Human protointestinal-like in-vitro model based on extraembryonic cells, construction method and application

Abstract

The invention discloses a human protogut-like in-vitro model based on embryonic cells, a construction method and application, and belongs to the technical field of protogut-like model culture. In order to solve the problem that the formation process of human original stripes cannot be simulated in vitro, a completely defined co-culture system is developed, embryonic stem cells and various extraembryonic cell types are cultured together, the regulation and control processes of in-vivo amniotic ectoderm, trophoblast and extraembryonic mesoderm are simulated, and the formation process of the human original stripes can be simulated in vitro. The invention also discloses the effect of different extraembryonic cells, which are not known before, in adjusting embryonic cells. Besides, by utilizing the advantages of the micro-engineering technology, the interaction between different extraembryonic cells and embryonic cells is reconstructed in space and molecule, and it is proved that the formation process of human original stripes can be summarized only through coordinated regulation and control of the extraembryonic cells. And a convenient in-vitro research platform is provided for analysis of human early-stage protointestine formation.

Description

Technical Field

[0001] The present invention belongs to the technical field of gastrula-like model culture, and in particular relates to a human gastrula-like in vitro model based on extraembryonic cells, a construction method and an application. Background Art

[0002] Early human embryos, especially at the stage when the primitive streak structure begins to form (shortly after implantation), are almost impossible to obtain, limiting the progress of serial studies on human embryos. Therefore, human embryos cultured in vitro have provided important insights into early embryonic lineage specification. However, due to sample scarcity and ethical restrictions, studying the interactions between various cell lineages in human embryos remains challenging. Previous studies in mice have shown that extraembryonic tissues play a crucial role in axis formation, morphogenesis and lineage specification. Despite this, there are significant differences in extraembryonic tissue specification and patterning between mammalian species. The details of how cells communicate in early human embryos are not specific, and the underlying mechanisms have long been a topic of research.

[0003] The recent emergence of in vitro stem cell-derived embryonic models has provided a new avenue for studying early human embryonic development. However, because the generation of these models relies on spontaneous aggregation and differentiation, various extraembryonic cells are often induced simultaneously in a mixed form, making it difficult to independently determine the specific regulatory role of each cell type. In addition, current methods for inducing human embryonic models often require the addition of serum, chemical inhibitors, growth factors, or gene overexpression, which may introduce unpredictable variables and thus affect further investigation of lineage interactions. Summary of the Invention

[0004] In response to the above-mentioned problems, the present invention provides a human gastrula model based on extraembryonic cells, a construction method and an application. By screening culture conditions and using a fully defined F4 co-culture system (serum-free, growth factor-free, chemical inhibitor-free and feeder-free), extraembryonic lineage cells and embryonic stem cells are co-cultured to reproduce the regulatory effects of different extraembryonic lineage cells on gastrulation in vitro. By integrating micro-engineering technology, the interaction between extraembryonic cells and embryonic cells is reconstructed spatially and molecularly, proving that the coordinated regulation of extraembryonic cells alone can summarize the formation of human primitive streaks. This model enables us to analyze the initial stage of gastrulation during the black box period of human embryonic development in vitro.

[0005] In order to achieve the above objectives, this application provides the following technical solutions:

[0006] One object of the present invention is to provide a method for constructing a human gastrula-like in vitro model based on extraembryonic cells, the method comprising the following steps:

[0007] Embryonic stem cells, trophoblast stem cells, amniotic-like cells, and extraembryonic mesoderm stem cells were inoculated into Matrigel-coated culture dishes and cultured using the F4 co-culture system to construct a human gastrula-like in vitro model.

[0008] In particular, the present invention utilizes extraembryonic mesodermal stem cells, trophoblast stem cells, amnion-like cells and embryonic stem cells to assemble a human gastrula-like culture model. The idea is as follows:

[0009] Using existing cell lines from sources such as embryonic stem cells, trophoblast stem cells, and amniotic cells, an in vitro co-culture system was constructed that simulates gastrulation during early human embryonic development. By optimizing the co-culture model, the type and concentration of the extracellular matrix, the cell ratio, and the spatial arrangement, the present invention achieved efficient induction of the initial stages of gastrulation and accurate expression of key markers, providing a new experimental platform for studying the mechanisms of gastrulation.

