Stem cell derived gastruloid models and methods for their construction and use

Abstract

A stem cell-derived gastruloid model, its construction method, and its use are provided. The gastruloid model is constructed in vitro from human pluripotent stem cells and mimics, to some extent, biological events and major embryonic structures of early embryonic development, such as the development of primitive endoderm, the establishment of a bilayered blastocyst, the emergence of the amniotic cavity and amniotic cells, and the emergence of the primitive streak. It has been verified at both the protein and transcriptome levels. This model can better reproduce the major characteristics of the embryo from the peri-implantation stage to the gastrula stage. Furthermore, this model can be induced in batches for use as a screening model for drugs and environmental toxicants in early embryos, thereby providing safety testing for drug administration in specific clinical early pregnancy patients.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, and in particular to an in vitro mimicking human peri-implantation gastruloid model derived from pluripotent stem cells, and methods for its derivation and use. [Background technology]

[0002] Human life begins with fertilization, when sperm and egg unite to form a zygote. This unique pluripotent cell undergoes continuous division and cell differentiation to form a blastocyst on day 5. The blastocyst is primarily composed of two cell groups: the inner cell mass (ICM) and the trophectoderm (TE). The ICM forms the embryo and extraembryonic tissues during later development, while the trophectoderm further develops to become the main component of the placenta. Before embryo implantation, the ICM begins to differentiate into the epiblast (EPI) and hypoblast (HYPO). After implantation, the EPI forms the amniotic sac, and the HYPO forms the yolk sac. The EPI then undergoes gastrulation, establishing a gastrula with three germ layers. Due to technical and ethical constraints and limited sample sizes, little research has been conducted on human peri-implantation embryonic development. Understanding the mechanisms of early human embryonic development is important for developmental biology and regenerative medicine. Currently, most of our knowledge of early human embryonic development comes from the Carnegie Histology and Anatomy of the Embryo study, and at this stage, there are still many unknown areas that need to be explored.

[0003] Currently, groundbreaking advances in human pre- and peri-implantation embryonic development have enabled researchers to culture human embryos in vitro up to embryonic day 14 before gastrulation and induce naive human pluripotent stem cells (nhPSCs) into pre-implantation embryoid bodies (PEBs). The combination of single-cell multi-omics sequencing and fluorescence imaging techniques has opened new avenues for human gastrula development and significantly expanded our understanding of the characteristics and mechanisms of early human gastrula development. While some studies have attempted to mimic post-implantation embryonic development at a 3D level by using hPSCs to establish amniote organs or reconstruct early development of the primitive ectoderm, these models lack key embryonic structures (such as the bilayered blastocyst and yolk sac) and do not observe the broad developmental stages of human primitive endoderm. Therefore, an ideal model for studying gastruloid development from the peri- to post-implantation stage remains to be established. Summary of the Invention [Problem to be solved by the invention]

[0004] In this study, we established a model and method for inducing human pluripotent stem cells (hPSCs) into gastruloids. This gastruloid model was constructed in vitro and mimics biological events and key embryonic structures during early embryonic development, such as the establishment of a bilayered blastocyst, the emergence of the amniotic cavity and amniocytes, and the appearance of the primitive streak. It has been validated at both the protein and transcriptome levels, demonstrating excellent recapitulation of key embryonic features from the peri-implantation stage to the gastrula stage. Furthermore, this model can be induced in batches for use as a drug screening model for early embryos, thereby providing safety testing for drug administration in specific clinical early pregnancy patients. [Means for solving the problem]

[0005] The human pluripotent stem cells used in the present invention include human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). The human induced pluripotent stem cells and human embryonic stem cells are established human induced pluripotent stem cells and human embryonic stem cells that are publicly available. In a specific embodiment, the human induced pluripotent stem cells were purchased from the Cell Bank / Stem Cell Bank of the Commission for the Preservation of Typical Cultures of the Chinese Academy of Sciences under the cell name DYR0100 and catalog number SCSP-1301, and the human embryonic stem cells were purchased from the Cell Bank / Stem Cell Bank of the Commission for the Preservation of Typical Cultures of the Chinese Academy of Sciences under the cell name H1 and catalog number SCSP-301.

[0006] The first object of the present invention is to provide a method for constructing a gastruloid model, specifically, a method for inducing pluripotent stem cells into a human gastruloid model in vitro, which method comprises the following steps:

[0007] (1) When the pluripotent stem cells grow to 80-90% confluence, digest the cells into single cells, resuspend the cells using PBS (phosphate buffer solution) to obtain cell suspension 1, and then digest the cells into single cells using TrypLE Select, preferably trypsin.

[0008] Cell suspension 1 was taken and G1ER medium containing Y-27632 was added to further resuspend the cells to obtain cell suspension 2.

[0009] Cell suspension 2 is inoculated onto an embryoid body culture plate that has been pre-coated with an anti-adhesion solution, and the plate is centrifuged.

[0010] On day 2 of culture, replace half of the medium with G1ER medium, and continue culture by replacing half of the medium every day until primitive endoderm and primitive ectoderm are formed.

[0011] Preferably, the ratio of the cell suspension 1 to the G1ER medium containing Y-27632 is 7.5 to 9.0 × 10 4 Add 2 mL of G1ER medium containing Y-27632 to each cell suspension containing 1 cell.

[0012] Preferably, the amount of cells inoculated into each microwell of the cell suspension 2 is 50 to 75 cells.

[0013] Preferably, the centrifugation is carried out at 800 rpm for 2 minutes using a horizontal centrifuge.

[0014] Preferably, the anti-adhesion solution is an Anti-adhension rinsing solution, and the embryoid body culture plate is an Agreewell 400™ culture dish.

[0015] Preferably, step (1) is performed on days 0 to 3 of the method for constructing a model of a post-implantation gastruloid.

