast SCs) and iPSCs. In Figure 12, left, electron micrograph of iEmbryoids from day 4 to day 8, right, cartoon illustration of corresponding developmental stage.

[0095] Figure 13: Immunofluorescence staining of embryoid model at different time points. In Figure 13, (a) immunofluorescence staining of iEmbryoids (day 1, day 2, day 3) at different time points (SOX2, SOX17) (n = 3 independent experiments, scale bar: 50 pm); (b) immunofluorescence staining of iEmbryoids (day 1, day 2, day 3) at different time points (OCT3 / 4, GATA6) (n = 3 independent experiments, scale bar: 50 pm).

[0096] Figure 14: Immunofluorescence staining of lineage-traced embryoid model (4CL ​Cell marker green fluorescence; 26#iHypoblast marker red fluorescence). In Figure 14, (a) representative immunofluorescence images of day 8 iEmbryoids (ISL1), arrows and dashed areas indicate putative EGFP-positive cells distributed in the parietal-like amnion-like cell region; (b) representative immunofluorescence images of day 8 iEmbryoids (BLIMP1), arrows and dashed areas indicate EGFP-positive cells distributed in the parietal-like germ cell-like (PGC-like) region; (c) representative immunofluorescence images of day 8 iEmbryoids (T), arrows and dashed areas indicate EGFP-positive cells involved in the formation of parietal-like primitive streak-like (PS-like) structure; (d) representative immunofluorescence images of day 8 iEmbryoids (GATA6), arrows and dashed areas indicate tdTomato-positive cells located in the parietal-like yolk sac-like cell region. (e) representative immunofluorescence images of day 8 iEmbryoids (BST2), arrows and dashed areas indicate EGFP-positive cells located in the parietal-like extraembryonic mesoderm-like (ExEM-like) cell region; (f) representative immunofluorescence images of day 8 iEmbryoids (CD34), arrows and dashed areas indicate tdTomato-positive cells distributed in the parietal-like hematoendothelial progenitor-like (HEP-like) region. a-f results n = 3 independent experiments, scale bar: 50 pm;

[0097] Figure 15: Different types of induced parietal-like cells and iPSCs construct human embryonic models. In Figure 15, (a) cartoon schematic diagram shows the cartoon-like embryonic assembly experimental process of human iEmbryoids; (b) bright field images during the assembly process of day 4 and day 8 iEmbryoids; (c) immunofluorescence staining images of day 4 and day 8 iEmbryoids at different time points (OCT3 / 4, GATA6), n = 3 independent experiments, scale bar: 50 pm).

[0098] FIG. 16: Single-cell sequencing analysis of embryoid model. In FIG. 16, (a) bright field images of pre-assembly cells of day 0 embryos, day 4 and day 8 iEmbryoids for single-cell RNA sequencing (sc-RNA seq); (b) UMAP demonstrates the clustering of cells from different lineages in day 0, 4, 8 iEmbryoids (colors correspond to cell type annotations; where, Epi, epiblast; Am, amnion; PGC, primordial germ cell; PS, primitive streak; Early meso, early mesoderm; Meso, mesoderm; ExEM, extraembryonic mesoderm; Intermediate, intermediate cell; VE / YS, visceral endoderm / yolk sac; SYS, secondary yolk sac; HEP, hemendothelial progenitor cell).

[0099] FIG. 17: Single-cell sequencing analysis of embryoid model. In FIG. 17, (a) proportion distribution of major cell populations in day 0, 4, 8 iEmbryoids. Each cell type includes: Epi (epiblast), Am (amnion), PGC (primordial germ cell), PS (primitive streak), Early meso (early mesoderm), Meso (mesoderm), ExEM (extraembryonic mesoderm), YS (yolk sac), SYS (secondary yolk sac), and HEP (hemendothelial progenitor cell); (b) cell fate trajectory reconstruction using Monocle3 software. Colors shown on the right of the figure correspond to annotation information of iEmbryoids at each time point (day 0, 4, 8); (c) analysis of integrated dataset by UMAP algorithm, which integrates day 0, 4, 8 iEmbryoids with in vitro cultured embryo samples and CS7 stage human primitive gut embryo samples. Colors shown on the left of the figure represent information according to cell type annotations in each dataset, and colors shown on the right of the figure distinguish iEmbryoids from each reference dataset source.

[0100] FIG. 18: Single-cell sequencing analysis of embryoid model. In FIG. 18, (a) based on UMAP clustering analysis of day 0, 4, 8 iEmbryoids, class hypoblast, yolk sac and secondary yolk sac-like cell populations are identified; (b) cell trajectory reconstruction analysis of class hypoblast and yolk sac related cell populations using Monocle3 tool. Colors shown on the right of the figure correspond to annotation information at each time point; (c) Dot Plot map demonstrates the expression characteristics of key markers of class hypoblast, yolk sac and secondary yolk sac-like cell populations in day 0, 4, 8 iEmbryoids.

[0101] FIG. 19: Embryo model single-cell sequencing analysis. In FIG. 19, (a) Expression of epiblast (Epi) markers (ESRG, POU5F1, NANOG and SOX2) in iHypoblast SCs UMAP; (b) Expression of amnion markers (GARBP, TFAP2A, ISL1 and VTCN1) in iHypoblast SCs UMAP; (c) Expression of primitive streak markers (EMOES, T, MIXL1 and WNT8A) in iHypoblast SCs UMAP; (d) Expression of primitive endoderm (Hypo) markers (ANXA3, FOXA1, LINC00261 and FOXA2) in iHypoblast SCs UMAP.

