Methods for constructing and using gastruloid stem cell lines

Abstract

The present invention relates to a method for constructing and using a gastruloid stem cell line. This study established stably passaged gastruloid stem cells. The cells retain some stem cell potential and express genes and proteins of the three germ layers (endodermal, mesodermal, and ectoderm) during gastrula development. Their characteristics are consistent with those of gastrula-stage cells, closely reproducing important features of gastrula-stage cells. Using this stem cell line, a three-dimensional gastruloid model can be constructed that can simulate gastrula development in vivo or in vitro. Important biological events during in vivo embryonic development, such as the separation of the endodermal and ectoderm lineages, the formation of the pre-amniotic cavity, the development of the primitive streak, and the specification of the mesoderm lineage, can be partially reproduced, closely reproducing important features of the gastrula-stage embryo after implantation. This model can be used to establish an in vitro drug screening platform that affects early embryonic development, providing reference for clinical drug use.

Description

[Technical Field]

[0001] The present invention belongs to the field of biotechnology and relates to a method for constructing and using a gastruloid stem cell line, specifically to a method for constructing a gastruloid stem cell line, a gastruloid stem cell line obtained by said method, and the construction and application of a model derived from said cell line. [Background technology]

[0002] During human embryonic development, the fertilized egg develops into tissues through cell division, proliferation, and specialization. organ These cells form a complex, individual organism. The first three weeks of embryonic development after fertilization are a crucial stage of human embryogenesis, particularly from the peri-implantation stage through the gastrula stage, during which cells undergo lineage specialization and rearrangement to form embryoid bodies with primordia. Problems with development during this stage can lead to miscarriage and birth defects. Understanding the mechanisms underlying early human development is crucial for both developmental biology and regenerative medicine. Due to technical and ethical constraints and limited sample sizes, research into early human embryonic development remains one-sided. Currently, much of our understanding of early human embryonic development comes from studies of the histology and anatomy of the Carnegie embryo. There are many unknowns about this process that remain to be explored.

[0003] Currently, groundbreaking advances have been made in the study of pre- and peri-implantation human embryonic development. Researchers are now able to culture human embryos in vitro up to embryonic day 14, before gastrulation, and induce human pluripotent stem cells (hPSCs) into pre-implantation blastocyst-like embryos. The combination of single-cell multi-omics sequencing and fluorescence imaging techniques has opened new avenues for human embryonic development research, greatly expanding our understanding of the characteristics and mechanisms of human embryonic development from implantation to pre-gastrulation.

[0004] Research has attempted to construct three-dimensional gastruloid models using hPSCs to simulate the mutually exclusive separation of cells of the three germ layers during gastrula development. However, the models lack essential embryonic structures (two blastodiscs, amniotic cavity, and yolk sac), and differentiation of neural cell lineages has not been observed. Therefore, an ideal gastruloid research model has not yet been established.

[0005] Therefore, there is an urgent need for a method to construct stably passaged gastruloid stem cells that can maintain stem cell pluripotency, express genes and proteins of the three germ layers of endoderm, mesoderm, and ectoderm cells at the gastrula development stage, obtain gastruloid stem cells with characteristics consistent with gastrula-stage cells, closely recapitulate important characteristics of gastrula-stage cells, and provide an ideal model for research on human embryos. Summary of the Invention

[0006] This study solved the above-mentioned technical problems in the prior art by establishing stably passaged gastruloid stem cells that retain a certain degree of stem cell pluripotency, express genes and proteins of the three germ layers of endoderm, mesoderm, and ectoderm during gastrula development, and have characteristics consistent with those of gastrula-stage embryonic cells, closely reproducing the important characteristics of gastrula-stage cells.

[0007] By inducing differentiation of these gastruloid stem cells, a three-dimensional gastruloid model capable of simulating gastrula development can be constructed in vitro or in vivo. This model partially reproduces key biological events during in vivo embryonic development, such as the separation of the endodermal and ectodermal lineages, the formation of the pre-amniotic cavity, the development of the primitive streak, and the specialization of the mesodermal lineage. Using single-cell multi-omics sequencing and fluorescence imaging techniques, these key biological events can be verified at both the protein and transcriptome levels, effectively reproducing key features of the postimplantation gastrula stage embryo. This model can be used to establish an in vitro drug screening platform that affects early embryonic development, providing reference for clinical drug use.

[0008] Furthermore, by inducing differentiation of these gastruloid stem cells, tissues derived from the three germ layers (ectoderm, mesoderm, and endoderm) such as neuroepithelium, smooth muscle, and intestinal tract can be produced. organ A primordium model can be formed.

[0009] A first object of the present invention is to provide a method for constructing a gastruloid stem cell line.

[0010] The construction method includes the following steps:

[0011] (1) Induction of stem cells to mesoderm: (1-1) Stem cells are digested into single cells, centrifuged, and resuspended to obtain cell suspension 1. The stem cells are human embryonic stem cells or human induced pluripotent stem cells, and human embryonic stem cells are established human embryonic stem cells derived from an embryo within 14 days of fertilization that has not undergone in vivo development.

[0012] (1-2) Cell suspension 1 is taken and centrifuged, the supernatant is discarded, and GK15-1 culture medium containing a ROCK inhibitor is added to further resuspend the cells, thereby obtaining cell suspension 2.

[0013] Preferably, the ratio of cell suspension 2 to the GK15-1 culture medium containing a ROCK inhibitor in step (1-2) is 1 × 10 per mL of GK15-1 culture medium containing a ROCK inhibitor. 6 It contains cells.

[0014] (1-3) The cell suspension 2 obtained in (1-2) is inoculated into a well plate pre-coated with Matrigel and cultured.

[0015] Preferably, the inoculation in step (1-3) is carried out by inoculating the cell suspension 2 at a density of 0.6 to 1 × 10 per square centimeter. 5 Inoculate at a density of cells.

[0016] Preferably, the culture conditions in step (1-3) are 37° C. and a volume concentration of carbon dioxide of 5.0 to 5.2%, more preferably 5.0%.

[0017] (1-4) On the second day of culture, the old medium is removed and replaced with GK15-1 medium, and this is continued daily until new mesoderm-like cells are obtained.

[0018] (2) Induction of newborn mesoderm-like cells toward primordial germ cell-like cells: (2-1) When the nascent mesoderm-like cells obtained in (1-4) grow to 60-90% confluence, digest the cells into single cells, centrifuge, and resuspend the cells to obtain cell suspension 3.

[0019] (2-2) Cell suspension 3 is taken and centrifuged, the supernatant is discarded, and then GK15-2 culture medium containing a ROCK inhibitor is added to further resuspend the cells, thereby obtaining cell suspension 4.

[0020] Preferably, the ratio of cell suspension 3 to the GK15-2 culture medium containing a ROCK inhibitor in step (2-2) is 1 × 10 per mL of GK15-2 culture medium containing a ROCK inhibitor. 5 It contains cells.

[0021] (2-3) The cell suspension 4 obtained in (2-2) is inoculated into a low-adhesion well plate for spheroid culture. From the second day of culture, remove the old medium and replace it with GK15-2 culture medium every day until cell spheroids containing primordial germ cell-like cells are obtained.

[0022] Preferably, the initial cell amount per well for spheroid culture in step (2-3) is 0.5 to 1 × 10 4 Each cell is an individual cell.

[0023] (3) Cell purification: (3-1) Digest the cell spheroids obtained in (2-3) into single cells and resuspend the cells using GK15-2 culture medium to obtain cell suspension 5.

[0024] (3-2) CD326 and CD49f double positive cells are selected from the cell suspension 5 obtained in (3-1), and the cells are resuspended by adding GK10 culture medium containing a ROCK inhibitor to obtain a cell suspension 6.

[0025] Preferably, 1 x 10 cells per mL of GK10 culture medium containing a ROCK inhibitor in step (3-2) 5 Contains double positive cells.

[0026] (4) Cell amplification: The cell suspension 6 obtained in (3-2) is inoculated into a well plate pre-plated with mitomycin C-treated mouse embryonic fibroblasts as feeder layer cells, and after 24 hours of culture, the medium is replaced with fresh GK10 medium, which is replaced daily, to obtain gastruloid stem cell lines.