[0016] (2) Discard 1 / 2 to 3 / 4 of the volume of G1ER medium in each well of the culture plate from step (1), add G2EE medium, and culture for pre-amniotic cavity induction. When adding the medium, add it slowly along the wall to prevent the formed gastruloids from floating up from the microwell and fusing.

[0017] In one particular embodiment, the culture plate is an Agreewell 400™ culture dish, and ¾ of the volume of G1ER medium in each well of the culture plate is discarded, ie, 1.75 mL / well.

[0018] Preferably, step (2) is performed on day 4 of the method for constructing a model of a post-implantation gastruloid.

[0019] (3) Discard 1 / 2 to 3 / 4 of the volume of G2EE medium in each well of the culture plate from step (2), and add aG2EE medium to induce amniotic cell formation. Add the medium slowly along the wall of the well to prevent the formed gastruloids from floating up and fusing with the microwell.

[0020] In one particular embodiment, the culture plate is an Agreewell 400™ culture dish, and ¾ of the volume of G2EE medium in each well of the culture plate is discarded, ie, 1.75 mL / well.

[0021] Preferably, step (3) is performed on day 5 of the method for constructing a model of a post-implantation gastruloid.

[0022] (4) Discard 1 / 2 to 3 / 4 of the volume of aG2EE medium from each well of the culture plate in step (3), add bG2EE medium, and culture until induction of amniocytes, primitive streak cells, and primordial germ cells occurs to obtain a post-implantation gastruloid model. When adding medium, add slowly along the wall to prevent the formed gastruloids from floating up and fusing from the microwells.

[0023] In one particular embodiment, the culture plate is an Agreewell 400™ culture dish, and ¾ of the volume of aG2EE medium in each well of the culture plate is discarded, ie, 1.75 mL / well.

[0024] Preferably, step (4) is performed on day 6 to 7 of the method for constructing a post-implantation gastruloid model.

[0025] Furthermore, the culture conditions for the culture in steps (1) to (4) are a temperature of 37°C and a volume concentration of carbon dioxide of 5% to 6%.

[0026] Furthermore, the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells, preferably human induced pluripotent stem cells or human embryonic stem cells.

[0027] Furthermore, the components of the G1ER medium in step (1) include: 50 vol% G-1™ Plus blastomere medium, 25 vol% Essential 8 medium and 25 vol% RACL medium.

[0028] The RACL medium contains the following: 95 vol% to 98.5 vol% basal medium RPMI 1640 (containing GlutaMAX), 1 vol% B27 supplement, 0.5 vol% penicillin-streptomycin, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, 100 ng / mL recombinant human activin A protein, 20 ng / mL recombinant human leukocyte factor protein, and 3 μM CHIR 99021.

[0029] The components of G1ER medium with Y-27632 include: 50% (vol / vol) G-1™ Plus Blastomere Medium, 25% (vol / vol) Essential 8 Medium, and 25% (vol / vol) RACL Medium, which contains the following: 95% to 98.5% basal medium RPMI 1640 (with GlutaMAX), 1% (vol / vol) B27 supplement, 0.5% (vol / vol) penicillin-streptomycin, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, 100 ng / mL recombinant human activin A protein, 20 ng / mL recombinant human leukocyte factor protein, 3 μM CHIR 99021, and 10 μM Y-27632.

[0030] Furthermore, the components of the G2EE medium in step (2) include: 50% by volume of G-2™ Plus Embryo Medium, 25% by volume of Essential 8 Medium, and 25% by volume of EBB Medium, which contains 100% Essential 6 Medium, 20 ng / mL of recombinant human bone morphogenetic protein 4, and 10 ng / mL of recombinant human fibroblast growth factor 2.

[0031] Furthermore, the components of the aG2EE medium in step (3) include: 50% by volume of G-2™ Plus Embryo Medium, 25% by volume of Essential 8 Medium, and 25% by volume of aEBB Medium, which contains 100% Essential 6 Medium, 100 ng / mL of recombinant human bone morphogenetic protein 4, and 10 ng / mL of recombinant human fibroblast growth factor 2.

[0032] Furthermore, the components of the bG2EE medium in step (4) include: 50% by volume of G-2™ Plus Embryo Medium, 25% by volume of Essential 8 Medium, and 25% by volume of bEBB Medium, which contains 100% Essential 6 Medium, 75 ng / mL of recombinant human bone morphogenetic protein 4, and 10 ng / mL of recombinant human fibroblast growth factor 2.

[0033] A second object of the present invention is to provide a gastruloid model constructed using the above method.

[0034] A third object of the present invention is to provide the use of the above gastruloid model, or tissues or organs derived from said model, or cultures thereof, in the study of the mechanisms of human early embryonic development.

[0035] A fourth object of the present invention is to provide the use of the above gastruloid model, or tissues or organs derived from said model, or cultures thereof, in diagnostic and / or therapeutic strategies for diseases of human early embryonic development.

[0036] A fifth object of the present invention is to provide the use of the above gastruloid model, or tissues or organs derived from the model, or cultures thereof, in screening, validating, evaluating, assessing or studying the effectiveness of drugs for the prevention and / or treatment of human early embryonic development disorders.

[0037] Furthermore, the human early embryonic development disorder is drug-induced human early embryonic teratogenesis.

[0038] The gastruloid model described in this invention is a model obtained by in vitro induction using pluripotent stem cells and has a final state similar to that of a Carnegie stage 6 embryo. The model can be induced in batches for use as an early embryo drug screening model, thereby providing safety testing for drug administration in specific clinical early pregnancy patients. The gastruloid model, or tissues or organs derived from the gastruloid model, or cultures thereof, cannot develop into an individual due to the lack of cell types such as trophoblasts.

[0039] The technical solution of the present invention has the following advantages: Feature 1: Human pluripotent stem cells can form cell spheres within 12 hours, have high induction stability, and exhibit stable cell proliferation characteristics in continuous gastrointestinal culture for 7 days.