[0102] FIG. 20: Embryo model single-cell sequencing analysis. In FIG. 20, (a) Expression of yolk sac / secondary yolk sac (YS / SYS) markers (APOA1, APOE, APOB and RSPO3) in iHypoblast SCs UMAP; (b) Expression of primitive germ cells (PGCs) (BLIMP1, NANOS3, CXCR4 and PDPN) in iHypoblast SCs UMAP; (c) Expression of early mesoderm (BMP4, MESP1, CFC1 and MESP2) in iHypoblast SCs UMAP; (d) Expression of mesoderm markers (ACTC1, HAND, GATA6 and SNAI2) in iHypoblast SCs UMAP; (e) Expression of hemogenic endothelial progenitor (HEP) markers (CD34, RUNX1, ERG, MEF2C and PECAM) in iHypoblast SCs UMAP; (f) Expression of extraembryonic mesoderm lineage (ExEM) markers (DCN, HGF, HAND2 and POSTN) in iHypoblast SCs UMAP.

[0103] FIG. 21: Embryo model immunofluorescence staining. In FIG. 21, immunofluorescence staining images of iEmbryoid SCs and iEmbryoids (day 4, day 8) embryo-like models before assembly (OCT3 / 4, GATA6 and APOA1) (n = 3 independent experiments, scale bar: 50 pm).

[0104] Figure 22: Immunostaining of embryoid-like hematopoietic features. In Figure 22, (a) cartoon schematic showing the experimental procedure of hematopoietic induction in vitro of iEmbryoids; (b) representative brightfield images taken at day 19 showing the formation of spheroid cell structures (dashed line box indicates); n = 3 independent replicates. Scale bar, 50 pm; (c) representative immunofluorescence images of hematopoietic differentiation at day 11 (CD34, CD43, and FOXA2) (n = 3 independent experiments, scale bar: 50 pm), dashed line box indicates the presence of CD34 + hematoendothelial progenitor cells co-localize with CD43 + hematoendothelial progenitor cells co-localize with CD43 + hematoendothelial progenitor cells co-localize with CD43 + hematoendothelial progenitor cells co-localize with CD43 + hematoendothelial progenitor cells co-localize with CD43 + hematoendothelial progenitor cells co-localize with CD43 + hematoendothelial progenitor cells co-localize with CD43 + hematoendothelial progenitor cells co-localize with CD43 + hematoendothelial progenitor cells co-localize with CD43

[0105] Figure 23: Immunostaining of embryoid-like hematopoietic features. In Figure 23, (a) flow cytometry analysis of iEmbryoids samples at day 11 of hematopoietic differentiation, detection of CD43 + hematoendothelial progenitor cells, CD31 + hematoendothelial progenitor cells, CD31 + hematoendothelial progenitor cells, CD31 + hematoendothelial progenitor cells, CD31 + hematoendothelial progenitor cells, CD31 + hematoendothelial progenitor cells, CD31 + hematoendothelial progenitor cells, CD31 + hematoendothelial progenitor cells, CD31 + hematoendothelial progenitor cells, CD31 +Megakaryocyte population. n = 3 independent replicates.

[0106] Figure 24: iEmbryoids exhibit yolk sac-like hematopoietic features. In Figure 24, (a) Representative phase-contrast microscopic images showing various colony types formed by single cells, including CFU-E (erythroid colony-forming unit), BFU-E (burst-forming unit-erythroid), CFU-GM (granulocyte-macrophage colony-forming unit), and CFU-GEMM (granulocyte-erythroid-macrophage-megakaryocyte multi-lineage colony) after colony-forming unit (CFU) assay of day 19 iEmbryoids. n = 3 independent replicates. Scale bar, 50 pm; (b) Quantification of the number of different types of CFU colonies formed per 10,000 cells in day 19 iEmbryoids. n = 3 independent replicates. DETAILED DESCRIPTION

[0107] The following examples are provided to better enable those skilled in the art to further understand the application, and are not intended to limit the scope of the application. Any product obtained by the combination of the application with other prior art features or by the modification of the application is intended to fall within the scope of the application.

[0108] The following examples were carried out according to the conventional experimental procedures described in the literature unless otherwise specified. The reagents or instruments used were commercially available unless otherwise specified.

[0109] The materials and reagents used in the following examples are listed in Table 1.

[0110] Table 1: Experimental materials and reagents

[0111] Example 1: An embryoid model and a method for constructing the same

[0112] The present example provides an embryoid model, which is spontaneously assembled from induced pluripotent stem cells (iPSCs) and induced hypoblast stem cells (iHypoblast SCs); the induced pluripotent stem cells (iPSCs) and the induced hypoblast stem cells (iHypoblast SCs) are both reprogrammed from somatic cells. The embryoid model is prepared as follows: induced pluripotent stem cells (iPSCs) and induced hypoblast stem cells (iHypoblast SCs) are both reprogrammed from somatic cells. The embryoid model is prepared as follows: induced pluripotent stem cells (iPSCs) and induced hypoblast stem cells (iHypoblast SCs) are both reprogrammed from somatic cells. The embryoid model is prepared as follows:

[0113] ​1. Constructing induced hypodermal stem cells through somatic cell reprogramming

[0114] Step 1: Using the CytoTune-iPS2.0 Sendai Reprogramming Kit, reprogram primary human adult skin fibroblasts (including donor 1 and donor 2) according to the instructions. The reprogramming process is as follows: human fibroblasts are reprogrammed at a rate of 5 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 500 cells in mouse embryonic fibroblast culture medium and transfected with Sendai virus mediated by multiplicity of infection (MOI) of 5 (KOS), 5 (c-Myc), and 6 (KLF4).