[0027] Preferably, the inoculation in step (4) is carried out by inoculating the cell suspension 6 at a density of 0.4 to 2 × 10 per square centimeter. 4 The cells are inoculated at a density of 1000 cells / well.

[0028] Preferably, the culture conditions in step (4) are 37°C and a volume concentration of carbon dioxide of 5.0%.

[0029] Furthermore, the components of the GK15-1 culture medium containing the ROCK inhibitor in (1-2) are: The medium contains 80-85% GMEM basal medium (volume percentage), 10-15% KOSR serum replacement (volume percentage), 1% penicillin-streptomycin double antibody (volume percentage), 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX additive (volume percentage), 1% 100 mM sodium pyruvate additive (volume percentage), 0.1 mM β-mercaptoethanol, 25-200 ng / mL recombinant human activin A factor, 1-10 μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021, and 5-20 μM ROCK inhibitor.

[0030] In a specific embodiment, the components of the GK15-1 culture medium containing a ROCK inhibitor in (1-2) are The medium contained 81% by volume of basal medium GMEM, 15% by volume of serum replacement KOSR, 1% by volume of penicillin-streptomycin double antibody, 1% by volume of 10 mM non-essential amino acids, 1% by volume of 200 mM GlutaMAX additive, 1% by volume of 100 mM sodium pyruvate additive, 0.1 mM β-mercaptoethanol, 50 ng / mL recombinant human activin A factor, 3 μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021, and 10 μM ROCK inhibitor.

[0031] Furthermore, the components of the GK15-1 culture medium in (1-4) are: The medium contains 80-85% GMEM basal medium (volume percentage), 10-15% KOSR serum replacement (volume percentage), 1% penicillin-streptomycin double antibody (volume percentage), 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX additive (volume percentage), 1% 100 mM sodium pyruvate additive (volume percentage), 0.1 mM β-mercaptoethanol, 25-200 ng / mL recombinant human activin A factor, and 1-10 μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021.

[0032] In a specific embodiment, the components of the GK15-1 culture medium in (1-4) are: The medium contained 81% by volume of basal medium GMEM, 15% by volume of serum replacement KOSR, 1% by volume of penicillin-streptomycin double antibody, 1% by volume of 10 mM non-essential amino acids, 1% by volume of 200 mM GlutaMAX additive, 1% by volume of 100 mM sodium pyruvate additive, 0.1 mM β-mercaptoethanol, 50 ng / mL recombinant human activin A factor, and 3 μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021.

[0033] Furthermore, the components of the GK15-2 culture medium containing the ROCK inhibitor in (2-2) are: The medium contains 80-85% GMEM basal medium (volume percentage), 10-15% KOSR serum replacement (volume percentage), 1% penicillin-streptomycin double antibody (volume percentage), 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX supplement (volume percentage), 1% 100 mM sodium pyruvate supplement (volume percentage), 0.1 mM β-mercaptoethanol, 100-500 ng / mL recombinant human bone morphogenetic protein 4, 50-200 ng / mL recombinant human stem cell factor, 1000-5000 U / mL recombinant human leukemia inhibitory factor, 50-250 ng / mL recombinant human epidermal growth factor, and 5-20 μM ROCK inhibitor.

[0034] In a specific embodiment, the components of the GK15-2 culture medium containing a ROCK inhibitor in (2-2) are The medium contains 81% GMEM basal medium (volume percentage), 15% KOSR serum replacement (volume percentage), 1% penicillin-streptomycin double antibody (volume percentage), 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX supplement (volume percentage), 1% 100 mM sodium pyruvate supplement (volume percentage), 0.1 mM β-mercaptoethanol, 200 ng / mL recombinant human bone morphogenetic protein 4, 100 ng / mL recombinant human stem cell factor, 1000 U / mL recombinant human leukemia inhibitory factor, 50 ng / mL recombinant human epidermal growth factor, and 10 μM ROCK inhibitor.

[0035] Furthermore, the components of the GK15-2 culture medium in (2-3) and (3-1) are: The medium contains 80-85% GMEM basal medium (volume percentage), 10-15% KOSR serum replacement (volume percentage), 1% penicillin-streptomycin double antibody (volume percentage), 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX supplement (volume percentage), 1% 100 mM sodium pyruvate supplement (volume percentage), 0.1 mM β-mercaptoethanol, 100-500 ng / mL recombinant human bone morphogenetic protein 4, 50-200 ng / mL recombinant human stem cell factor, 1000-5000 U / mL recombinant human leukemia inhibitory factor, and 50-250 ng / mL recombinant human epidermal growth factor.

[0036] In a specific embodiment, the components of the GK15-2 culture medium in (2-3) and (3-1) are: The medium contains 81% GMEM basal medium (volume percentage), 15% KOSR serum replacement (volume percentage), 1% penicillin-streptomycin double antibody (volume percentage), 10 mM non-essential amino acids (volume percentage), 200 mM GlutaMAX supplement (volume percentage), 1% 100 mM sodium pyruvate supplement (volume percentage), 0.1 mM β-mercaptoethanol, 200 ng / mL recombinant human bone morphogenetic protein 4, 100 ng / mL recombinant human stem cell factor, 1000 U / mL recombinant human leukemia inhibitory factor, and 50 ng / mL recombinant human epidermal growth factor.

[0037] Furthermore, the components of the GK10 culture medium containing the ROCK inhibitor in (3-2) are: The medium contains 80-85% GMEM basal medium (volume percentage), 10% KOSR serum replacement (volume percentage), 2.5% FBS (fetal bovine serum), 1% penicillin-streptomycin double antibody (volume percentage), 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX additive (volume percentage), 1% 100 mM sodium pyruvate additive (volume percentage), 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, 50-200 ng / mL recombinant human stem cell factor, and 5-20 μM ROCK inhibitor.

[0038] In a specific embodiment, the components of the GK10 culture medium containing a ROCK inhibitor in (3-2) are The medium contained 83.5% GMEM basal medium (volume percentage), 10% KOSR serum replacement (volume percentage), 2.5% FBS (fetal bovine serum), 1% penicillin-streptomycin double antibody (volume percentage), 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX additive (volume percentage), 1% 100 mM sodium pyruvate additive (volume percentage), 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, 100 ng / mL recombinant human stem cell factor, and 10 μM ROCK inhibitor.

[0039] Furthermore, the components of the GK10 culture medium in (4) are: The medium contains 80-85% GMEM basal medium (volume percentage), 10% KOSR serum replacement (volume percentage), 2.5% FBS (fetal bovine serum), 1% penicillin-streptomycin double antibody (volume percentage), 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX additive (volume percentage), 1% 100 mM sodium pyruvate additive (volume percentage), 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, and 50-200 ng / mL recombinant human stem cell factor (HRF).

[0040] In a specific embodiment, the components of the GK10 culture medium in (4) are: The medium contains 83.5% GMEM basal medium (volume percentage), 10% KOSR serum replacement (volume percentage), 2.5% FBS (fetal bovine serum), 1% penicillin-streptomycin double antibody (volume percentage), 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX additive (volume percentage), 1% 100 mM sodium pyruvate additive (volume percentage), 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, and 100 ng / mL recombinant human stem cell factor (HRF).

[0041] A second object of the present invention is to provide a gastruloid stem cell line constructed using the above method.

[0042] Additionally, a gastruloid stem cell line designated human gastruloid-like stem cell line CCRM-hGOSC-1, with accession number CCTCC NO. C2022114 and date of deposit April 27, 2022, has been deposited at the China Center for Typical Cultures Depositary. The gastruloid stem cell line was constructed using human embryonic stem cells.

[0043] Additionally, a gastruloid stem cell line designated human gastruloid-like stem cell line DYR0100-hGOSC-1, with accession number CCTCC NO. C2022115 and date of deposit April 27, 2022, has been deposited at the China Center for Typical Cultures Depositary. The gastruloid stem cell line was constructed using human induced pluripotent stem cells.

[0044] A third object of the present invention is to provide a use of the gastruloid stem cell line in constructing a gastruloid model.