[0040] Characteristic feature 2: On day 1 of gastruloid induction, lineage separation between primitive endoderm and primitive ectoderm begins, and primitive endoderm-like cells that express both pluripotency markers (OCT4) and early primitive endoderm markers (OTX2, GATA6) appear.

[0041] Feature 3: On day 3, the typical bilayered scutellum structure of the embryo appears, with the epiblast side being columnar cells that are OCT4 and SOX2 positive, and the hypoblast side being cuboidal cells that are OTX2, GATA6, and SOX17 positive.

[0042] Feature 4: On the 4th to 5th day of gastruloid induction, amniotic cavity structures begin to form on the epiblast side, and several amniotic cells that are TFAP2a and CDX2 positive, weakly OCT4 positive, and SOX2 negative appear.

[0043] Feature 5: On day 6–7 of gastruloid induction, amniocytes isolated from the epiblast become thinner and form distinct squamous amniotic epithelium that expresses amniotic cell markers such as TFAP2a and CDX2 but negatively expresses pluripotency markers such as OCT4 and SOX2.

[0044] Feature 6: On days 6-7 of gastruloid induction, some T and EOMES-positive cells were isolated from the epiblast, and the expression of pluripotency markers (OCT4) further weakened, indicating that they were undergoing an epithelial-mesenchymal transition (EMT) process. These findings are consistent with the formation of a primitive streak, demonstrating the onset of gastruloid gastrulation after implantation.

[0045] Characteristic feature 7: On day 6–7 of gastruloid induction, a small number of primordial germ cells emerge that are positive for pluripotency markers (OCT4, NANOG, NANOS3), primordial germ cell markers (SOX17, AP2C, BLIMP1), and characteristically negative for SOX2.

[0046] Feature 8: The final state of gastruloids after 7 days of induction culture resembles that of Carnegie stage 6 embryos.

[0047] Feature 9: The model can be derived in batches for use as an early embryo drug screening model, thereby providing safety testing of drug administration in selected clinical early pregnancy patients. [Effects of the Invention]

[0048] Beneficial Effects: The model provides a platform for in vitro studies of human early embryonic development, aiming to understand the complexity of human early embryonic development, and provides a research platform for developing clinical treatments for early embryonic peri-implantation disorders. [Brief explanation of the drawings]

[0049] [Figure 1] Figure 1 is a white light diagram showing the culture process of a gastruloid model induced for 1 to 7 days, provided in one embodiment of the present invention. Figure 1A shows a post-implantation gastruloid induced by human induced pluripotent stem cells (hiPSCs), and Figure 1B shows a post-implantation gastruloid induced by human embryonic stem cells (hESCs). The scale bar is 100 µm. [Figure 2]Figure 2 is a white light diagram showing the sampling process of postimplantation gastruloids induced by human induced pluripotent stem cells (hiPSCs) on days 3 and 7 of induction, with scale bars of 50 μm and 100 μm. [Figure 3] Figure 3 is a growth curve diagram of gastruloids provided by one embodiment of the present invention for 1 to 7 days of induction, where each point represents the cell mass of one blastoid, and the sample size at each time point is 20 or more. Figure 3A is a growth curve diagram of human induced pluripotent stem cells (hiPSCs) for 1 to 7 days of induction, and Figure 3B is a growth curve diagram of human embryonic stem cells (hESCs) for 1 to 7 days of induction. [Figure 4] Figure 4 shows IF images showing the process from the appearance of primitive endoderm to the appearance of a bilayered scutellum structure during days 1 to 3 of gastruloid induction provided by one embodiment of the present invention. The scale bar is 20 μm. Figure 4A is an immunofluorescence image of GATA6 and OCT4, and Figure 4B is an immunofluorescence image of SOX2 and SOX17. [Figure 5] Figure 5 shows IF images showing the process from the formation of the amniotic cavity to the appearance of amniotic cells during days 4 to 7 of gastruloid induction provided by one embodiment of the present invention. The scale bar is 20 μm. Figure 5A shows CDX2, OCT4, and GATA6, and Figure 5B shows SOX2, TFAP2a, and SOX17.

[0050] [Figure 6] Figure 6 shows IF images of the migration process of the primitive streak during gastrulation in induced gastruloids for 7 days, provided by one embodiment of the present invention. Figure 6A shows that OCT4+T+ gastrulation motor cells gradually transition into T+EOMES+ positive definitive endoderm fate cells and ultimately differentiate into EOMES+ definitive endoderm cells. Figure 6B shows that OCT4+T+ gastrulation motor cells gradually transition into T+MIXL1+ positive mesoderm fate cells and ultimately differentiate into MIXL1+ mesoderm cells. The scale bar is 50 μm. [Figure 7]Figure 7 shows the results of bioinformatics analysis provided by one embodiment of the present invention, in which A is a UMAP clustering diagram of single-cell sequencing data of gastruloids at 7 days of induction, in which Epiblast is primitive ectoderm, Endoderm is primitive endoderm, Primitive Streak is primitive streak, Primitive Streak anlage Epiblast is primitive streak anlage ectoderm, PGC is primordial germ cell, Amnion is amnion, and Mesoderm is mesoderm, and B shows the basis for the associated clustering. [Figure 8] Figure 8 shows the results of drug application in a gastruloid model provided by one embodiment of the present invention. A shows white light images of samples from the control group, dimethyl sulfoxide, 5 μM THD, and 10 μM THD on day 7 of gastruloid induction, and DAPI (4',6-diamidino-2-phenylindole) staining results, with scale bars of 200 μm and 100 μm, respectively. B shows the cell volume of a single gastruloid from the control group, dimethyl sulfoxide, 5 μM THD, and 10 μM THD on day 7 of gastruloid induction. C shows the volume of a single gastruloid from the control group, dimethyl sulfoxide, 5 μM THD, and 10 μM THD on day 7 of gastruloid induction. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention discloses a method for inducing human pluripotent stem cells into a gastruloid model. The reagents, instruments, cell lines, etc. used in the method for inducing human pluripotent stem cells into a gastruloid model provided by the present invention are all commercially available. The human induced pluripotent stem cells were purchased from the Cell Bank / Stem Cell Bank of the Committee for the Preservation of Endothelial Cell Cultures, Chinese Academy of Sciences, under the cell name DYR0100 and catalog number SCSP-1301. The human embryonic stem cells were purchased from the Cell Bank / Stem Cell Bank of the Committee for the Preservation of Endothelial Cell Cultures, Chinese Academy of Sciences, under the cell name H1 and catalog number SCSP-301.