[0115] Step 2: On day 7 of transfection, transfected human fibroblasts in the fibroblast culture medium were dissociated using TryPLE Select at room temperature (25°C) for 5 minutes (0.5 mL / well). The cells were then sputtered at 6 × 10⁻⁶ ppm. 4 The cells were re-inoculated into cell culture plates that had been pre-coated with feeder cells (mouse embryonic fibroblasts were used as feeder cells; for feeder cell plating instructions, please refer to the literature "Liu, X. et al. Reprogramming roadmap reveals route to human induced trophoblast stem cells. Nature 586, 101-107 (2020)"). The cells were then cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2.

[0116] Step 3: After culturing for 24 hours, replace the culture medium in the cell culture plate with the culture medium for inducing hypodermal stem cells, and continue culturing in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2.

[0117] Step 4: After 20 days of culture, hypodermal-like stem cells with high PDGFRA expression are enriched and screened using FACS (flow cytometry) based on PDGFRA expression.

[0118] Step 5: Select PDGFRA-positive hypodermal-like cells and use 6×10 4 The inoculum of cells / well was re-inoculated into cell culture plates with feeder cells laid 1 day in advance (in addition to feeder cells, the cell culture plates also contain 1 mL / well of additional 10 μM ROCK-I / II inhibitor Y27632-inducing hypoblastocyst culture medium), and cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2.

[0119] Step six: After 24 hours of culture, replace the medium in the cell culture plate with the induced hypoblast stem cell medium without additional supplement of the ROCK-I / II inhibitor Y27632, and continue to culture in a 37 °C cell culture incubator with 5% (v / v) CO2, 5% (v / v) O2.

[0120] Step seven: After 5 days of culture, pick the hypoblast-like colonies in the cell culture plate and transfer them to a cell culture plate with feeder cells prepared 1 day in advance (1 mL / well of the induced hypoblast stem cell medium supplemented with 10 mM of the ROCK-I / II inhibitor Y27632 was added to the cell culture plate in addition to the feeder cells) at a seeding amount of 6 x 105cells / well, and culture in a 37 °C cell culture incubator with 5% (v / v) CO2, 20% (v / v) O2. Replace the medium every day during the culture. 4

[0121] Step eight: When the hypoblast-like cells in the cell culture plate reach 80-90% confluence, dissociate the hypoblast-like cells in the cell culture plate with Accutase at room temperature (25 °C) for 5 minutes (0.5 mL / well of Accutase was used) and transfer them to a cell culture plate with feeder cells prepared 1 day in advance (1 mL / well of the induced hypoblast stem cell medium supplemented with 10 mM of the ROCK-I / II inhibitor Y27632 was added to the cell culture plate in addition to the feeder cells) at a seeding amount of 6 x 105cells / well, and culture in a 37 °C cell culture incubator with 5% (v / v) CO2, 20% (v / v) O2. After 24 hours of culture, the construction of the induced hypoblast stem cells (iHypoblast SCs) is successful. 4

[0122] Step nine: After the construction of the induced hypoblast stem cells (iHypoblast SCs) is successful, subculture the induced hypoblast stem cells (iHypoblast SCs) at a subculture ratio of 1:5 every 3 days, and the subculture process is the same as step eight.

[0123] The mouse embryonic fibroblast medium (MEF medium) is a 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.

[0124] ​​The culture medium for inducing hypodermal stem cells was N2B27 basal medium containing 10 ng / mL human PDGFAA, 10 ng / mL human LIF, 25 ng / mL human FGF4, 1 μg / mL heparin (to promote the binding of FGF4 to its receptor and optimize the biological function of FGF4), 3 μM CHIR99021, 1 μM A83-01 and 10 ng / mL human BMP4; N2B27 basal medium was 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 consisted of Neurobasal medium and DMEM / F12 in a 1:1 volume ratio.

[0125] 2. Culture of induced pluripotent stem cells

[0126] Step 1: Referring to the literature "Mazid, MA et al. Rolling back human pluripotent stem cells to an eight-cell embryo-like stage. Nature 605, 315-324 (2022)", primed induced pluripotent stem cell lines (primed iPSCs) were cultured in cell culture plates using E8 complete medium to convert the primed induced pluripotent stem cell lines (primed iPSCs) into... Induced pluripotent stem cells (i.e., stem cells) (iPSCs) (2 mL of E8 complete medium per well).

[0127] Step 2: Convert the obtained Induced pluripotent stem cells at 1×10 5 The cells were re-seeded one day in advance into cell culture plates containing feeder cells (mouse embryonic fibroblasts were used as feeder cells; for feeder cell plating methods, please refer to the literature "Liu, X. et al. Reprogramming roadmap reveals route to human induced trophoblast stem cells. Nature 586, 101-107 (2020)"). The cells were cultured in a 37°C cell culture incubator with 2 mL / well of additional supplemented 10 μM Y27632.

[0128] The E8 complete medium is an E8 basal medium containing 1% (v / v) E8 supplement and 1% (v / v) penicillin-streptomycin.

[0129] 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; the mixed medium consists of Neurobasal medium and DMEM / F12 in a 1:1 volume ratio.

[0130] 3. Assembly of induced embryonic-like structures (iEmbryoids)

[0131] Step 1: Take the first generation from Part 2. Induced pluripotent stem cells (iPSCs) are cultured until the cells reach a confluence density of 70-80%. The culture medium in the cell culture plate is then discarded, and the plate is first washed with DPBS buffer. Induced pluripotent stem cells (1 mL DPBS buffer per well, wash once), then transfer the cells from the cell culture plate... Induced pluripotent stem cells were dissociated using TryPLE Select at room temperature (25°C) for 5 minutes (1 mL of TryPLE Select per well, dissociating into single cells).

[0132] Step 2: After dissociation, centrifuge the cell culture plate and discard the supernatant. Resuspend the cell pellet in the cell culture plate using mouse embryonic fibroblast culture medium supplemented with 0.1% (v / v) CEPT (1 mL / well of culture medium) to obtain the cell resuspension.