[0010] The inventors have established a fully defined F4 co-culture system. The culture medium uses APEL exogenous factor-free basal medium (commercially available from STEMCELL Technologies), which is serum-free, growth factor-free, chemical inhibitor-free, and feed-free, and supports the differentiation of embryonic stem cells into multiple lineages. Culture plates are coated with a defined extracellular matrix (Matrigel) substitute to promote cell adhesion and growth, and then embryos and extraembryonic cells are seeded at a specified density. The F4 co-culture system is maintained under standard conditions (37°C, 5% CO2), and the medium is regularly replaced to ensure a stable environment.

[0011] When embryonic stem cells reached 80% confluence in mTeSRPLUS medium, the cells were cultured with TrypLE TM Express dissociation into single cells or use ReleSR to dissociate into small cell clumps. Resuspend the cells in mTeSR PLUS and then test the starting cell density at a seeding density of 1:10-1:40. Inoculation tests were also conducted with different concentrations of matrix gels such as Matrigel and Collagen 1. At the same time, different seeding ratios of three extraembryonic cells, namely, trophoblast stem cells, amniotic-like cells, and extraembryonic mesoderm stem cells, were compared. After the co-seeding was completed, basal culture medium without exogenous factors such as E6, APEL, and N2B27 was added for culture to screen for the gastrulation system that depends on the regulation of extraembryonic cells.

[0012] Through the above research, the present invention combines microchip technology with the systematic construction of F4 co-culture system to establish an experimental platform with clear components, strong controllability, and stable induction of gastrulation. The ideas are as follows:

[0013] Microengineering technology is used to reconstruct the relative positions of extraembryonic cells and the ectoderm in vitro. Using micropatterning, ESCs are confined to an area of uniform width in the CS6-7 human embryonic disc, approximately 700 μm, to construct an embryonic disc-like structure. Matrigel and other matrix gels are used to simulate the basement membrane secreted by the hypoblast, and the embryonic disc-like structure is surrounded by extraembryonic mesoderm stem cells and trophoblast stem cells. In addition, Transwell is used to distribute amniotic-like cells above the embryonic disc-like structure. Under F4 (no feeder layer, no chemical inhibitors, no growth factors, and no serum) culture conditions, the coordinated interactions of extraembryonic cells with embryonic cells can be spatially and molecularly summarized.

[0014] After approximately 72 hours of co-culture, a groove-like primitive streak-like structure gradually formed in the midline region of the embryonic disc. The inventors subsequently referred to this model as TEAPS (trophoblast stem cells, extraembryonic mesoderm stem cells, and amnion-like cells induced primitive streak-like structures). Consistent with the formation of the primitive streak in natural embryos, cells migrating along the groove-like region exhibited reduced cell-cell junctions and specifically expressed T and MIXL1, recognized markers of gastrulating cells.

[0015] In native embryos, a conserved feature of primitive streak formation is the establishment of the anterior-posterior (AP) axis prior to gastrulation. The primitive streak emerges exclusively in the posterior region of the embryonic disc. This process is driven by the precise regulation of signaling factors (such as WNT and NODAL) through the formation of gradients of secreted antagonists (such as DKK1 and CER1). To regulate the anterior region of the epiblast, DKK1 and CER1 proteins were added to the TEAPS model. In the presence of DKK1 or CER1, cells were T-negative, with no detectable cell migration, representing the anterior blastoderm.

[0016] Leveraging the standardized nature of microengineering techniques, the TEAPS model can be robustly established, enabling detailed investigation of the process of human primitive streak formation. Immunofluorescence staining was performed on TEAPS at different stages of formation. Results showed that at 24 hours, N-cadherin, an early epithelial-mesenchymal transition (EMT) marker, began to be expressed, while another marker, SLUG, remained in the cytoplasm, with nearly undetectable T signal. By 36 hours, cell migration had begun, T expression was detectable at the edge of the forming primitive streak-like structure, and SLUG began to enter the nucleus. By 48 hours, cell migration was more pronounced, with SLUG prominently localized in the nucleus. T was detected not only within the primitive streak-like structure but also in some cells migrating toward the edge of the embryonic disc. Overall, the TEAPS model recapitulates the formation of the human primitive streak through the spatial and molecular coordination of extraembryonic cells.