[0052] (1) The sources of the medium components used to induce the human pluripotent stem cells into gastruloids in vitro are as follows: G-1™ Plus Blastomere Medium: 10128, purchased from Vitrolife, Sweden; G-2™ Plus embryo medium: 10132, purchased from Vitrolife, Sweden; Essential 8 medium: A1517001, purchased from Gibco, USA; Essential 6 medium: A1516401, purchased from Gibco, USA; RPMI 1640 (containing GlutaMAX): 61870036, purchased from Gibco, USA; Anti-adhension rinsing solution: 07010, purchased from STEMCELL, USA; B27 additive: 12587-010, purchased from Gibco, USA; Non-essential amino acids, Gibco® MEM Non-Essential Amino Acids, 11140076, purchased from Gibco, USA; β-mercaptoethanol: 21985023, purchased from Gibco, USA; Recombinant human bone morphogenetic protein 4:314-BP, purchased from R&D Systems, USA; Recombinant human leukemia inhibitory factor: 225-SC, purchased from R&D Systems, USA; Recombinant human activin A protein: 338-AC, purchased from R&D Systems, USA; Recombinant human fibroblast growth factor 2:3718-FB, purchased from R&D Systems, USA; ROCK inhibitors: specific types are Y27632 and HY-10071, purchased from MCE, USA; CHIR99021: The specific type is Laduviglusib trihydrochloride, HY-10182B, purchased from MCE, USA.

[0053] (2) Preparation of G1ER medium: 50 vol% G-1™ Plus blastomere medium, 25 vol% Essential 8 medium, and 25 vol% RACL medium; the RACL medium contains the following: 98.5 vol% basal medium RPMI 1640 (with GlutaMAX), 1 vol% B27 supplement, 0.5 vol% penicillin-streptomycin, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, 100 ng / mL recombinant human activin A protein, 20 ng / mL recombinant human leukocyte factor protein, and 3 μM CHIR 99021.

[0054] (3) Preparation of G1ER medium containing Y-27632: 50 vol% G-1™ Plus blastomere medium, 25 vol% Essential 8 medium, and 25 vol% RACL medium; the RACL medium contains the following: 98.5% basal medium RPMI 1640 (with GlutaMAX), 1 vol% B27 supplement, 0.5 vol% penicillin-streptomycin, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, 100 ng / mL recombinant human activin A protein, 20 ng / mL recombinant human leukocyte factor protein, 3 μM CHIR 99021, and 10 μM Y-27632.

[0055] (4) Preparation of G2EE medium: 50 vol% G-2™ Plus Embryo Medium, 25 vol% Essential 8 Medium, and 25 vol% EBB Medium; the EBB Medium contains 100% Essential 6 Medium, 20 ng / mL recombinant human bone morphogenetic protein 4, and 10 ng / mL recombinant human fibroblast growth factor 2.

[0056] (5) Preparation of aG2EE medium: 50 vol% G-2™ Plus Embryo Medium, 25 vol% Essential 8 Medium, and 25 vol% aEBB Medium; the aEBB Medium contains 100% Essential 6 Medium and multiple cytokines (100 ng / mL recombinant human bone morphogenetic protein 4, 10 ng / mL recombinant human fibroblast growth factor 2).

[0057] (6) Preparation of bG2EE medium: 50 vol% G-2™ Plus Embryo Medium, 25 vol% Essential 8 Medium, and 25 vol% bEBB Medium; the bEBB Medium contains 100% Essential 6 Medium and multiple cytokines (75 ng / mL recombinant human bone morphogenetic protein 4, 10 ng / mL recombinant human fibroblast growth factor 2).

[0058] Example 1 Induction of Gastruloids from Human Induced Pluripotent Stem Cells (iPSCs) (Stage 1): Days 0-3, Formation of Primitive Endoderm and Primitive Ectoderm When human induced pluripotent stem cells DYR0100 were grown to 80-90% confluence, cells were digested into single cells using TrypLE Select and resuspended using PBS to obtain cell suspension 1.

[0059] 9.0x10 4 Cell suspension 1 containing 100 cells was taken, and 2 mL of G1ER medium containing Y-27632 was added to resuspend the cells, thereby obtaining cell suspension 2.

[0060] Cell suspension 2 was inoculated into one well of an Agreewell 400™ culture dish that had been previously coated with anti-adhension rinsing solution. After centrifugation at 800 rpm for 2 minutes using a horizontal centrifuge, the cell density per microwell was 75 cells (each well of the Agreewell 400™ culture dish contained 1200 microwells, i.e., the cell density per microwell was 9.0 x 10 4 / 1200=75 pieces).

[0061] On the second day of culture, G1ER medium was used, and half of the medium was replaced every day until the third day of culture, during which time primitive endoderm and primitive ectoderm were formed.