[0133] Step 3: Add 0.1% Gelatin to the cell culture plate at a rate of 1.5 mL / well and let it stand at room temperature (25)℃ for 10 minutes to obtain a cell culture plate treated with 0.1% gelatin.

[0134] Step 4: Count the cells in the cell resuspension. Induced pluripotent stem cells (inoculated with cell suspension) at 1×10 6The cell pellet in the cell culture plate was centrifuged to discard the supernatant, and the cell pellet in the cell culture plate was resuspended with the mouse embryonic fibroblast culture medium supplemented with 0.1% (v / v) CEPT (1 mL / well of the medium) to obtain a cell resuspension. Feeder cells were maintained in culture with stem cells.

[0135] Step five: After the incubation, the cell culture plate was centrifuged to discard the supernatant, and the cell pellet in the cell culture plate was resuspended with the embryonic assembly basal medium additionally supplemented with 10 ng / mL human LIF and 0.1% (v / v) CEPT (1 mL / well of the medium) to obtain resuspension A.

[0136] Step six: The induced hypoblast stem cells (iHypoblast SCs) in the first passage in part one were taken and cultured until the cells reached 70-80% density, the medium in the cell culture plate was discarded, the induced hypoblast stem cells in the cell culture plate were first washed with DPBS buffer (1 mL / well of the DPBS buffer, once), and then the induced hypoblast stem cells in the cell culture plate were dissociated with Accutase at room temperature (25°C) for 5 minutes (1 mL / well of the Accutase, dissociated into single cells).

[0137] Step seven: After the dissociation, the cell culture plate was centrifuged to discard the supernatant, and the cell pellet in the cell culture plate was resuspended with the mouse embryonic fibroblast culture medium additionally supplemented with 0.1% (v / v) CEPT (1 mL / well of the medium) to obtain a cell resuspension.

[0138] Step eight: 0.1% Gelatin was added to the cell culture plate at an addition amount of 1.5 mL / well, and the cell culture plate was left to stand at room temperature (25°C) for 10 minutes to obtain a 0.1% gelatin-treated cell culture plate.

[0139] Step nine: The cell resuspension was counted, and the induced hypoblast stem cells in the cell resuspension (seeded with the cells and the resuspension) were reseeded in the 0.1% gelatin-treated cell culture plate at a seeding amount of 1 × 10 6 The cell pellet in the cell culture plate was centrifuged to discard the supernatant, and the cell pellet in the cell culture plate was resuspended with the mouse embryonic fibroblast culture medium supplemented with 0.1% (v / v) CEPT (1 mL / well of the medium) to obtain a cell resuspension.

[0140] Step 10: After the culture is completed, discard the culture medium in the cell culture plate. First, wash the induced hypoblast stem cells in the cell culture plate with DPBS buffer (1 mL DPBS buffer per well, wash once). Then, dissociate the induced hypoblast stem cells in the cell culture plate with Accutase at room temperature (25°C) for 5 minutes (1 mL Accutase per well, dissociate into single cells).

[0141] Step 11: After incubation, centrifuge the cell culture plate and discard the supernatant. Resuspend the cell pellet in the cell culture plate using embryo assembly basal medium supplemented with 10 ng / mL human LIF and 0.1% (v / v) CEPT (1 mL of medium per well) to obtain resuspension B.

[0142] Step 12: Perform cell counting on resuspension A and resuspension B, and separate the cells in resuspension A... Induced pluripotent stem cells (inoculated along with resuspension) and induced hypoblastomere stem cells in resuspension B were co-seeded at a rate of 50 cells / well into each well of an AggreWell 400 embryologie culture plate (with 1 mL / well of embryo assembly basal medium supplemented with 10 ng / mL human LIF and 0.1% (v / v) CEPT). The plates were first centrifuged at 100 rcf for 3 minutes to ensure even cell distribution, and then cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 20% (v / v) O2. The culture medium was changed daily during culture. When changing the medium, the embryo assembly basal medium used from day 2 of culture only needed to be supplemented with 10 ng / mL human LIF; additional 0.1% (v / v) CEPT was not required.

[0143] Step Thirteen: After 4 days of culture, replace the culture medium in the AggreWell 400 embryoid culture plate with embryo assembly basal medium supplemented with 200 ng / mL human BMP4, and continue culturing in a 37°C cell culture incubator with 5% (v / v) CO2 and 20% (v / v) O2. Change the culture medium daily during the culture period. After 8 days of culture, the embryo model is successfully constructed and is called induced embryoids (iEmbryoids).

[0144] The embryo assembly basal medium was 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 consisted of Neurobasal medium and DMEM / F12 in a 1:1 volume ratio.

[0145] Experimental Example 1: Exploration of induced embryoid (iEmbryoids) construction conditions and verification of induced embryoid performance

[0146] This experimental example provides an experiment of exploring the construction conditions of induced embryoids (iEmbryoids) and verifying the performance of induced embryoids, and the experimental process is as follows:

[0147] 1、 iPSCs and iHypoblast SCs spontaneously assemble to form induced embryoids

[0148] Understanding the cellular and molecular processes of human post-implantation development is one of the fundamental problems in developmental and stem cell biology. In the post-implantation stage of human embryogenesis, The ectoderm (Epi) acquires apical-basal polarity to build the amnion cavity and eventually forms the amnion and post-implantation ectoderm. At the same time, the hypoblast (Hypo) differentiates into the visceral endoderm (VE) and yolk sac (YS) endoderm, forming the primary yolk sac and secondary yolk sac cavity. Epi and VE generate a bilayer disc structure, followed by the embryo entering the primitive gut movement stage (Fig. 1d), which is considered a milestone in mammalian embryonic development. Therefore, this study attempts to develop a robust model to simulate human peri-gastrulation embryonic development by combining iPSCs and iHypoblast SCs to develop a robust model to simulate human peri-gastrulation embryonic development.