[0045] A fourth object of the present invention is to provide a gastruloid model obtained by inducing differentiation using a gastruloid stem cell line.

[0046] A fifth object of the present invention is to provide a method for constructing a gastruloid model by inducing differentiation of a gastruloid stem cell line, where the differentiation induction can be in vivo or in vitro.

[0047] Furthermore, in vivo differentiation induction is The method includes the steps of obtaining a gastruloid stem cell line, resuspending it in GK10 culture medium, injecting the cell suspension into the testis of a mouse, and culturing it for 10 to 20 days to obtain a gastruloid model. Preferably, mouse are immunodeficient mice, More preferably, mouse is a BALB / c nude mouse.

[0048] Furthermore, the injection is carried out by injecting a cell suspension into the testes of the mouse via the vas deferens of the mouse.

[0049] Furthermore, the injection dose was 2 to 8 × 10 4 cells are injected.

[0050] Furthermore, in vitro differentiation induction (1) When gastruloid stem cells grew to 60-90% confluence, the cells were digested into single cells and resuspended in GK10 medium to obtain cell suspension 7. CD326 and CD49f double-positive cells in cell suspension 7 were selected and centrifuged, and 1.5-1.75 × 10 cells were cultured in mTR medium. 4 Resuspend the cells at a concentration of 6.0-7.0 x 10 cells / mL per well in a low-adhesion well plate. 3 seeding the cells and culturing them until the gastruloid stem cells are organized into a three-dimensional structure; (2) Without discarding the mTR culture medium in (1), E6BIN medium is added, and the cells are cultured until the amniotic cavity is formed and the specification of the mesendoderm lineage is complete, thereby obtaining a gastruloid model.

[0051] Preferably, the amount of E6BIN medium added is 50 to 200 μL / well.

[0052] Preferably, the culture conditions in steps (1) and (2) are 37°C and a volume concentration of carbon dioxide of 5.0%.

[0053] Furthermore, the components of the mTR culture medium in step (1) were 99% mTeSR by volume. TM 1 Complete medium, 1% penicillin-streptomycin double antibody by volume, 5-20 μM ROCK inhibitor.

[0054] In one particular embodiment, the components of the mTR culture medium in step (1) are 99% by volume of mTeSR. TM 1 complete medium, 1% penicillin-streptomycin double antibody by volume, and 10 μM ROCK inhibitor.

[0055] Furthermore, the components of the E6BIN medium in step (2) include, by volume, 100% Essential 6 medium, 20 ng / mL recombinant human fibroblast growth factor 2, 50 ng / mL recombinant human noggin protein, and 5 μM IWP-2.

[0056] Sixth Object of the Invention is a gastruloid The model was obtained by injecting a gastruloid stem cell line into the testes of animals and culturing them. An organ primordium model, the organ primordium comprising: Tissues derived from three germ layers: ectoderm, mesoderm, and endoderm organ primordium and Preferably, the culture period is 30 to 90 days. , providing an organ primordium model .

[0057] The seventh object of the present invention is to provide a method for producing a gastruloid stem cell line, comprising the steps of: obtaining a gastruloid stem cell line, resuspending it in GK10 culture medium, injecting the cell suspension into the testis of a mouse, and culturing the cell suspension for 30 to 90 days to obtain an organ primordium model; Preferably, mouse are immunodeficient mice, More preferably, mouse The present invention aims to provide a method for constructing organ primordium models in BALB / c nude mice.

[0058] Furthermore, the injection is carried out by injecting a cell suspension into the testes of the mouse via the vas deferens of the mouse.

[0059] In addition, 2 to 8 × 10 4 cells are injected.

[0060] Furthermore, after 30 to 40 days of culture, a neuroectoderm model, and / or a primordial germ cell model, and / or an amniotic epithelial cell model is obtained. After 40 to 50 days of culture, a neuroepithelial cell model is obtained. After 70 to 90 days of culture, a gut organ primordium model, and / or a muscle primordium model, and / or a cartilage primordium model, and / or a neuron primordium model, and / or a skin primordium model is obtained.

[0061] The eighth object of this patent is to provide a gastruloid stem cell line, a gastruloid model, an organ primordium model, or tissue or tissues derived from said cell line, gastruloid model, or organ primordium model. organ Alternatively, the cultures may be used to study the mechanisms of early human embryonic development.

[0062] The ninth object of this patent is to provide a gastruloid stem cell line, a gastruloid model, an organ primordium model, or tissue or tissues derived from said cell line, gastruloid model, or organ primordium model. organ Alternatively, the present invention provides a method for the diagnosis and / or treatment of diseases of human early embryonic development.

[0063] The tenth object of this patent is to provide a gastruloid stem cell line, a gastruloid model, an organ primordium model, or tissue or tissues derived from said cell line, gastruloid model, or organ primordium model. organ Alternatively, the present invention provides a method for the preparation of a culture of the same for use in screening, verifying, evaluating, assessing or researching the efficacy of drugs for preventing and / or treating disorders of human early embryonic development.

[0064] The penicillin-streptomycin double antibody solution according to the present invention contains 10,000 units / mL of penicillin and 10,000 μg / mL of streptomycin.

[0065] ROCK inhibitors according to the present invention include, but are not limited to, Y-27632, ROCK-IN-1, and Chroman.

[0066] The gastruloid stem cell line CCRM-hGOSC-1 described in the present invention is a novel cell line obtained by in vitro induction of human embryonic stem cells, and its cellular characteristics are similar to those of human gastruloid stem cells. In a specific embodiment, a gastruloid model obtained 10 to 20 days after injection of the gastruloid stem cell line CCRM-hGOSC-1 into the testes of nude mice has the characteristics of a human gastrula-stage embryo and can simulate the formation of a gastrula. 30 to 90 days after injection of CCRM-hGOSC-1 into the testes of nude mice, the formation of organ primordia can be simulated, and a model similar to a human organ primordium can be obtained. The gastruloid stem cell line CCRM-hGOSC-1, gastruloid model, organ primordium model, or tissue or tissues derived from the cell line or gastruloid model can be used. organAlternatively, the cultures are unable to develop into human or animal individuals due to the lack of cell types such as trophoblasts.

[0067] The CCRM-hGOSC-1 cells provided by the present invention have at least the following characteristics:

[0068] Feature 1: CCRM-hGOSC-1 cells have a rapid proliferation rate, and clone formation can be observed 3 days after single cell passage. The cells are relatively uniform in size and morphology, being round or oval, and the boundaries of cell clones are clearly defined.

[0069] Feature 2: CCRM-hGOSC-1 cells grow actively, have good cell activity, are highly stable in cell culture, and have stable cell growth characteristics in in vitro culture.

[0070] Feature 3: CCRM-hGOSC-1 cells were immunofluorescently stained using cell slides, and cell groups within the same clone simultaneously expressed three germ layer proteins: pluripotency genes OCT4 and SOX2, mesodermal genes EOMES and TBXT, and endodermal genes GATA4 and GATA6.

[0071] Feature 4: The chromosome structure and number of CCRM-hGOSC-1 cells were normal, with a chromosome number of 44+XY, belonging to a diploid male cell line.

[0072] Feature 5: RNA sequencing showed that CCRM-hGOSC-1 cells expressed protein markers for three germ layers, including mesodermal genes MIXL1, EOMES, MESP1, WNT3, TBXT, and GSC, endodermal genes ELF3, FOXA2, CXCR4, GATA4, GATA6, and SOX17, and pluripotency genes POU5F1 (OCT4), NANOG, KLF4, and TFCP2L1. The proliferated cell line exhibited multilineage cell specialization while retaining some stem cell pluripotency and exhibiting multidermal lineage gene expression characteristics similar to those of gastrula-stage cells.