[0062] Example 2 Induction of Gastruloids from Human Induced Pluripotent Stem Cells (hiPSCs) (Stage 2): Day 4, Pre-induction of the Amniotic Cavity After discarding 3 / 4 of the volume of the first-stage G1ER medium per well, i.e., 1.75 mL / well, the second-stage G2EE medium was slowly added to the well along the wall to prevent the formed gastruloids from floating up from the microwell and fusing, and pre-amniotic cavity induction was performed.

[0063] Example 3 Induction of gastruloids from human induced pluripotent stem cells (hiPSCs) (Stage 3): Day 5, Induction of amniotic cells After discarding 3 / 4 of the volume of the second-stage G2ER medium per well, i.e., 1.75 mL / well, the third-stage aG2EE medium was slowly added to the well along the wall to induce amniotic cell formation, in order to prevent the formed gastruloids from floating up from the microwell and fusing.

[0064] Example 4 Induction of gastruloids from human induced pluripotent stem cells (hiPSCs) (Stage 4): Days 6-7, induction of amniocytes, primitive streak cells, and primordial germ cells After discarding 3 / 4 of the volume of the third-stage aG2EE per well, i.e., 1.75 mL / well, the fourth-stage bG2EE medium was slowly added to the well along the wall to prevent the formed gastruloids from floating up from the microwell and fusing. After 24 hours, 1.75 mL of the medium (bG2EE medium) was continuously replaced, and the cells were cultured until the 7th day to induce amniocytes, primitive streak cells, and primordial germ cells, thereby obtaining a model of the post-implantation gastruloid.

[0065] Example 5: Induction of gastruloids from human embryonic stem cells (hESCs). The experimental procedures of Examples 1 to 4 were repeated. (1) On days 0 to 3, primitive endoderm and primitive ectoderm are formed. When human embryonic stem cells H1 were grown to 80-90% confluence, cells were digested into single cells using TrypLE Select and resuspended using PBS to obtain cell suspension 1.

[0066] 9.0x10 4 Cell suspension 1 containing 100 cells was taken, and 2 mL of G1ER medium containing Y-27632 was added to resuspend the cells, thereby obtaining cell suspension 2.

[0067] Cell suspension 2 was inoculated into one well of an Agreewell 400™ culture dish that had been previously coated with anti-adhension rinsing solution. After centrifugation at 800 rpm for 2 minutes using a horizontal centrifuge, the cell density per microwell was 75 cells (each well of the Agreewell 400™ culture dish contained 1200 microwells, i.e., the cell density per microwell was 9.0 x 10 4 / 1200=75 pieces).

[0068] On the second day of culture, G1ER medium was used, and half of the medium was replaced every day until the third day of culture, during which time primitive endoderm and primitive ectoderm were formed.

[0069] (2) Day 4, pre-induction of the amniotic cavity: After discarding 3 / 4 of the volume of the first-stage G1ER medium per well, i.e., 1.75 mL / well, the second-stage G2EE medium was slowly added to the well along the wall to prevent the formed gastruloids from floating up from the microwell and fusing, and pre-amniotic cavity induction was performed.

[0070] (3) Day 5, amniocyte induction: After discarding 3 / 4 of the volume of the second-stage G2EE medium per well, i.e., 1.75 mL / well, the third-stage aG2EE medium was slowly added to the well along the wall to induce amniotic cell formation, in order to prevent the formed gastruloids from floating up from the microwell and fusing.

[0071] (4) Days 6-7, induction of amniocytes, primitive streak cells, and primordial germ cells: After discarding 3 / 4 of the volume of the third-stage aG2EE per well, i.e., 1.75 mL / well, the fourth-stage bG2EE medium was slowly added to the well along the wall to prevent the formed gastruloids from floating up from the microwell and fusing. After 24 hours, 1.75 mL of the medium (bG2EE medium) was continuously replaced, and the cells were cultured until the 7th day to induce amniocytes, progenitor streak cells, and primordial germ cells, thereby obtaining the gastruloid model.

[0072] Example 6: Verification of the biological properties of gastruloids derived from human induced pluripotent stem cells (hiPSCs) 1.1 Morphological observation of induced gastruloids Observation of gastruloid models induced for 1 to 7 days in the culture dishes of Examples 1 to 4 using an upright fluorescence microscope revealed rapid cell proliferation and a gradual increase in gastruloid volume. A clear structure of two layers of scutellum was observed on the third day, an amniotic cavity structure appeared on the fourth or fifth day, and a yolk sac structure appeared in some of the gastruloids on the fourth to seventh day. The diameter of the gastruloids on the seventh day was approximately 250 μm to 350 μm. The results of the morphological observations are shown in Figures 1A and 2.

[0073] 1.2 Measurement of the growth curve of induced post-implantation gastruloids 1.2.1 Growth curve measurement procedure Samples were taken every 24 hours for days 1 to 7 of induction in the culture dishes of Examples 1 to 4.

[0074] After collecting the samples, they were fixed with 4% paraformaldehyde for 30 minutes, the fixative was discarded, and the samples were washed with PBS three times for 5 minutes each time.

[0075] Samples were then blocked with 5% bovine serum albumin (containing 1% Triton) for 4 hours at room temperature.

[0076] Next, DAPI was diluted at a ratio of 1:200 and stained for 4 hours at room temperature.

[0077] After discarding the DAPI dilution solution, PBS was added and the sample was washed three times for 5 minutes each time. The sample was then transferred to a Chamber staining chamber, and a certain amount of iohexol solution prepared for clearing was added. The slide was then lightly covered with the Chamber and photographed using a confocal microscope.

[0078] After imaging, the parameters were adjusted using the IMARIS software to perform single sample counting, and the samples from each day were counted. 21 There were ~36 replicate samples.

[0079] 1.2.2 Growth curve measurement results The growth curve data shown in Table 1 below were obtained over seven consecutive days of measurements.