[0149] Leukemia inhibitory factor (LIF) plays an important role in maintaining mammalian embryonic stem cells and is included in iPSCs ( medium 4CL) and iHypoblast SCs (Hypo medium 26#). Therefore, this study first introduced LIF in the basal aggregation medium to promote iPSCs and iHypoblast SCs survival and aggregation from day 0 to day 4 (Fig. 1a). Bone morphogenetic protein (BMP) signaling has been shown to initiate primitive gut movement, including downstream WNT and NODAL signaling, to simulate human peri-gastrulation embryonic development. Therefore, this study introduced exogenous BMP4 in the basal aggregation medium from day 4 to day 8 to promote the formation of a peri-gastrulation embryonic model (Fig. 1a, Fig. 1b, Fig. 1e).

[0150] In human early embryonic development, the inner cell mass will separate into ectoderm and hypoblast lineages from day 5 to day 7 of embryonic development. In the post-implantation stage, the ectoderm forms a ring-shaped layer expressing OCT3 / 4 and SOX2, while the hypoblast is located below the ectoderm, expressing GATA6 and SOX17. Consistent with this, this study observed that Upon introduction of iPSCs and iHypoblast SCs into 3D AggreWell400 plates, Epi-like and hypoblast-like cells segregated into two distinct regions by day 4. Day 4 aggregates clearly expressed Epi and hypoblast lineage markers such as OCT3 / 4, SOX2, and GATA6, SOX17, while showing signs of early cavity formation (Figures 2a-b, 4a, 5a).

[0151] 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 the epiblast-like and hypoblast-like cells gradually expanded, forming an OCT3 / 4+SOX2lowamnion-like cavity and GATA6+SOX17+ yolk sac-like cavity, i.e., a bilayer disc structure, by day 6, and further expanding by day 8 (Figures 2a-b). In addition, this study formed aggregates from each cell line separately to understand the contribution of iPSCs and iHypoblast SCs to the aggregates. Contribution of iPSCs and iHypoblast SCs to the aggregates. In In the individual aggregates of iPSCs or iHypoblast SCs, no distinct amnion-like and YS-like cavities were observed by day 4, and these aggregates gradually collapsed by day 8, indicating that Cell-cell interactions between iPSCs and iHypoblast SCs were critical for inducing morphological changes (Figures 3a-d).

[0152] Considering that the day 8 aggregates were derived from human skin fibroblasts, this study named this induced stem cell-based embryonic model as “iEmbryoids”. The efficiency of forming well-structured day 8 iEmbryoids was estimated to be 17.09% (Figure 1c). Overall, these findings suggest that iEmbryoids are consistent with the human post-implantation embryonic development characteristics.

[0153] 2. Induced embryoids recapitulate key developmental hallmarks of human peri-gastrulation period

[0154] Next, we examined whether iEmbryoids could mimic key developmental hallmarks of pre-implantation and peri-gastrula stage embryos by immunofluorescent staining analysis. The amnion plays a pivotal role in regulating the epiblast (Epi) and initiating the primitive streak movement. At day 6, ISL1+TFAP2A+ cells exhibited reduced SOX2 expression compared to the other side, confirming their amnion-like identity by morphology and gene expression (Fig. 4b). In primate development, primordial germ cells (PGCs) first appear in the amnion and subsequently migrate to the visceral endoderm (VE). At day 8, co-immunofluorescent staining of PGC markers (OCT4+SOX17+BLIMP1+ and NANOG+SOX17+TFAP2C+) indicated the presence of PGC-like cells in some iEmbryoids (Fig. 4c, Fig. 5c), a result consistent with the location and expression pattern in human embryonic models.

[0155] Subsequently, we verified the initiation process of the primitive streak movement by the formation of the anterior-posterior (A-P) axis. The early establishment of the A-P axis is a common feature of mammals, occurring when a subset of cells in the epiblast begins to express the T gene in the posterior region, while their antagonists CER1 and LEFTY are expressed in the anterior region of the blastoderm. At day 8, T+ELCs were located on the posterior side of iEmbryoids epiblast-like cells (Fig. 5d-5e). At the same time, anterior VE-like cells of CER1 and LEFTY were located under the anterior side of iEmbryoids epiblast-like cells, forming the anterior signaling center of the epiblast pattern, which together with the posterior T+epiblast-like cells constituted the putative A-P axis and symmetry breaking (Fig. 5d-5e). Further immunofluorescent staining analysis confirmed the position information of anterior VE marker OTX2 on the adjacent side of epiblast-like cells (Fig. 4d). Notably, a group of cells expressed GATA6 and GATA4 but not SOX17 (Fig. 6a-6c), surrounding the YS-like cells, consistent with the ExEM pattern in the marmoset embryo. At the same time, these cells also expressed multiple ExEM markers, including BST2, FOXF1, and VIM, further verifying the ExEM-like cell identity in day 8 iEmbryoids (Fig. 6a-6c). In addition, we observed a group of flat epithelial cells on the YS-like structure, co-expressing CD34 and ERG (Fig. 6d), indicating that these cells represent the hematopoietic endothelial progenitor (HEP) fate.

[0156] In summary, day 8 iEmbryoids recapitulated key milestone events of human peri-gastrula stage embryonic development, including amnion and yolk sac cavity formation, PGC specification, primitive streak movement initiation, A-P axis formation, ExEM expansion, and HEP appearance, suitable as a model to understand the process of primitive streak formation and human peri-gastrula stage embryonic development.