[0017] The specific steps are as follows:

[0018] Fabrication of PDMS stamp micropatterns;

[0019] Transfer the Matrigel-coated PDMS stamp micropattern to a culture dish;

[0020] Using TrypLE TM Express dissociated embryonic stem cells and cultured at 3 × 10 5 cells / cm 2 The density of cells was seeded into the circular area of the PDMS stamp micropattern with a diameter of 700 μm to cultivate a simulated embryonic disc-like structure;

[0021] Extraembryonic mesodermal stem cells and trophoblast stem cells were seeded around the periphery of the embryonic disc structure. The density of the seeding was 3×10 5 cells / cm 2 ;

[0022] Amniotic cells were collected at a rate of 3×10 5 cells / cm 2 The density of cells was seeded on the top area of the Transwell membrane, which was placed above the culture dish and on top of the embryonic disc structure to achieve spatial positioning of the cells inside and outside the embryo;

[0023] The cells were cultured in an F4 co-culture system at 37°C and 5% CO2 for 72 hours, and the F4 co-culture system was replaced once a day.

[0024] In particular, the quantitative ratio of extraembryonic mesoderm stem cells, trophoblast stem cells, amniotic cells and embryonic stem cells during the construction of the human gastrula-like in vitro model was 1:1:1:1.

[0025] In particular, the preparation process of PDMS stamp micropatterns is as follows:

[0026] (1) The stamp was designed by AutoCAD, and then the CAD file was imported into Fusion360 software to generate a CNC machine program, which was transferred to a micro CNC milling machine and milled into a plastic block;

[0027] (2) Prepare a PDMS stamp by mixing the PDMS curing agent with the base polymer in a ratio of 1:10;

[0028] (3) The mixture was poured onto a mold and vacuumed, then the mold was baked at 66 °C for 4 h and the PDMS stamp was peeled off from the mold.

[0029] The second object of the present invention is to provide a human gastrula-like in vitro model based on extraembryonic cells, which is obtained using the above-mentioned construction method.

[0030] A third objective of the present invention is to provide the application of the human gastrula model constructed above for in vitro analysis of the initial stages of gastrulation. This invention constructs a highly physiologically relevant in vitro gastrula model focused on human gastrulation and early body axis establishment, focusing on the spatial and signaling regulatory role of extraembryonic lineages in this process. This model can simulate the key developmental events of anterior-posterior axis establishment and primitive streak formation in natural embryos, exhibiting embryo-like spatial structure and molecular expression characteristics.

[0031] Studies have shown that gastrulation-related cells in this model can spontaneously generate in the absence of exogenous inducing factors and exhibit marker gene expression consistent with in vivo embryonic development, confirming the important role of extraembryonic lineages in embryonic stem cell fate transformation and the initiation of gastrulation. Further analysis confirmed that this model not only reconstructs the patterning process of early body axis formation, but also achieves the coordinated differentiation of multiple lineage cells, including neural, mesoderm, endoderm, and myocardium.

[0032] This study highlights the potential of gastrula-like models for simulating the spatiotemporal patterns of early embryonic development, elucidating extraembryonic cell signaling, and reconstructing functional tissue architecture, filling the gap in experimental systems for the "black box" stages of human development. The constructed system provides a new in vitro platform for studying the mechanisms of gastrulation, organogenesis, and modeling related developmental diseases, with significant theoretical and practical value.

[0033] A fourth objective of the present invention is to provide an extraembryonic cell-based human gastrula-like in vitro model for studying the in vitro differentiation of multi-lineage cells, including neural, mesodermal, endodermal, and cardiac lineages. The differentiated structures exhibit specific expression of markers such as SOX2, SOX17, PAX6, TBX6, GATA4, KRT8, ISL1, and cTnT, and can form a cardiac-like cavity-like structure with rhythmic contraction.

[0034] Definition of terms:

[0035] The gastrula referred to in the present invention refers to an embryonic model of the gastrula constructed in vitro.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] (1) The present invention uses microchip patterning technology to accurately simulate the spatial relationship between the embryonic ectoderm and extraembryonic lineage cells, and constructs a three-dimensional gastrula-like structure with the characteristics of the anterior-posterior axis and the dorso-ventral axis. This breaks through the limitations of the traditional gastrula-like model, which lacks extraembryonic tissue participation and spatial structure control, and improves the model's biomimetic properties and developmental restoration.

[0038] (2) The present invention clarifies the key role of extraembryonic lineage cells in the induction of gastrulation, and utilizes their synergistic signaling effect on embryonic stem cells without exogenous induction conditions to induce the differentiation of multiple lineages such as neural, endoderm and myocardium, and form tissue structures with functional characteristics (such as beating myocardium-like structures), significantly enhancing the multi-lineage functional expression ability and research application value of the model. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1A An exemplary diagram of a gastrula-like model designed based on embryonic structure is shown.