[0073] Feature 6: Human gastruloid formation can be simulated 10-20 days after injection of CCRM-hGOSC-1 cells into nude mouse testes. In samples taken on day 10, a double blastocyst-like structure, as well as amniotic cavity and yolk sac structures, were observed to form within the testicular lumen. Amniotic cavity-like structures developed in the OCT4- and SOX2-positive epidermal-like cell cluster. GATA6 / GATA4 / EOMES-positive cells indicated the migration and organization of primitive endoderm-like cells into primary yolk sac-like structures. The epidermal and hypodermal cells were neatly arranged between the amniotic cavity and yolk sac, forming embryonic structures similar to those in CS5b and CS5c embryos. Furthermore, some embryonic structures began to develop into gastruloids. Epithelial-mesenchymal transition (EMT) was observed in epidermal cells, resulting in the development of gastruloid-like cells that were EOMES / T-positive, had reduced OCT4 expression, and were SOX2-negative. On day 20, gastrula cells developed and formed a gastruloid structure. OCT4-positive cells surrounded the cavity and further formed an amniotic cavity-like structure. At the top of the amniotic cavity, they gradually differentiated into KRT7 / GATA2 / GATA3-positive amniotic epithelial-like cells, and EOMES / T-positive gastrula cells developed. In some embryos, the yolk sac was gradually covered by proliferating and migrating mesendodermal cells.

[0074] Feature 7: Organ primordium formation can be simulated 30-90 days after injection of CCRM-hGOSC-1 into nude mouse testes. 30-40 days later, the amniotic cavity proliferates and expands, and neuroectoderm develops. 50-90 days later, hGOSCs differentiate to form tissues derived from the three germ layers: ectoderm, mesoderm, and endoderm, such as neuroepithelium, smooth muscle, and intestine. organ Endoderm: Immunofluorescent CDX2 & GATA6 labeling and HE staining morphological analysis showed that hGOSCs gradually formed an intestinal structure over time after injection, and by day 90, an intestinal tube surrounded by a muscle layer had formed. organMesoderm: Immunofluorescence analysis revealed that SOX9 labeled cartilage and ACTA2 labeled muscle. Morphological analysis using HE staining revealed that hGOSCs developed muscle and cartilage 90 days after injection. Ectoderm: SOX17, BLIMP1, and TFAP2C labeling revealed the presence of a small number of primordial germ cells 30 days after injection of hGOSCs into the testicular lumen. GATA2, GATA3, and KRT7 labeling revealed amniotic epithelial cells near the primordial germ cells. HE staining morphology and KER15, which indicates keratinocytes, and ACTA2, which indicates muscle, revealed the development of skin morphology. HE and immunofluorescence staining revealed the development of stem neuroepithelial cells after 40-50 days, and the neuroepithelial cells differentiated to form neurons after 70-90 days. OTX2 and SOX2 label neuroepithelial or radial glial cells, while TUJ1 and DCX label neuronal cells.

[0075] The gastruloid stem cell line DYR0100-hGOSC-1 described in the present invention is a novel cell line obtained by in vitro induction of pluripotent stem cells, and its cellular characteristics are similar to those of gastruloid stem cells. In a specific embodiment, a gastruloid model differentiated from the gastruloid stem cell line DYR0100-hGOSC-1 has the characteristics of a human gastrula stage embryo, can simulate the formation of a gastrula embryo, and is used to study the morphological developmental characteristics and gene functions of the gastrula stage. The gastruloid stem cell line DYR0100-hGOSC-1, gastruloid model, organ primordium model, or tissues or tissues derived from the cell line, gastruloid model, or organ primordium model are also useful. organ Alternatively, the cultures may lack cell types such as trophoblasts and therefore fail to develop into individuals.

[0076] DYR0100-hGOSC-1 provided by the present invention has at least the following characteristics:

[0077] Feature 1: DYR0100-hGOSC-1 has a rapid proliferation rate, and clone formation can be observed 3 days after single cell passage. The cells are relatively uniform in size and morphology, round or oval, with clear boundaries between cell clones.

[0078] Feature 2: DYR0100-hGOSC-1 has vigorous growth, good cell activity, high stability in cell culture, and stable cell growth characteristics in in vitro culture.

[0079] Feature 3: DYR0100-hGOSC-1 was immunofluorescently stained using a cell slide. Cell groups within the same clone simultaneously expressed three germ layer proteins: pluripotency genes OCT4 and SOX2, mesodermal genes EOMES, TBXT, CDX2, and MIXL1, and endoderm genes GATA4, GATA6, SOX17, and OTX2.

[0080] Feature 4: DYR0100-hGOSC-1 had normal chromosome structure and number, with a 44+XY chromosome count, belonging to a diploid male cell line.

[0081] Feature 5: DYR0100-hGOSC-1 cells exhibited multilineage cell specialization while retaining some stem cell pluripotency and exhibited gene expression profiles similar to those of gastrula-stage cells. RNA sequencing demonstrated that DYR0100-hGOSC-1 cells expressed protein markers for three germ layers, including mesodermal genes MIXL1, EOMES, MESP1, WNT3, TBXT, and GSC, endodermal genes ELF3, FOXA2, CXCR4, GATA4, GATA6, and SOX17, and pluripotency genes POU5F1 (OCT4), NANOG, KLF4, TFCP2L1, and SOX2.

[0082] Feature 6: DYR0100-hGOSC-1 can form spheroids within 12 hours, has good cell activity, high induction stability, and exhibits stable cell growth characteristics in gastroscopy cultures lasting 4 days.

[0083] Feature 7: During gastruloid induction, on days 0-1, gastruloid stem cells begin to organize into a stable three-dimensional structure, within which epidermal (expressing OCT4 and SOX2), primitive streak (expressing TBXT and MIXL1), and endoderm (expressing SOX17, OTX2, and FOXA2) cells coexist. A small amount of amniotic epithelial cells (expressing CDX2) and extraembryonic mesodermal cells (expressing LUM) also exist.

[0084] Feature 8: On days 2 to 4 of gastruloid induction, the epidermal lineages (expressing OCT4 and SOX2), mesoderm (expressing EOMES and MESP1) that developed from the primitive streak, and endoderm (expressing SOX17, OTX2, and FOXA2) began to separate within the three-dimensional model formed by the organization of gastruloid stem cells, and the OCT4- and SOX2-positive epidermal cells formed an amniotic cavity-like structure.

[0085] Feature 9: After 4 days of induction culture, the gastruloid state resembles that of Carnegie stage 7 embryos.

[0086] Beneficial Effects: The present invention's method for constructing a gastruloid stem cell line, the gastruloid stem cell line obtained by the method, and the construction of a model derived from the cell line are intended to provide a platform for in vitro research into human gastruloid development, to understand and study the complexity of human early embryonic development, and to provide a research platform for the development of clinical treatments for diseases surrounding early embryo implantation. [Brief explanation of the drawings]