[0157] 3. Induction of single-cell transcriptomic profiling of embryoid-like structures

[0158] To further understand the cellular composition and transcriptional landscape of iEmbryoids, beyond 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 7a). UMAP analysis revealed a total of 11 distinct cell clusters (Figure 7b). This study annotated these cell clusters based on the expression pattern of lineage-specific markers, enabling this study to classify all identified cell clusters (Figures 7c-d). This annotation identified a broad range of embryonic and extraembryonic cell types present during human peri-gastrulation embryonic development (Figures 7c-d).

[0159] Specifically, this study observed implantation-posterior Epi-like cells expressing core pluripotency markers in the Epi cluster, such as POU5F1 (also known as OCT3 / 4), SOX2, NANOG, TERF1, and ESRG (Figure 9a). In addition, this study observed a cell cluster expressing ISL1, TFAP2A, GARBP, HEY1, and VTCN1, which was defined as the amnion cluster (Figure 9b). 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 10b). 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 9d), while cells within the SYS cluster showed high expression of endoderm markers AFP and the nutrient transport marker TTR, as well as various apolipoprotein markers (APOA1, APOA2, APOB), supporting its classification as a SYS-like region (Figure 10a).

[0160] Next, the study identified the primitive streak marker TBXT (also known as Brachyury) that binds to CDH1, WNT8A, GAL, and MSGN1 (Figure 9c). In primitive streak dorsal cells, early mesoderm markers such as MESP1, MESP2, EOMES, MIXL1, and BMP4 were increased in expression, annotating early mesoderm (Figure 10c). In contrast, mesoderm clusters exhibited a broad heterogeneity of marker gene profiles, similar to CS7 primitive gut embryos, expressing multiple mesoderm types of genes, including ACTC1, GATA6, HAND1, PDGFRA, and SNAI2 (Figure 10d). Co-expression of HAND1 and GATA6 marked the anterior cardiac lateral plate mesoderm. The study observed this co-expression pattern in multiple clusters, including early mesoderm, mesoderm, and ExEM clusters, and amnion clusters (Figure 11b), suggesting that these embryoid mesoderm clusters do not represent specific mesoderm subtypes, but rather transitional mesoderm states. In mice, POSTN is a marker of ExEM, marking not only the amnion but also the yolk sac. The study detected multiple clusters expressing ExEM markers POSTN, DCN, and HAND2, which were defined as ExEM clusters (Figure 11b). There are multiple reports suggesting that ExEM originates from hypoblast, while the data from the study suggest that ExEM-like cells are closer to mesoderm lineage. This finding can support the hypothesis that ExEM originates from primitive streak cells in iEmbryoids. In addition, the study verified the expression of human hematopoietic progenitor markers CD34 and ERG by immunofluorescence staining (Figure 6d). Consistent with the immunofluorescence staining results of day 8 iEmbryoids, HEP clusters expressed hematopoietic and endothelial markers CD34, ERG, RUNX1, MEF2C, and PECAM1, located near mesoderm clusters (Figure 11a).

[0161] To further assess the transcriptional similarity between iEmbryoids and human embryos, this study integrated iEmbryoid scRNA-seq data with three previously reported scRNA-seq datasets from human CS6 in vitro cultured embryos and real CS7 gastrula. UMAP analysis revealed a high degree of identity between iEmbryoid cells and human embryonic cells (Fig. 8a). Significant similarity was observed between iEmbryoids and multiple cell lineages in CS7 gastrula, while the similarity was lower compared to in vitro cultured CS6 pre-gastruloid embryos, indicating similarity between iEmbryoids and CS6b human embryos (Fig. 8a). Furthermore, ExEM and PGC annotations of CS7 gastrula, as well as heatmap analysis of ExEM-like and PGC-like cells in vitro, revealed similar expression patterns to iEmbryoid cluster counterparts (Fig. 8b). Notably, subpopulation analysis of the integrated blood cell sample revealed five distinct subpopulations, labeled as hematopoietic endothelial cells, blood progenitor cells, erythromycin progenitor cells, and myeloid progenitor cells (Figure 8c). In summary, the aforementioned single-cell RNA sequencing (scRNA-seq) analysis supports the reconstruction of major cell types in CS6b human embryos by iEmbryoids.

[0162] Experimental Example 2: Electron Microscopic Analysis of an Embryo Model

[0163] We investigated the effects of combining homologous induced hypoblast stem cells (iHypoblast SCs) and Electron microscopy was performed on human embryonic models (iEmbryoids) constructed using iPSCs. Electron micrographs of the iEmbryoids from day 4 to day 8 show that the cellular structure and tissue morphology of the embryonic models gradually changed with the passage of culture time, exhibiting characteristics similar to human perigastrulation embryonic development. These electron micrographs visually demonstrate the developmental process of the embryonic models at the cellular ultrastructural level, further confirming the accuracy and reliability of the embryonic model presented in this application in simulating human embryonic development (Figure 12).

[0164] Experimental Example 3: Immunofluorescence staining analysis of embryo models at different time points

[0165] Immunofluorescence staining analysis at early time points (day 1, day 2, day 3)

[0166] We performed immunofluorescence staining analysis on iEmbryoids at different time points (day 1, day 2, day 3) to detect the expression of key markers such as SOX2, SOX17, OCT3 / 4 and GATA6. The results showed that at early time points, cells in the embryonic model had begun to express these markers, and as the culture time extended, the expression of markers gradually increased and the distribution became more clear. This indicates that the embryonic model of the application can simulate the specialization trajectory of different cell lineages in the early stage of human embryonic development, providing a powerful tool for studying the mechanism of early embryonic development (Figure 13).