[0080] Table 1 shows the growth curve data obtained over seven consecutive days. 21~36 There are replicate samples. [Table 1]

[0081] Based on the cell growth curve data in Table 1, a schematic diagram of the growth curve of induced post-implantation gastruloids was obtained, as shown in Figure 3A. The horizontal axis of Figure 3 represents the induction culture time (days), and the vertical axis represents the cell mass (cells) within a single gastruloid.

[0082] Referring to the growth curve data in Table 1 and the schematic diagram of the growth curve in Figure 3A, it can be seen that cells within the gastruloid steadily proliferated from 50–75 cells / cell on day 0 to 1040 cells / cell on day 7.

[0083] In summary, the induced gastruloid cells proliferate rapidly, grow actively, have good cell activity, and exhibit high stability of cell induction, showing stable cell proliferation characteristics in vitro.

[0084] 1.3 Identification by immunofluorescence (IF) 1.3.1 Immunofluorescence Identification Process Samples were taken every 24 hours for days 1 to 7 of induction in the culture dishes of Examples 1 to 4.

[0085] The sections were fixed with 4% paraformaldehyde for 30 minutes, the fixative was discarded, and the sections were washed with PBST three times for 5 minutes each time.

[0086] Samples were blocked with 5% bovine serum albumin (containing 1% Triton) for 4 hours at room temperature.

[0087] The blocking solution was discarded, and an appropriate amount of proportionally diluted antibody was added and incubated at 4°C for 24 hours.

[0088] The primary antibody was discarded, and the sections were washed three times with PBS (containing 1% Triton) for 5 minutes each time. Diluted secondary antibody and DAPI were then added, and the sections were incubated at room temperature for 4 hours.

[0089] The secondary antibody was discarded, and the slides were washed three times with PBS (containing 1% Triton) for 5 minutes each time. The slides were then transferred to a Chamber staining chamber, and a certain amount of iohexol solution prepared for clearing was added. The slides were then lightly covered with the Chamber and photographed using a confocal microscope.

[0090] 1.3.2 Identification results by immunofluorescence The results of identification by immunofluorescence are shown in Figures 4 to 6.

[0091] On day 1 of induction, a small number of GATA6 and OCT4 double-positive cells appeared among the OCT4 and SOX2-positive primitive ectoderm, indicating the initiation of differentiation of the primitive endoderm lineage. On day 2 of induction, the number of GATA6-positive cells began to increase, and simultaneously, SOX17-positive cells appeared. This type of cell gradually separated from the primitive ectoderm, further indicating an increase in the number of primitive endoderm cells. On day 3 of induction, the GATA6- and SOX17-positive primitive endoderm cells were completely separated from the OCT4- and SOX2-positive primitive ectoderm cells, forming a neatly arranged bilayered blastocyst structure between the two cell types. Among these, the primitive ectoderm cell nuclei formed columnar three-dimensional cells, while the primitive endoderm cell nuclei formed small columnar cells (Figure 4).

[0092] On days 4–5 of induction, a cavity-like structure consistent with the characteristics of the amniotic cavity gradually appeared on the side of the primitive ectoderm, and cells positive for transcription factor 2a (TFAP2a), CDX2, and OCT4, but negative for SOX2, emerged, becoming early amniocytes. On days 6–7 of induction, mature amniocytes positive for TFAP2a and CDX2, but negative for OCT4 and SOX2, appeared, and the nuclei became flattened, resembling mature squamous amniocytes (Figure 5).

[0093] On day 6–7 of induction, T, EOMES, and MIXL1-positive cells appeared in the OCT4-positive columnar primitive ectoderm, and the OCT4 positivity gradually weakened, resembling the primitive streak, demonstrating that gastruloids had initiated epithelial–mesenchymal transition (EMT) and gastrulation (Figure 6 ).

[0094] 1.4 RNA sequencing identification 1.4.1 RNA sequencing identification procedure Gastruloids induced in the culture dishes of Examples 1 to 4 were collected on the 7th day.

[0095] The medium was removed, and the gastruloids were washed at least twice with PBS (0.01M, pH 7.4) to remove old medium and debris from detached dead cells. They were then treated with 2 mL of 0.24% trypsin for digestion, placed in an environment at 37°C for 3 minutes, and gently pipetted with a pipette gun to disperse the gastruloids into single cells. The digestion was then terminated using serum-containing medium.

[0096] The supernatant was removed by centrifugation at 1000 rpm for 5 minutes, and the cells were resuspended in PBS and transferred to enzyme-free EP tubes for sequencing.

[0097] 1.4.2 Results of identification by RNA sequencing We analyzed and clustered the gastruloid data on day 7 of induction. The results showed that the gastruloids obtained by culture could be divided into seven major groups: primitive ectoderm, primitive endoderm, primitive streak precursor cells, primitive streak, primordial germ cells, amniotes, and mesoderm. This was verified by the colocalization of three known related specific markers. Here, POIU5F1, SOX2, and DNMT3B define the primitive ectoderm; HNF4A, FOXA2, and PDGFRA define the primitive endoderm; TFAP2A, ISL1, and ABCG2 define the amniotes; TBXT, EOMES, and MIXL1 define the primitive streak precursor cells and the primitive streak; HAND1, BMP4, and SNAI2 define the mesoderm; and PRDM1, TFAP2C, and NANOG define the primordial germ cells (Figure 7).

[0098] Example 7: Verification of the biological properties of gastruloids induced by human embryonic stem cells (hESCs) 1.1 Morphological observation of induced post-implantation gastruloids Observation of gastruloid models induced in the culture dishes of Example 5 for 1 to 7 days using an upright fluorescence microscope revealed rapid cell proliferation and a gradual increase in gastruloid volume. A clear structure of two layers of scutellum was observed on day 3, an amniotic cavity structure appeared on days 4 to 5, and a yolk sac structure appeared in some of the gastruloids on days 4 to 7. The diameter of the gastruloids on day 7 was approximately 250 μm to 350 μm. The results of the morphological observations are shown in Figure 1B.