[0040] Figure 1B The process of constructing a gastrula-like model based on microchip co-culture technology and the cell growth morphology diagram are demonstrated exemplarily.

[0041] Figure 2 The primitive streak structure appearing in the TEAPS gastrula model and the corresponding staining analysis and identification are shown as examples.

[0042] Figure 3 Schematic diagram showing the cell regulation of the TEAPS model.

[0043] Figure 4A A schematic diagram of the subsequent extended culture of the TEAPS model in a 3D environment is shown as an example.

[0044] Figure 4B The test results of the TEAPS model after 3D development are exemplified, showing multiple cell lineages such as neural and mesoderm.

[0045] Figure 4C We exemplify the overshoot of the TEAPS model during prolonged 3D culture, demonstrating the emergence of cardiomyocyte populations and endoderm lineages. DETAILED DESCRIPTION

[0046] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0047] Example 1

[0048] This example mainly introduces a gastrulation-like system for co-culturing extraembryonic lineages and embryonic stem cells, including the following parts:

[0049] like Figure 1AThe present invention adopts a step-by-step inoculation co-culture strategy. First, human embryonic stem cells are inoculated on a culture matrix containing different concentrations of matrix gel components (including Matrigel, Collagen 1, etc.) to form a sheet-like adherent growth state. Subsequently, extraembryonic cells such as trophoblast stem cells and amniotic cells are added to simulate the spatial distribution and signal transduction relationship between different lineage cells in the early stages of embryonic development. This sequential inoculation mode helps embryonic stem cells establish a stable growth structure and reduces the interference of extraembryonic cells on their early adherence and proliferation states. At the same time, the addition of different types of extraembryonic cells can respectively exert the inductive function of the in vivo signal center, promoting the transformation of intraembryonic cells to a fate related to gastrulation.

[0050] In terms of matrix condition screening, the present invention systematically compared the effects of different types and concentrations of Matrigel and Collagen 1 on the cell state and induction effect in the co-culture system. The results showed that Matrigel of appropriate concentration supported the sheet-like distribution of embryonic stem cells while also allowing extraembryonic cells to attach, thereby forming a three-dimensional microenvironment that was closer to the in vivo developmental structure. In terms of cell ratio, the present invention set up multiple groups of co-culture experiments to compare the inoculation density and ratio between different embryonic stem cells and extraembryonic cells, and found that the appropriate intraembryonic: extraembryonic ratio can significantly increase the frequency of occurrence of gastrulation-related cell populations. Too high an extraembryonic cell ratio may cause embryonic stem cells to be inhibited or differentiated chaotically, while if the ratio is too low, the induction effect is not obvious. Therefore, reasonable control of cell ratio and inoculation time is a key parameter for constructing a gastrulation-like model.

[0051] Finally, after a series of tests, the inventors found that embryonic stem cells with a growth density of 80% were seeded at a density of 1:40 on a 12-well cell culture plate covered with 0.5% Matrigel. After culturing at 37°C for 24 hours, relatively stable cell clones could be obtained. Then, TrypLE TM Express dissociated three types of extraembryonic cells: blastoderm stem cells, amnion-like cells, and extraembryonic mesoderm stem cells, and inoculated them into wells around embryonic stem cells at a ratio of 1:1:1:1. Amnion cells were inoculated in Transwells and placed above embryonic stem cells. APEL was then used for co-culture, and Y-2763 was added for 24 hours. The cells were maintained in APEL medium (fully defined, i.e., F4 co-culture system) for 1-3 days, and the formation of primitive streaks in vitro was observed.

[0052] Immunofluorescence staining and other methods were used to evaluate the induced co-culture system. The results demonstrated that, without the need for exogenous growth factors, inhibitors, or serum, embryonic stem cells were able to effectively initiate gastrulation under the influence of extraembryonic cells. Typical gastrulation marker genes, including TBXT (Brachyury), MIXL1, and CDX2, were expressed in the co-culture system, with their expression patterns displaying temporal and spatial patterns consistent with in vivo developmental stages.