[0167] Experimental Example 4: Immunofluorescence staining of lineage tracing embryonic model

[0168] To further trace the origin and differentiation trajectory of each lineage in iEmbryoids, this study used fluorescence labeling method, marking cells derived from original pluripotent induced stem cells (iPS cells) as EGFP positive, and induced Hypoblast-like stem cells (iHypoblast SCs) as td-Tomato positive, so as to distinguish the progeny cell types in iEmbryoids in vivo and clarify the development trajectory of each lineage. The experimental results showed that EGFP positive cells mainly contributed to the following types of cell lineages: ISL1 positive amnion-like cells, BLIMP1 positive PGC-like cells, T positive primitive streak-like cells and BST2 positive ExEM-like cells. The results suggest that the above lineages are derived from iPS cell population. In contrast, td-Tomato positive cells mainly contributed to GATA6 positive yolk sac-like cells and CD34 positive HEP-like cells, indicating that these lineages are mainly derived from iHypoblast-like stem cell population. In general, the 8-day-old iEmbryoids reproduced the key milestone events of human peri-gastrulation period embryonic development, including amnion and yolk sac cavity formation, PGC specification, primitive streak initiation, A-P axis formation, ExEM expansion and HEP appearance, making it suitable as a model for understanding primitive streak formation and human gastrulation process. (Figure 14). Experimental Example 5: Experiment of constructing embryonic model by different types of induced hypoblast stem cells

[0169]

[0170] ​We tried to utilize other different types of hypoblast-like cells and ESCs to construct human embryonic models. The experimental results showed that the combination of different types of hypoblast-like cells and ESCs was not able to successfully assemble into embryonic models with different developmental characteristics. These embryonic models did not show organized diversity in morphology and cell marker expression, further demonstrating the uniqueness of the method of the present application (Figure 15).

[0171] Experimental Example 6: Single-cell sequencing analysis of embryonic models

[0172] (1) Single-cell sequencing of embryonic models at different time points

[0173] We performed single-cell RNA sequencing (sc-RNA seq) analysis on the cells before embryonic assembly on day 0, iEmbryoids on day 4 and day 8. UMAP algorithm analysis results showed that from day 0 to day 8, the cells in the embryonic models gradually differentiated into a variety of different lineage cells, including ectoderm (Epi), amnion (Am), primordial germ cells (PGC), primitive streak (PS), early mesoderm (Early meso), mesoderm (Meso), extraembryonic mesoderm (ExEM), visceral endoderm / yolk sac (VE / YS), secondary yolk sac (SYS), and hematopoietic endothelial progenitor cells (HEP). These results indicate that the embryonic models of the present application can simulate the cell differentiation and lineage specification processes at different time points during human embryonic development, providing rich data resources for studying the molecular mechanisms of embryonic development (Figure 16).

[0174] (2) Reconstruction of cell fate trajectories

[0175] We reconstructed the cell fate trajectories using Monocle3 software, and the results showed the process of cell differentiation from early undifferentiated state to different lineage cells in the embryonic models. This analysis further revealed the time sequence and lineage relationship of cell differentiation in the embryonic models, providing important clues for understanding the cell fate determination mechanisms during embryonic development (Figure 17a-b).

[0176] (3) Integrated analysis of embryonic models and human embryonic data

[0177] We integrated the single-cell sequencing data of the embryoid model with in vitro cultured embryo samples and CS7 human primitive gut embryo samples for integrated analysis. The UMAP algorithm analysis results showed that the cells in the embryoid model had high similarity with the corresponding cell types in human embryos, indicating that the embryoid model of the application can highly simulate the cell state and lineage characteristics during human embryonic development at the transcriptome level, providing a more accurate in vitro model for studying human embryonic development (Figure 17c). In addition, we showed the distribution of hypoblast-like, yolk sac-like and secondary yolk sac-like cell populations in the iEmbryoids on day 0, day 4 and day 8 through UMAP clustering analysis. The results showed that the embryoid model of the application can simulate the cell differentiation and lineage specialization process at different time points during human embryonic development, accurately identify specific cell populations, and further confirm the accuracy and reliability of the embryoid model in simulating human embryonic development (Figure 18).

[0178] (4) Key marker expression feature analysis

[0179] We showed the key marker expression features of hypoblast-like, yolk sac-like and secondary yolk sac-like cell populations in the iEmbryoids embryoid model on day 0, day 4 and day 8 through Dot Plot and UMAP clustering analysis. The expression of these markers further confirmed the formation of different cell types and structures in the embryoid model, providing detailed molecular marker information for studying cell differentiation and tissue formation during embryonic development (Figures 19 and 20).

[0180] In addition, we showed the expression of three markers, OCT3 / 4, GATA6 and APOA1, in the iEmbryoid SCs before assembly and in the iEmbryoids embryoid model on day 4 and day 8 through immunofluorescence staining. The results showed that as the culture time passed, cell populations expressing these markers appeared in the iEmbryoids, and their distribution had certain regularity. This indicates that the embryoid model of the application can successfully simulate the formation of multiple key cell types and structures during human embryonic development, reflecting the dynamic process of cell differentiation and tissue formation in the embryoid model during culture, providing strong support for studying embryonic development.

[0181] Experimental Example 7: Yolk sac-like hematopoietic feature study of embryoid model

[0182] (1) Immunofluorescence staining analysis

[0183] The embryoid model (iEmbryoids) was subjected to immunofluorescence staining analysis of yolk sac-like hematopoietic features. The results showed that on day 11 of hematopoietic differentiation, CD34 + Hypohemogenic endothelial progenitor cells and CD43 +Hematopoietic progenitor-like cells co-localized with FOXA2 + Endoderm-like structure region; at day 14 of hematopoietic differentiation, cell population expressing erythroid marker CD235a + and megakaryocytic marker CD42b + was observed, surrounded by CD34 + Hematopoietic endothelial progenitor-like cells; at day 19 of hematopoietic differentiation, cell population expressing erythroid marker CD235a + and megakaryocytic marker CD42b + was still apparent, and surrounded by CD34 + Hematopoietic endothelial progenitor-like cells. These results demonstrated that the embryonic model of the present application was able to mimic the yolk sac-like hematopoiesis, and provided a powerful tool for studying the mechanism of embryonic hematopoiesis (Figure 22).