[0099] 1.2 Measurement of the growth curve of induced post-implantation gastruloids 1.2.1 Growth curve measurement procedure Samples were taken every 24 hours for days 1 to 7 of induction in the culture dishes of Example 5.

[0100] After collecting the samples, they were fixed with 4% paraformaldehyde for 30 minutes, the fixative was discarded, and the samples were washed with PBS three times for 5 minutes each time.

[0101] Samples were then blocked with 5% bovine serum albumin (containing 1% Triton) for 4 hours at room temperature.

[0102] Next, DAPI was diluted at a ratio of 1:200 and stained for 4 hours at room temperature.

[0103] After discarding the DAPI diluted solution, PBS was added and washed three times for 5 minutes each time. The sample was then transferred to the Chamber staining chamber, and a certain amount of iohexol solution prepared for clearing was added. The slide was then lightly covered with the Chamber and photographed under a confocal microscope;

[0104] After the imaging, the parameters are set and adjusted using the IMARIS software to perform counting of single samples. 30~37 There were replicate samples.

[0105] 1.2.2 Growth curve measurement results The growth curve data shown in Table 2 below were obtained over seven consecutive days of measurements.

[0106] Table 2 shows growth curve data obtained over 7 consecutive days of measurements, with 30–37 replicate samples for each day. [Table 2]

[0107] Based on the cell growth curve data in Table 2, a schematic diagram of the growth curve of induced gastruloids was obtained, as shown in Figure 3B. The horizontal axis of Figure 3B represents the induction culture time (days), and the vertical axis represents the cell mass (cells) within a single gastruloid.

[0108] Referring to the growth curve data in Table 2 and the schematic diagram of the growth curve in Figure 3, it can be seen that the cells in the gastruloids steadily proliferated from 50–75 cells / cell on the initial day 0 to 986 cells / cell on the 7th day.

[0109] In summary, the induced gastruloid cells proliferate rapidly, grow actively, have good cell activity, and exhibit high stability of cell induction, showing stable cell proliferation characteristics in vitro.

[0110] Example 8 Use of an in vitro derived gastrointestinal model from human pluripotent stem cells Thalidomide was selected for model testing of drugs that exhibit teratogenic effects on early embryos. Two gradients of 5 μM and 10 μM were selected, and a blank control group and a dimethyl sulfoxide group (the solvent for thalidomide) were added for testing.

[0111] When human induced pluripotent stem cells DYR0100 were grown to 80-90% confluence, cells were digested into single cells using TrypLE Select and resuspended using PBS to obtain cell suspension 1.

[0112] 9.0x10 4Cell suspension 1 containing 100 cells was taken, and 2 mL of G1ER medium containing Y-27632 was added to resuspend the cells. Then, 5 μM and 10 μM 1‰ thalidomide, dimethyl sulfoxide solution, and a blank control group were added, for a total of four groups, to obtain cell suspension 2.

[0113] Cell suspension 2 was reseeded into one well of an Agreewell 400™ culture dish that had been previously coated with anti-adhension rinsing solution. After centrifugation at 800 rpm for 2 minutes using a horizontal centrifuge, the cell density per microwell was 50-75 cells (each well of the Agreewell 400™ culture dish contains 1200 microwells, i.e., the cell density per microwell was 9.0 x 10 cells). 4 / 1200=75 pieces).

[0114] On the second day of culture, half of the medium was replaced with G1ER medium, and 5 μM and 10 μM 1‰ thalidomide, dimethyl sulfoxide solution, and blank control were added, respectively. Half of the medium was replaced every day until the third day of culture.

[0115] At the end of the first stage of induction, samples were taken from the four groups and fixed in 4% paraformaldehyde for 30 min after white light recording, followed by relevant immunofluorescence staining for statistical validation.

[0116] On the third day of culture, 1.75 mL of the first-stage G1ER medium was discarded, and then the second-stage G2EE medium was slowly added along the wall to prevent the formed gastruloids from floating up from the microwell and fusing, and 5 μM and 10 μM 1‰ thalidomide, dimethyl sulfoxide solution, and a blank control were added, respectively.

[0117] On the fourth day of culture, after discarding 1.75 mL of the second-stage G2EE medium, the third-stage aG2EE medium was slowly added along the wall to prevent the formed gastruloids from floating up from the microwell and fusing, and 5 μM and 10 μM 1‰ thalidomide, dimethyl sulfoxide solution, and blank control were added, respectively.

[0118] After culturing for 5 days, 1.75 mL of the third stage aG2EE medium was discarded, and then the fourth stage bG2EE medium was slowly added along the wall to prevent the formed gastruloids from floating up from the microwell and fusing, and 5 μM and 10 μM 1‰ thalidomide, dimethyl sulfoxide solution, and blank control were added, respectively. After 24 hours, 1.75 mL of medium (bG2EE medium) was replaced and the culture was continued until the 7th day.

[0119] Upon completion of the four stages of induction, samples were taken from the four groups, and after white light recording, the cells were fixed with 4% paraformaldehyde for 30 min and nuclear staining of relevant cells was performed for statistical validation.

[0120] White light imaging of samples collected over 7 days clearly showed that the gastruloi in the drug-treated group were smaller in volume than those in the control group and lacked a cavity structure. Further marking of the nuclei with DAPI staining revealed that, compared with the control group, the gastruloi in the drug-treated group exhibited a relatively disorganized internal structure, failing to form the amniotic cavity and yolk sac, and only germ layer separation had occurred (Figure 8A).

[0121] At the same time, further statistical analysis of the cell mass and volume of single gastruloi in the control and drug-treated groups revealed that the cell mass and volume of gastruloi decreased with increasing drug concentration, with significant statistical differences compared with the blank control and dimethyl sulfoxide control groups (Figure 8B, C).