[0087] [Figure 1] Figure 1A shows cell morphology diagrams provided in Example 2 of the present invention, including human embryonic stem cells, neo-mesoderm-like cells, primordial germ cell-like cell spheroids, and gastruloid stem cells. [Figure 2] FIG. 1 is a growth curve diagram provided in Example 2 of the present invention. [Figure 3]This is a diagram of the results of immunofluorescence identification provided in Example 2 of the present invention. In the figure, OCT4 and SOX2 are pluripotency genes, EOMES and TBXT are mesoderm genes, and GATA4 and GATA6 are endoderm genes. The scale is 100 μm. [Figure 4] FIG. 1 is a diagram of chromosome karyotype analysis provided in Example 2 of the present invention. [Figure 5] FIG. 1 is a diagram of RNA sequencing analysis provided in Example 2 of the present invention. [Figure 6] This is HE of the in vivo gastruloid provided in Example 3 of the present invention. In the figure, the asterisk * indicates the amniotic cavity, the thick arrow ↑ indicates amniotic epithelial cells, the square ■ indicates the yolk sac, the thin arrow ↑ indicates epidermal cells, the triangle ▲ indicates primitive endoderm cells, and the five-pointed star ★ indicates gastrulation cells. The scale is 100 μm. [Figure 7] IF of the in vivo gastruloid provided in Example 3 of the present invention. In the figure, OCT4 / SOX2 is used to label epidermal cells, GATA6 / GATA4 is used to label primitive endoderm cells, T+EOMES is used to label gastrulation cells, and KRT7 / GATA2 / GATA3 is used to label amniotic epithelial cells. The scale is 20 μm. [Figure 8] 8A shows the HE of the internal organ development prototype provided in Example 4 of the present invention, in which Fig. 8A shows the digestive tract organ primordium derived from the endoderm, Fig. 8B shows the skin organ primordium derived from the ectoderm, and Fig. 8C shows the cartilage organ primordium derived from the mesoderm. [Figure 9] IF of the in vivo organ development prototype provided in Example 4 of the present invention. In the figure, GATA6 and CDX2 mark the digestive tract, KRT15 and ACTA2 mark the skin, and SOX9 marks the cartilage. [Figure 10] 10A, 10B, 10C, and 10D are cell morphology diagrams of different growth generations P1 (FIG. 10A), P7 (FIG. 10B), P20 (FIG. 10C), and P30 (FIG. 10D) provided in Example 6 of the present invention. The scale is 100 μm. [Figure 11] FIG. 1 is a growth curve diagram provided in Example 6 of the present invention. [Figure 12] 1 is a diagram of the results of immunofluorescence identification provided in Example 6 of the present invention. The scale is 100 μm. [Figure 13] FIG. 1 is a diagram of chromosome karyotype analysis provided in Example 6 of the present invention. [Figure 14] FIG. 1 shows RNA sequencing analysis of gastruloid stem cell lines provided in Example 6 of the present invention. [Figure 15] 1 shows white light images of the gastruloid induction culture provided in Example 9 of the present invention during the culture process from day 0 to day 4. The scale is 100 μm. [Figure 16] 1 shows white light images of the sampling process from day 0 to day 4 of the gastruloid induction culture provided in Example 9 of the present invention. The scale is 100 μm. [Figure 17] FIG. 1 is a growth curve diagram of gastruloid induction from days 1 to 4 provided in Example 9 of the present invention. [Figure 18] 1 is an IF diagram of the process of gastruloid stem cells organizing into a three-dimensional structure on day 1 of gastruloid induction provided in Example 9 of the present invention. The scale is 50 μm. [Figure 19] This is an IF image of the process in which gastruloid model induction is completed on days 2 to 4 of Example 9 of the present invention, and the formation of the pre-amniotic cavity and the specialization of the mesendoderm lineage occur. The scale is 50 μm. [Figure 20] FIG. 1 is a diagram of RNA sequencing analysis of the gastruloid model provided in Example 9 of the present invention.

[0088] Biological material deposit information The gastruloid stem cell line with accession number CCTCC NO. C2022114 was deposited at the China Center for Typical Cultures Depositary on April 27, 2020, with the depositor's address at Wuhan University, Wuhan City, China, and its classification name is human gastruloid-like stem cell line CCRM-hGOSC-1.

[0089] The gastruloid stem cell line with accession number CCTCC NO. C2022115 was deposited at the China Center for Typical Cultures Depositary on April 27, 2020, with the depositor's address at Wuhan University, Wuhan City, China, and its classification name is human gastruloid-like stem cell line DYR0100-hGOSC-1. DETAILED DESCRIPTION OF THE INVENTION

[0090] 1. Method for inducing gastruloid stem cell lines from human embryonic stem cells The present invention discloses a method for inducing gastruloid stem cells from human embryonic stem cells and constructs a human-like in vitro post-implantation gastruloid model derived from the cell line. The reagents, instruments, cell lines, etc. used in the present invention are all commercially available.

[0091] Human embryonic stem cells were provided by the Reproductive Medicine Center of Jiangsu Provincial People's Hospital, and the cell name was CCRM-hESCs-22(46,XY).

[0092] (1) The sources of the components of the culture medium used to induce human embryonic stem cells to human gastruloid stem cell lines in vitro are as follows: GMEM (Glasgow's MEM) medium: 11710035, purchased from Gibco, USA. Serum substitute (Thermo Fisher Scientific confidential formulation): 10828028, purchased from Gibco, USA. Fetal bovine serum: 12483020, purchased from Gibco, USA. Gibco® MEM Non-Essential Amino Acids: 11140076, purchased from Gibco, USA. GlutaMAX TM Additive: 35050061, purchased from Gibco, USA. Sodium pyruvate additive: 11360070, purchased from Gibco, USA. Penicillin-streptomycin double antibody: 15140122, 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 stem cell factor: 7734-LF, purchased from R&D Systems, USA. Recombinant human leukemia inhibitory factor: 225-SC, purchased from R&D Systems, USA. Recombinant human epidermal growth factor: 236-EG, purchased from R&D Systems, USA. Recombinant human activin A protein: 338-AC, purchased from R&D Systems, USA. Glycogen synthase kinase-3 (GSK-3) inhibitors: a specific class is CHIR 99021. ROCK inhibitors: Specific types are Y-27632 and HY-10071, and are purchased from MCE, USA. Adenylate cyclase activator: A specific type is Forskolin 1099, purchased from R&D Systems, USA. PDE4 inhibitors: A specific type is Rolipram, 0905, purchased from R&D Systems, USA.

[0093] (2) The sources of the components of the culture medium used to induce human pluripotent stem cells in vitro into post-implantation blastocyst-like embryos are as follows: mTeSR TM 1 Medium: #85850, purchased from STEMCELL Technologies, Canada. Essential 6 medium: A1516401, purchased from Gibco, USA. Recombinant human fibroblast growth factor 2:3718-FB, purchased from R&D Systems, USA. Recombinant human Noggin protein: HY-P7051A, purchased from MCE, USA. ROCK inhibitors: Specific types are Y-27632 and HY-10071, and are purchased from MCE, USA. WNT inhibitors: specific types are IWP-2 and S7085, purchased from Selleck, USA.

[0094] (3) Preparation of GK15-1 culture medium containing ROCK inhibitor Y-27632 for mesoderm induction of embryonic stem cells (stage 1): The medium consisted of 81% basal medium GMEM (GMEM), 15% serum replacement KOSR, 1% penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10 mM non-essential amino acids, 1% 200 mM GlutaMAX additive, 1% 100 mM sodium pyruvate additive, 0.1 mM β-mercaptoethanol, 50 ng / mL recombinant human activin A, 3 μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021, and 10 μM ROCK inhibitor Y-27632.

[0095] (4) Preparation of GK15-1 culture medium for inducing mesoderm-directed embryonic stem cells (stage 1): The medium consisted of 81% basal medium GMEM (Genetical Medium), 15% serum replacement KOSR (Kosher Systemic Sodium Chloride), 1% penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10 mM non-essential amino acids, 1% 200 mM GlutaMAX supplement, 1% 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, and several cytokines: 50 ng / mL recombinant human activin A factor, 3 μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021.

[0096] (5) Preparation of GK15-2 culture medium containing ROCK inhibitor Y-27632 for induction of primordial germ cell-like cells from neonatal mesoderm-like cells (stage 2) and cell purification: The medium consisted of 81% GMEM basal medium (volume percentage), 10% KOSR serum replacement (volume percentage), 1% penicillin-streptomycin double antibody (volume percentage) (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX supplement (volume percentage), 1% 100 mM sodium pyruvate supplement (volume percentage), 0.1 mM β-mercaptoethanol, 100 ng / mL recombinant human stem cell factor, 200 ng / mL recombinant human bone morphogenetic protein 4, 1000 U / mL recombinant human leukemia inhibitory factor, 50 ng / mL recombinant human epidermal growth factor, and 10 μM ROCK inhibitor Y-27632.

[0097] (6) Preparation of GK15-2 culture medium for inducing primordial germ cell-like cells from neonatal mesoderm-like cells (stage 2) and cell purification: The medium consisted of 81% GMEM basal medium (volume percentage), 15% KOSR serum replacement (volume percentage), 1% penicillin-streptomycin double antibody (volume percentage containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX additive (volume percentage), 1% 100 mM sodium pyruvate additive (volume percentage), 0.1 mM β-mercaptoethanol, 200 ng / mL recombinant human bone morphogenetic protein 4, 100 ng / mL recombinant human stem cell factor, 1000 U / mL recombinant human leukemia inhibitory factor, and 50 ng / mL recombinant human epidermal growth factor.