[0053] Example 2

[0054] This example mainly introduces a method of constructing an in vitro culture model of gastrula-like cells using a microchip and the culture conditions optimized in Example 1. Figure 1B ,include:

[0055] The mold for the microchip stamp was made by a micro CNC milling machine 97. The stamp was designed in AutoCAD, and the CAD file was then imported into Fusion 360 software to generate a CNC machine program. The program was transferred to the micro CNC milling machine and a plastic block was milled. A polydimethylsiloxane (PDMS)-based stamp was then prepared by mixing a PDMS curing agent with a base polymer in a ratio of 1:10. The mixture was then cast onto the mold and vacuumed, and the mold was then baked at 66°C for 4 hours. The PDMS stamp was then peeled off from the mold for subsequent experiments. The PDMS stamp with the accurate pattern was immersed in 1% Matrigel for 30 minutes at room temperature. Next, it was treated with UV ozone for 45 seconds. The Matrigel-coated PDMS stamp was then dried under nitrogen and pressed into a UV ozone-treated cell culture dish for 1 minute to transfer the PDMS stamp micropattern to the dish with Matrigel adhesive.

[0056] Using TrypLE TM Express dissociated embryonic stem cells and cultured at 4 × 10 5 cells / cm 2 The cells were reseeded at a density of 100 μg / mL in mTeSR PLUS medium containing Y-27632 on the prepared Matrigel-coated PDMS stamp micropattern. After 10 minutes, the wells were washed with PBS to remove unattached cells and refilled with mTeSR PLUS medium. After 24 hours, the medium was replaced with mTeSR PLUS containing 1% Matrigel for 1 hour to coat the gaps within the wells. Then, a mixture of extraembryonic mesoderm stem cells and trophoblast stem cells was added at a ratio of 1:1 at a rate of 3 × 10 5 cells / cm 2 The density of 10 × 10 cells was seeded into the wells, while amniotic cells were seeded at a density of 3 × 10 5 cells / cm 2After all cells in the TEAPS model were inoculated, the culture medium was changed to APEL medium. The culture medium was changed once a day, and the cells were incubated at 37°C and 5% CO2 to observe the formation process of the primitive streak. The inventors analyzed the expression of primitive streak characteristic genes such as TBXT (Brachyury), MIXL1 and CDX2 by immunofluorescence staining, and confirmed the establishment of the microchip-based gastrula model. Figure 2 shown.

[0057] Example 3

[0058] This example mainly introduces the application of the human gastrula model constructed above in in vitro analysis of the initial stage of gastrulation, including the following parts:

[0059] The gastrula model constructed in the present invention is consistent with the formation of the primitive streak in natural embryos. Cells migrating along the furrow-shaped area show reduced cell connections and specifically express T and MIXL1, which are recognized markers of gastrulation cells. In natural embryos, a conserved feature of primitive streak formation is the establishment of the anterior-posterior (AP) axis before gastrulation. The primitive streak appears only in the posterior region of the embryonic disc-like structure. This process is driven by the precise regulation of the gradient formed by signaling factors (such as WNT and NODAL) through the secretion of antagonists (such as DKK1 and CER1). Figure 3 To regulate the anterior region of the epiblast, DKK1 and CER1 proteins were added to the TEAPS model (gastruloid model). In the presence of DKK1 or CER1, cells were T-negative and no cell migration was detected, representing the anterior blastoderm.

[0060] Immunofluorescence staining was performed on TEAPS at different stages of formation. The results showed that at 24 hours, the early EMT marker protein N-cadherin began to be expressed, while another marker protein SLUG remained in the cytoplasm, and the T signal was almost undetectable. By 36 hours, cell migration had begun, T expression could be detected at the edge of the formed primitive streak-like structure, and SLUG began to enter the cell nucleus. By 48 hours, cell migration was more obvious, and SLUG was clearly located in the cell nucleus. T could be detected not only in the primitive streak-like structure, but also in some cells migrating to the edge of the embryonic disc-like structure. In summary, the TEAPS model can recapitulate the formation of the human primitive streak through the spatial and molecular coordination of extraembryonic cells.