[0122] It is understood that said gastruloids, or tissues or organs derived from said models, or cultures thereof, can be used to screen, validate, evaluate, assess or study drugs for the prevention and / or treatment of disorders of early embryonic development in humans.

[0123] Although the present invention has been described to some extent, it is clear that suitable modifications to individual requirements can be made without departing from the spirit and scope of the present invention. It should be understood that the present invention is not limited to the described embodiments, but falls within the scope of the claims, including equivalents of each described element. The specification of the present invention and its drawings are for illustrative purposes, not limiting the scope of the claims. The scope of protection of the present invention is limited by the claims and their equivalents. The specification of the present invention includes multiple inventive concepts, such as "preferably," "according to a preferred embodiment," or "optionally," all of which mean that the corresponding paragraphs disclose independent concepts. The applicant reserves the right to file divisional applications based on each inventive concept. In the present specification, features preceded by "preferably" are merely optional and should not be construed as essential, and the applicant reserves the right to waive or delete related preferred features at any time.

Claims

1. 1. A method for constructing a model of a gastruloid, comprising: (1) When the pluripotent stem cells grow to 80-90% confluence, the cells are digested into single cells and resuspended using PBS to obtain cell suspension 1; Cell suspension 1 was taken, and G1ER medium containing Y-27632 was added to further resuspend the cells to obtain cell suspension 2. The cell suspension 2 is inoculated onto an embryoid body culture plate pre-coated with an anti-adhesion solution, and centrifuged. On the second day of culture, half of the medium is replaced with G1ER medium, and culture is continued with half of the medium replaced every day until primitive endoderm and primitive ectoderm are formed; Preferably, the ratio of the cell suspension 1 to the G1ER medium containing Y-27632 is 7.5 to 9.0 × 10 ４ To each cell suspension containing 100 cells, 2 mL of G1ER medium containing Y-27632 was added. Preferably, the amount of cells inoculated into each microwell of the cell suspension 2 is 50 to 75 cells; (2) discarding ½ to ¾ of the volume of the G1ER medium in each well of the culture plate in step (1), adding G2EE medium and culturing the cells to perform pre-amniotic cavity induction; (3) discarding ½ to ¾ of the volume of the G2EE medium in each well of the culture plate in step (2), adding aG2EE medium and culturing the cells to induce the formation of amniotic cells; (4) discarding 1 / 2 to 3 / 4 of the volume of the aG2EE medium in each well of the culture plate in step (3), adding bG2EE medium, and culturing until induction into amniotic cells, primitive streak cells, and primordial germ cells, thereby obtaining a post-implantation gastruloid model.

2. The method according to claim 1, wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells, preferably human induced pluripotent stem cells or human embryonic stem cells.

3. The components of the G1ER medium in step (1) are: 50 vol% G-1™ Plus Blastomere Medium, 25 vol% Essential 8 Medium, and 25 vol% RACL Medium; The RACL medium contains 95 vol% to 98.5 vol% of basal medium RPMI 1640 (containing GlutaMAX), 1 vol% of B27 supplement, 0.5 vol% of penicillin-streptomycin, 0.1 mM of non-essential amino acids, 0.1 mM of β-mercaptoethanol, 100 ng / mL of recombinant human activin A protein, 20 ng / mL of recombinant human leukocyte factor protein, and 3 μM of CHIR 99021; The components of G1ER medium containing Y-27632 are:

2. The method of claim 1, comprising 50 vol% G-1™ Plus Blastomere Medium, 25 vol% Essential 8 Medium, and 25 vol% RACL Medium, wherein the RACL Medium contains 98.5% basal medium RPMI 1640 (containing GlutaMAX), 1 vol% B27 supplement, 0.5 vol% penicillin-streptomycin, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, 100 ng / mL recombinant human activin A protein, 20 ng / mL recombinant human leukocyte factor protein, 3 μM CHIR 99021, and 10 μM Y-27632.

4. The components of the G2EE medium in step (2) are:

2. The method of claim 1, comprising 50 vol% G-2™ Plus Embryo Medium, 25 vol% Essential 8 Medium, and 25 vol% EBB Medium, wherein the EBB Medium contains 100% Essential 6 Medium, 20 ng / mL recombinant human bone morphogenetic protein 4, and 10 ng / mL recombinant human fibroblast growth factor 2.

5. The components of the aG2EE medium in step (3) are:

2. The method of claim 1, comprising 50 vol% G-2™ Plus Embryo Medium, 25 vol% Essential 8 Medium, and 25 vol% aEBB Medium, wherein the aEBB Medium contains 100% Essential 6 Medium, 100 ng / mL recombinant human bone morphogenetic protein 4, and 10 ng / mL recombinant human fibroblast growth factor 2.

6. The components of the bG2EE medium in step (4) are:

2. The method of claim 1, comprising 50 vol% G-2™ Plus Embryo Medium, 25 vol% Essential 8 Medium, and 25 vol% bEBB Medium, wherein the bEBB Medium contains 100% Essential 6 Medium, 75 ng / mL recombinant human bone morphogenetic protein 4, and 10 ng / mL recombinant human fibroblast growth factor 2.

7. A model of a gastruloid, characterized in that it is obtained by constructing it using the method of claim 1.

8. Use of the gastruloid model according to claim 7, or tissues or organs derived from said model, or cultures thereof, in the study of the mechanisms of early human embryonic development.

9. Use of the gastruloid model according to claim 7, or tissues or organs derived from said model, or cultures thereof, in diagnostic and / or therapeutic strategies for diseases of human early embryonic development.

10. Use of the gastruloid model described in claim 7, or tissues or organs derived from said model, or cultures thereof, in screening, verifying, evaluating, assessing, or researching the effectiveness of drugs for the prevention and / or treatment of human early embryonic developmental diseases, preferably drug-induced human early embryonic teratogenesis.

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