[0098] (7) Preparation of GK10 culture medium containing ROCK inhibitor Y-27632 for in vitro expansion of gastruloid stem cell lines: The medium consisted of 83.5% basal medium GMEM (by volume), 10% serum replacement KOSR (by volume), 2.5% fetal bovine serum (FBS), 1% penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10 mM non-essential amino acids, 1% 200 mM GlutaMAX supplement, 1% 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, 100 ng / mL recombinant human stem cell factor, and 10 μM ROCK inhibitor Y-27632.

[0099] (8) Preparation of GK10 medium for in vitro expansion of gastruloid stem cell lines: The medium consisted of 83.5% basal medium GMEM (by volume), 10% serum replacement KOSR (by volume), 2.5% fetal bovine serum (FBS), 1% penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10 mM non-essential amino acids, 1% 200 mM GlutaMAX additive, 1% 100 mM sodium pyruvate additive, 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, and 100 ng / mL recombinant human stem cell factor. [Example]

[0100] Construction of gastruloid stem cell line CCRM-hGOSC-1 An embodiment of the present invention provides a gastruloid stem cell named human gastruloid-like stem cell line CCRM-hGOSC-1, which has been deposited in the China Center for Type Culture Collection under accession number CCTCC NO. C2022114.

[0101] (1) Induction of mesoderm direction from embryonic stem cells (Step 1): (1-1) Human embryonic stem cells (CCRM-hESCs-22(46,XY)) were provided by the Reproductive Medicine Center of Jiangsu Provincial People's Hospital. When the human embryonic stem cells reached 80-90% confluence, they were digested into single cells using TrypLE Select. After centrifugation at 1300 rpm for 3 minutes in a horizontal centrifuge, the cells were resuspended in PBS to obtain cell suspension 1.

[0102] (1-2) Cell suspension 1 was centrifuged, the supernatant was discarded, and the cells were resuspended in GK15-1 culture medium containing Y-27632 to obtain cell suspension 2. 1 × 10 cells were collected per mL of GK15-1 culture medium containing Y-27632. 6 It contains cells.

[0103] (1-3) Cell suspension 2 was seeded into a 6-well plate pre-coated with Matrigel, at a density of 8 × 10 cells per square centimeter. 5 The cells were inoculated at a cell density of 1000 cells / well, and the cells in the 6-well plate were shaken uniformly and then transferred to an incubator at 37°C and 5% CO2 for culture.

[0104] (1-4) On the second day of culture, the old medium was removed and replaced with GK15-1 medium, which was replaced daily until newborn mesoderm-like cells were obtained.

[0105] The above step of inducing embryonic stem cells toward mesoderm occurs between days 0 and 2 of the gastruloid stem cell line construction method.

[0106] (2) Induction of newborn mesoderm-like cells toward primordial germ cell-like cells (stage 2): (2-1) When the nascent mesoderm-like cells obtained in (1-4) grew to 80-90% confluence, the cells were digested into single cells using TrypLE Select, centrifuged at 1300 rpm for 3 minutes using a horizontal centrifuge, and resuspended in PBS to obtain cell suspension 3.

[0107] (2-2) Centrifuge cell suspension 3, discard the supernatant, add GK15-2 culture medium containing Y-27632, and further resuspend the cells. The cells were then diluted to 1 × 10 per mL of GK15-2 culture medium containing Y-27632. 5 A cell suspension containing 4 cells was obtained.

[0108] (2-3) Add the cell suspension 4 obtained in (2-2) to a round-bottom, transparent, low-adhesion U-bottom 96-well plate at 5 x 10 cells per well. 3 The cells were reseeded and cultured as spheroids. On the second day of culture, the old medium was removed and replaced with GK15-2 medium. Half of the medium was replaced every day until cell spheroids containing primordial germ cell-like cells were obtained.

[0109] The above-mentioned step of inducing mesoderm-like cells toward endoderm occurs on days 3 to 6 of the gastruloid stem cell line construction method.

[0110] (3) Cell purification: (3-1) The cell spheroids obtained in (2-3) were digested into single cells using collagenase IV and 0.25% Trypsin-EDTA trypsin, centrifuged at 1300 rpm for 3 minutes using a horizontal centrifuge, and the cells were resuspended in GK15-2 culture medium to obtain cell suspension 5.

[0111] (3-2) Using a flow cell sorter, CD326 and CD49f double-positive cells were selected from the cell suspension 5 obtained in (3-1), centrifuged at 200 g for 3 minutes, and resuspended in GK10 medium containing Y-27632. The cells were then diluted to 1 × 10 per mL of GK10 medium containing Y-27632. 5 A cell suspension containing 6 cells was obtained.

[0112] (4) Cell amplification: Mitomycin C-treated mouse embryonic fibroblasts were revived the day before and spread as feeder layer cells. The cell suspension 6 obtained in (3-2) was added to the previously prepared feeder layer cells at a density of 2 × 10 per square centimeter. 4The cells were seeded at a suitable density, shaken uniformly, and placed in an incubator at 37°C with 5% CO2. After 24 hours, the medium was replaced with fresh GK10 medium, which was changed daily, allowing for observation of clone formation. The gastruloid stem cell line CCRM-hGOSC-1 was obtained and deposited at the China Center for Genetic Cell Cultures (CCTCC NO. C2022114). [Example]

[0113] Explanation of each biological property of a cell 1.1 Cell morphology observation Human gastrostomy stem cells (CCRM-hGOSC-1) were observed under an inverted microscope. The cells proliferated rapidly, and clone formation was observed two days after single cell passage. The cells were relatively uniform in size and morphology, with round or oval shapes and clear boundaries between cell clones. The results of the morphological observations are shown in Figure 1.

[0114] 1.2 Growth curve measurement 1.2.1 Growth curve measurement procedure When human gastroblastoid stem cells CCRM-hGOSC-1 reached 70-90% confluence, the medium was removed and the cells were washed at least twice with PBS (0.01M, pH 7.4) to remove old medium and any cells that had shed. Then, the cells were digested with TrypLE Select and observed under a microscope. The digested cells were collected during digestion. Once all the cells were digested, the collected cells were added to GK10 medium to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and GK10 medium was added. The cells were then diluted to 1 x 10 per mL of GK10 medium. 5 It contains cells.

[0115] 1 x 10 cells per well (12-well plate) were cultured on the previously prepared feeder layer cells. 5The cells were seeded at a density of 100 cells / well, shaken evenly, and placed in an incubator at 37°C with 5% CO2. After 24 hours, the medium was replaced with fresh GK10 medium, which was changed daily. From the time of seeding, the medium in three wells was discarded every 24 hours, TrypLE Select was added, the cells were digested, the cells were suspended, and the average number of cells in the three wells was counted. Each well was counted three times and the average was taken. The cells in the remaining wells were continued to be cultured until day 5, with the medium changed once daily.

[0116] 1.2.2 Growth curve measurement results Measurements were taken over five consecutive days, and the growth curve data shown in Table 1 below was obtained.

[0117] Table 1 shows the growth curve data obtained over five consecutive days.

[0118] [Table 1]

[0119] Based on the cell growth curve data in Table 1, a schematic diagram of the cell growth curve shown in Figure 2 was obtained. The horizontal axis of Figure 2 represents the culture time (days), and the vertical axis represents the number of cells (×10 5 (pieces).

[0120] Referring to the growth curve data in Table 1 and the growth curve diagram shown in Figure 2, it can be seen that CCRM-hGOSC-1 cells grew well for 5 consecutive days.

[0121] From the above, it can be seen that CCRM-hGOSC-1 cells have a fast proliferation rate, vigorous cell growth, good cell activity, high cell culture stability, and stable cell growth characteristics in vitro.

[0122] 1.3 Immunofluorescence identification 1.3.1 Immunofluorescence identification procedure Small circular glass slides were disinfected with 75% ethanol and sterilized with ultraviolet light, then transferred to cell culture dishes and coated with fibronectin to enhance the adhesion of the slides. CCRM-hGOSC-1 cells were then seeded and cultured according to the usual cell subculture procedure.