[0061] In addition, if Figure 4A 、 4B, 4C, the present invention tested the developmental potential of TEAPS. By isolating the blastoderm-like structure formed in TEAPS and transferring it to a low-adhesion 96-well plate, it was continued to be cultured under basic culture conditions to induce it to form a three-dimensional tissue structure with axial and multi-lineage differentiation characteristics. During the extended culture process, most of the structures spontaneously developed into elongated, curved, asymmetric and multi-layered complex tissue configurations on the 4th day. In these structures, SOX2-positive epithelial-like cells were mainly distributed in the outer layer, SOX17-positive cells formed an inner tubular structure, and neural mesodermal progenitor cells co-expressing SOX2 and T were detected at one end. The tail marker CDX2 showed obvious asymmetric expression, suggesting that the body axis direction has been established. Immunofluorescence results showed that the outer layer SOX2+ cells co-expressed the neural marker PAX6, while the inner cells expressed the mesoderm marker TBX6, simulating the anterior-posterior axis and dorsal-ventral axis characteristics of the early embryo. In addition, neuronal-like cells express markers such as SOX1, PAX3, and PAX7, while SOX17+ cells co-express endoderm and intestinal markers GATA4 and KRT8. More importantly, the anterior region of some structures forms a lumen-like region composed of cardiac progenitor cells that co-express ISL1 and cTnT, and exhibits rhythmic contractions. This invention can reconstruct gastrulation-like structures in vitro without the intervention of exogenous factors, providing a highly physiologically relevant model platform for studying early human embryonic development, body axis establishment, and the origin of the cardiointestinal system.

[0062] It can be understood that the above specific description of the present invention is only used to illustrate the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents to achieve the same technical effects; as long as the use requirements are met, they are within the scope of protection of the present invention.

Claims

1. A method for constructing a human gastrula-like in vitro model based on extraembryonic cells, characterized in that: The construction method includes: Human embryonic stem cells, trophoblast stem cells, amniotic-like cells, and extraembryonic mesoderm stem cells were inoculated into Matrigel-coated culture dishes and cultured using the F4 co-culture system to construct a human gastrula-like in vitro model.

2. The method for constructing a human gastrula-like in vitro model based on extraembryonic cells according to claim 1, characterized in that: The specific process of constructing the human gastrula-like in vitro model is as follows: Transfer the Matrigel-coated PDMS stamp micropattern to a culture dish; Using TrypLE TM Express dissociated embryonic stem cells and seeded them into the circular area of a 700 μm diameter PDMS stamp micropattern to cultivate embryonic disc-like structures; Extraembryonic mesodermal stem cells and trophoblast stem cells are seeded around the periphery of the embryonic disc-like structure; Amnion-like cells were seeded on the top area of the Transwell membrane, which was placed above the culture dish and on top of the embryonic disc structure to achieve spatial positioning of the intra-embryonic and extra-embryonic cells; The cells were cultured in an F4 co-culture system at 37°C and 5% CO2 for 72 hours, and the F4 co-culture system was replaced once a day.

3. The method for constructing a human gastrula-like in vitro model based on extraembryonic cells according to claim 2, characterized in that: The seeding density of embryonic stem cells was 3×10 5 cells / cm 2 .

4. The method for constructing a human gastrula-like in vitro model based on extraembryonic cells according to claim 2, characterized in that: The density of the mixture of extraembryonic mesoderm stem cells and trophoblast stem cells was 3×10 5 cells / cm 2 .

5. The method for constructing a human gastrula-like in vitro model based on extraembryonic cells according to claim 2, characterized in that: The seeding density of amniotic cells was 3×10 5 cells / cm 2 .

6. The method for constructing a human gastrula-like in vitro model based on extraembryonic cells according to claim 2, characterized in that: During the construction of the human gastrula-like in vitro model, the quantitative ratio of extraembryonic mesoderm stem cells, trophoblast stem cells, amniotic membrane-like cells and embryonic stem cells was 1:1:1:

1.

7. A human gastrula-like in vitro model based on extraembryonic cells, characterized in that: The method for constructing a human gastrula-like in vitro model based on extraembryonic cells is used as described in any one of claims 1 to 6.

8. Use of the extraembryonic cell-based human gastrula in vitro model according to claim 7 in in vitro analysis of simulating primitive streak formation.

9. The use according to claim 8, characterized in that After 72 hours of culture, a groove-shaped primitive streak-like structure was formed in the central area of the human gastrula in vitro model. The central area showed reduced cell connections in the embryonic disc-like structure and specific expression of T and MIXL1.

10. Use of the extraembryonic cell-based human gastrula-like in vitro model according to claim 7 in studying the in vitro differentiation process of multi-lineage cells.

11. The use according to claim 10, characterized in that Multilineage cells include neural, mesodermal, endodermal and cardiac lineages.

12. The use according to claim 10, characterized in that Differentiation is manifested by the specific expression of SOX2, SOX17, PAX6, TBX6, GATA4, KRT8, ISL1 and cTnT markers, and the formation of myocardial-like cavity-like structures with rhythmic contraction ability.

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