[0184] (2) Flow cytometry analysis

[0185] We performed flow cytometry analysis on the cells of the embryonic model at different time points of hematopoietic differentiation (day 11, day 14, day 19). The results showed that at day 11 of hematopoietic differentiation, CD43 + Hematopoietic progenitor-like cells, CD31 + Endothelial-like cells and CD34 + Hematopoietic endothelial progenitor-like cells were detected; at day 14 of hematopoietic differentiation, CD43 + Hematopoietic progenitor-like cells, CD31 + Endothelial-like cells and CD34 + Hematopoietic endothelial-like cells further differentiated; at day 19 of hematopoietic differentiation, CD43 + population further differentiated into CD235ab + Erythroid cells, CD14 + Myeloid cells and CD42b + Megakaryocytic-like cells. These results further confirmed the occurrence and development of yolk sac-like hematopoiesis in the embryonic model, and provided detailed information on cell populations for studying the mechanism of embryonic hematopoiesis (Figure 23).

[0186] (3) Colony forming unit (CFU) analysis

[0187] We performed colony-forming unit (CFU) assay on day 19 embryo model. The results showed that single cell was able to form multiple colony types, including erythroid colony-forming unit (CFU-E), burst-forming erythroid colony-forming unit (BF-E), granulocyte-macrophage colony-forming unit (CFU-GM), and granulocyte-erythroid-macrophage-megakaryocyte multi-lineage colony (CFU-GEMM). The number of different types of CFU colonies formed per 10,000 cells was further demonstrated the differentiation and function of hematopoietic cells in the embryo model, which provided strong support for the study of cell differentiation and function during embryonic hematopoiesis (Figure 24).

[0188] The above supplementary experimental data further verified the strong advantages and wide applicability of the embryo model of the present application in simulating various key characteristics and mechanisms of human peri-gut stage embryonic development.

[0189] Obviously, the above embodiments are only examples for the sake of clarity, and are not intended to limit the embodiments. Based on the above description, those skilled in the art can make other different forms of changes or modifications. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or modifications derived therefrom are still within the protection scope of the present application.

Claims

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

2. The embryo model of claim 1, wherein, The method for preparing the embryonic model comprises the following steps of: After the induced pluripotent stem cells and the induced hypoblast stem cells are dispersed, the two types of cells are inoculated into a culture container, and are sequentially cultured by using embryonic assembly basal medium additionally supplemented with leukemia inhibitory factor and CEPT, embryonic assembly basal medium additionally supplemented with leukemia inhibitory factor, and embryonic assembly basal medium additionally supplemented with bone morphogenetic protein 4, so as to obtain an embryonic model.

3. The embryo model of claim 2, wherein, The components of the embryonic assembly basal medium comprise GlutaMAX, non-essential amino acids, penicillin-streptomycin, sodium pyruvate, 2-mercaptoethanol, N2 supplement, B27 supplement, and bovine serum albumin.

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

5. The embryo model according to claim 3 or 4, wherein The components of the embryonic assembly basal medium further comprise a matrix; the matrix comprises Neurobasal medium and DMEM / F12 medium; in the matrix, the volume ratio of the Neurobasal medium to the DMEM / F12 medium is 0.5-1:0.5-1.

6. The embryo model according to any one of claims 1 to 5, wherein The method for preparing the induced hypoblast stem cells comprises: first, using Yamanaka factors to transfect somatic cells, and then using an induced hypoblast stem cell culture medium to induce and culture the transfected somatic cells, to obtain induced hypoblast stem cells.

7. A method of 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 the induced hypoblast stem cells are dispersed, the two are inoculated into a culture container, and are sequentially cultured using embryonic assembly basal medium additionally supplemented with leukemia inhibitory factor and CEPT, embryonic assembly basal medium additionally supplemented with leukemia inhibitory factor, and embryonic assembly basal medium additionally supplemented with bone morphogenetic protein 4, to obtain an embryonic model.

8. An embryo assembly medium, characterized by, The components of the embryonic assembly medium comprise 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 embryonic assembly medium comprise 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 embryonic assembly medium comprise 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 of screening for a drug for preventing and / or treating a disease, characterized by, The method comprises: using the embryonic model according to any one of claims 1-6 to screen drugs for preventing and / or treating diseases; the diseases include genetic diseases and / or developmental disorders.

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

11. Use of the embryonic model according to any one of claims 1 to 6 or the method according to claim 7 or the embryonic assembly basal medium according to claim 8 for the screening of drugs for the prevention and / or treatment of diseases or for the evaluation of drug toxicity, characterized in that, The disease includes genetic disease and / or developmental disorder.

12. A cell therapeutic agent for preventing and / or treating a disease, characterized by, The components of the cell therapy drug comprise the embryonic model according to any one of claims 1 to 6, or the cell therapy drug is prepared from the embryonic model according to any one of claims 1 to 6.

13. Use of the embryonic model according to any one of claims 1 to 6 in the preparation of a cell therapy drug for preventing and / or treating a disease.

14. An organ transplant, comprising, The organ transplant is prepared from the embryonic model according to any one of claims 1 to 6.

15. Use of the embryonic model according to any one of claims 1 to 6 in the preparation of an organ transplant.

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