[0123] CCRM-hGOSC-1 cells were cultured until passage, and then small circular glass slides were removed, placed in a dish, and fixed with 4% PFA for 40 minutes.

[0124] The 4% PFA fixative was discarded, and the sections were washed three times with PBS. 5% BSA was added and the sections were blocked at room temperature for 2 hours.

[0125] The 5% BSA blocking solution was discarded, and the antibody diluted according to the ratio was added and incubated overnight at 4°C.

[0126] After discarding the primary antibody, the dish was washed three times with PBS, and diluted secondary antibody and live cell staining solution Hoechst 33342 were added to the dish and incubated at room temperature for 2 hours.

[0127] After discarding the secondary antibody, the slides were washed three times with PBS, glycerol was added to the slides, a small circular slide was removed from the dish, and placed upside down on the slide with the glycerol drop. The position of the small slide was fixed using nail polish, and photographs were taken using a confocal fluorescence microscope.

[0128] 1.3.2 Results of immunofluorescence identification The results of immunofluorescence identification are shown in Figure 3. The expanded cell lines were immunofluorescently stained using cell slides to identify genes for each germ layer. We found that the pluripotency genes OCT4 and SOX2, the mesoderm genes EOMES and TBXT, and the endoderm genes GATA4 and GATA6 were partially expressed in the cell clones.

[0129] 1.4 Chromosome karyotype analysis and identification 1.4.1 Chromosome karyotyping and identification procedures cell culture When the cells reached 70-90% confluence, the medium was removed and the cells were washed at least twice with PBS (0.01 M, pH 7.4) to remove old medium and any unhealthy cells. The cells were then digested with 1-2 mL of TrypLE Select and observed under a microscope. The digested cells were collected during digestion. Once all the cells were digested, the collected cells were added to GK10 medium to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 minutes and the supernatant was removed.

[0130] Colchicine treatment 20 μg / mL colchicine was added to the cell culture medium at a ratio of 1:200 to a final concentration of 0.1 μg / mL, and the cells were incubated in a 37°C incubator for 3 hours to obtain colchicine-treated gastruloid stem cells CCRM-hGOSC-1.

[0131] Hypotonic treatment A hypotonic 0.56% KCl solution was preheated to 37°C. The culture dish was removed, and the colchicine-treated gastruloid stem cells CCRM-hGOSC-1 in the dish were digested into a single-cell suspension. The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 2000 rpm / min for 10 minutes. The supernatant was discarded. 9 mL of hypotonic 0.56% KCl solution preheated to 37°C was added to the cell pellet, and the pellet was gently squirted 50 times with a rubber-tipped glass dropper. The cell pellet was then hypotonicized at 37°C for 40 minutes.

[0132] fixed A fixative solution (methanol:glacial acetic acid = 4:1) was prepared and mixed uniformly at room temperature. 1 mL of fixative solution was added to the cells, gently mixed by spraying, and then centrifuged at 300 g for 10 minutes. The supernatant was discarded, 10 mL of fresh fixative solution was added, and the cells were gently sprayed to form a single-cell suspension. The cells were then fixed at room temperature for 1 hour. The cells were centrifuged at 300 g for 10 minutes. The supernatant was discarded, and 10 mL of fresh fixative solution was added, gently sprayed to resuspend the cells, and the cells were fixed at room temperature for 30 minutes. The cells were then centrifuged at 300 g for 10 minutes. The supernatant was discarded, and a small amount of fixative solution (0.2-0.6 mL) was added depending on the amount of cell sediment to resuspend the cells.

[0133] Specimen preparation Illuminate the alcohol lamp and remove a clean glass slide from the distilled water. No draining is necessary. With one hand, tilt the undrained glass slide over the waste tank. With the other hand, aspirate the cell suspension with a pipette gun and dispense a drop of cell suspension onto each slide from 30–60 cm above the slide. Place one drop at each of three different positions on each slide. Then, immediately bake the back of the slide five times over the alcohol lamp. After marking the area, transfer the slide to a 37°C oven and bake overnight.

[0134] staining Human gastroblastoma stem cell slide specimens were placed in a 37°C dryer and then placed in an 80°C oven for 2.5 hours before banding. 0.25% Trypsin-EDTA was preheated to ensure the temperature reached 37°C before use. The slide specimens were immersed in 0.25% Trypsin-EDTA trypsin and treated for 30–40 s. The slides were then removed and rinsed 2–3 times on the front and back of the slide under a thin stream of water. The specimens were then immersed in Giemsa stain preheated to 37°C for approximately 10 minutes. The front and back of the slides were rinsed 2–3 times with tap water, and the slides were then dried using lens paper to absorb the moisture from the surface.

[0135] Microscopy Under a low-magnification microscope, medium-length, well-dispersed mitotic phases were selected, observed with an oil objective, and photographed to obtain the results of chromosome karyotype analysis and identification.

[0136] 1.4.2 Results of chromosome karyotype analysis and identification The results of karyotype analysis and identification are shown in Figure 4. Referring to Figure 4, karyotype analysis and identification showed that the chromosome structure and number of the cells were normal, and the chromosome number of the cells was 44+XY, which belonged to a male cell line with a diploid karyotype.

[0137] 1.5 RNA sequencing and identification 1.5.1 RNA sequencing and identification procedures When CCRM-hGOSC-1 cells reached 70-90% confluence, the medium was removed and the cells were washed at least three times with PBS (0.01M, pH 7.4) to remove old medium and detached cells. Then, the cells were cultured using TrypLE Select. CCRM-hGOSC-1 The cells were digested into single cells, collected, and terminated with GK10 medium. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, 1 mL of Trizol was added, and the cells were stored in a low-temperature (-80°C) refrigerator.

[0138] Phenol-chloroform method CCRM-hGOSC-1 RNA was extracted from the cell samples.

[0139] The cell sample was removed from the low-temperature refrigerator and thawed on ice. 200 μL of chloroform was added, and the mixture was vigorously shaken until homogenous. The mixture was then left to stand at room temperature for 3 minutes and centrifuged at 12,000 rpm for 15 minutes using a tabletop high-speed centrifuge precooled to 4°C.

[0140] The centrifuged sample was transferred to ice, the supernatant was removed, and depending on the volume of the supernatant, isopropanol was added at a ratio of 1:1 and liver glycogen was added at a ratio of 200:1. After shaking evenly, the sample was stored in a low-temperature (-80°C) refrigerator for 30 minutes. The sample was then removed, thawed on ice, and centrifuged at 12,000 rpm for 15 minutes using a tabletop high-speed centrifuge pre-cooled to 4°C.

[0141] The supernatant was discarded, and the cell pellet was washed once with enzyme-free 75% alcohol and centrifuged at 12,000 rpm for 10 minutes using a tabletop high-speed centrifuge precooled to 4°C. The supernatant was discarded, and the remaining 75% alcohol was allowed to air dry in a fume hood.

[0142] Air-dried cell samples are redissolved by adding 10–20 μL of enzyme-free water depending on the amount of RNA, and then transferred to enzyme-free EP tubes for sequencing.

[0143] 1.5.2 RNA sequencing and identification results The results of RNA sequencing and characterization are shown in Figure 5. Transcriptome comparison between CCRM-hGOSC-1 proliferated cells and hESCs (human embryonic stem cells, hESCs) revealed elevated levels of multilineage cell (ectoderm, endoderm, mesoderm, amniote, and primordial germ cell) marker genes in the proliferated cells compared with hESCs. Specifically, expression of mesoderm genes MIXL1, EOMES, MESP1, WNT3, and TBXT, and GSC and endoderm genes ELF3, FOXA2, CXCR4, GATA4, GATA6, and SOX17 were significantly increased. Expression of SOX2 was decreased, but POU5F1 (OCT4) and NANOG remained expressed. Furthermore, elevated expression of naive pluripotency factors KLF4 and TFCP2L1 indicates that the proliferated cell line remains pluripotent. The proliferated cell line undergoes multilineage cell specialization while retaining some degree of stem cell pluripotency and undergoing gene expression of multidermal lineage cells, the characteristics of which are similar to those of cells at the gastrula stage. [Example]

[0144] Formation of a gastruloid model by injecting the cell line into mice CCRM-hGOSC-1 cells have characteristics of the gastrula stage, can simulate gastrula formation, and can be used to study morphological developmental characteristics and gene functions at the gastrula stage.When these cells are injected into mouse testes, gastruloids can be formed after 10 to 20 days.

[0145] 1. Injection into Nude Mouse Testes Preparation: Prepare appropriate capillary glass needles using a micropipette puller. The micropipette puller parameters were Heat 515, Pull 100, Trip 75, and Delay 75.

[0146] When CCRM-hGOSC-1 cells reached 70-90% confluence, the medium was removed and the cells were washed at least twice with PBS (0.01 M, pH 7.4) to remove old medium and any detached cells. Then, the cells were digested with 1-2 mL of TrypLE and observed under a microscope. The digested cells were collected during digestion. Once all the cells were digested, the collected cells were added to GK10 medium to terminate the digestion. The cells were centrifuged at 1000 rpm for 5 minutes and the supernatant was removed.

[0147] Resuspend the cells in GK10 medium to a cell density of 2 x 10 6 BALB / c nude mice were injected with 5-6 × 10 5 cells were injected.

[0148] After injection, mice were fed normally.

[0149] 2. Verification of Gastrulation 2.1 Testicular tissue sampling and specimen preparation Testicular tissues from mice injected in 1 were sampled on days 10 and 20 after injection, respectively.

[0150] The sampled testicular tissue was fixed in 4% PFA or mDF fixative at room temperature for 6 hours, and then the tissue was cut in half.

[0151] After 42 hours, the fixative was discarded, and the testes were dehydrated at room temperature as follows: 70% alcohol for 24 hours, followed by 80% alcohol for 2 hours, 90% alcohol for 2 hours, 100% alcohol for 1 hour, and then dehydrated in 1:1 alcohol:xylene for 25 minutes. The tissue was then permeabilized in xylene for 25 minutes.

[0152] The tissue blocks were transferred to an embedding frame and infiltrated with paraffin, and then infiltrated with paraffin (1) and paraffin (2) for 45 minutes each.

[0153] After tissue embedding, 5-μm serial sections were prepared and placed in a water bath to flatten the paraffin slides, after which intact sections free of tissue were selected and scooped up, and the sections were attached.

[0154] Sections were dried overnight at 65°C, and slides were equilibrated at 37°C for 30 min before being transferred to room temperature for long-term storage.

[0155] 2.2 HE staining (hematoxylin and eosin) Paraffin sections were dewaxed and incubated in xylene (1) and xylene (2) at 37°C for 15 minutes each.

[0156] The tissue was hydrated at room temperature in an alcohol gradient (100% alcohol (1), 100% alcohol (2), 90% alcohol, 80% alcohol, and 70% alcohol). Each step lasted 2 minutes, and the tissue was finally transferred to tap water for 10 minutes.

[0157] The sections were placed in a hematoxylin staining tank for 40 seconds and then rinsed in running water for 5 minutes.

[0158] The sections were rinsed once in 1% HCl and rinsed with running water for 10 minutes.

[0159] The sections were placed in an eosin staining tank and stained for 3 minutes.

[0160] After staining, the tissue sections were dehydrated in gradient alcohol (70% alcohol, 80% alcohol, 90% alcohol, 100% alcohol (I), 100% alcohol (II)) for 2 minutes each, and then in xylene (I) and xylene (II) for 15 minutes each.

[0161] The tissue was dripped with resin, covered with a coverslip, placed in a 37°C oven for 2 hours, removed, stored at room temperature, and photographed under an upright microscope.

[0162] 2.3 Immunofluorescence staining (IF) The tissue sections were dewaxed, hydrated, and transferred to tap water using the same procedure as for HE staining.

[0163] Antigen repair: 200 mL of acidic antigen repair solution was prepared and added to the antigen repair box. The sections were transferred to the repair box and heated in a microwave oven on high heat for 3 minutes and on low heat for 7 minutes to repair the antigens, and then allowed to cool to room temperature.

[0164] Blocking: The slides were washed with PBS, a circle was drawn around the tissue using an immunohistochemistry pen, 100 μL of 5% BSA solution was added to the circle, and the slides were incubated at room temperature for 2 hours.

[0165] Primary antibody incubation: Aspirate as much liquid as possible from the tissue, then re-cover the tissue with antibody diluted in 5% BSA and incubate overnight at 4°C.

[0166] The antibody on the tissue was removed and washed three times with PBS, for 5 minutes each time.

[0167] Fluorescent secondary antibodies and Hoechst 33342 were diluted 1:1000 in 5% BSA, added to the tissue slides, and incubated for 2 hours at room temperature. The slides were washed three times with PBS, for 5 minutes each time.

[0168] Slides were mounted with glycerol and photographed under a confocal fluorescence microscope.

[0169] 2.4 Results of HE and IF validation of gastrula embryos After injecting the gastruloid stem cell line CCRM-hGOSC-1 described in this invention into nude mouse testes, the formation of a double blastocyst-like structure, amniotic cavity, and yolk sac structure in the lumen was observed in the samples taken on day 10. A cavity similar to the pre-amniotic cavity was formed in the OCT4- and SOX2-positive epidermal-like cell mass. GATA6 / GATA4 / EOMES-positive cells teeth, Primitive endoderm-like cells migrated and organized into primary yolk sac-like structures This shows thatThe epidermal and hypodermal layers were neatly arranged between the amniotic cavity and the yolk sac, forming embryonic structures similar to those in CS5b and CS5c embryos. Furthermore, within the testicular lumen at day 10, some embryonic structures began to develop into gastruloids. Epi-like cells underwent epithelial-mesenchymal transition (EMT), generating gastrulation cells that were EOMES / T-positive, had reduced OCT4 expression, and were SOX2-negative.

[0170] On day 20, gastruloid cells developed and formed a gastruloid structure, resulting in a gastruloid model. OCT4-positive cells surrounded the amniotic cavity, forming a structure similar to that of the amniotic cavity. At the top of the amniotic cavity, these cells gradually differentiated into KRT7 / GATA2 / GATA3-positive amniotic epithelial-like cells, and EOMES / T-positive gastrulation cells also developed. In some embryos, the yolk sac was gradually covered by proliferating and migrating mesendodermal cells (Figures 6 and 7). [Example]

[0171] Formation of organ primordium models by injecting the cell line into mice. Organ primordium development can be simulated 30 to 90 days after injection of the gastruloid stem cell line CCRM-hGOSC-1 cells described in the present invention into the testes of mice.

[0172] 1. Nude Mouse Testicular Injection Preparation: Prepare appropriate capillary glass needles using a micropipette puller. The micropipette puller parameters were Heat 515, Pull 100, Trip 75, and Delay 75.

[0173] When CCRM-hGOSC-1 cells reached 70-90% confluence, the medium was removed and the cells were washed at least twice with PBS (0.01 M, pH 7.4) to remove old medium and any unhealthy cells. Then, the cells were digested with 1-2 mL of EDTA-Trypsin and observed under a microscope. The digested cells were collected and, once all the cells were digested, the digestion of the collected cells was terminated. The cells were centrifuged at 1,000 rpm for 5 minutes and the supernatant was removed.

[0174] Resuspend the cells in GK10 medium to a cell density of 2 x 10 6 / mL, and 5-6 × 10 5 cells were injected.

[0175] After injection, mice were fed normally.

[0176] 2. Verification of organ primordium formation 2.1 Testicular tissue sampling and specimen preparation Testicular tissue samples were taken from mice injected in 1. every 10 days from day 30 to day 90 after injection.

[0177] The sampled testicular tissue was placed in 4% PFA or mDF fixative and fixed at room temperature for 6 hours, after which the tissue was cut in half.

[0178] After 42 hours, the fixative was discarded, and the testes were dehydrated at room temperature as follows: 70% alcohol for 24 hours, followed by 80% alcohol for 2 hours, 90% alcohol for 2 hours, 100% alcohol for 1 hour, and then dehydrated in 1:1 alcohol:xylene for 25 minutes. The tissue was then permeabilized in xylene for 25 minutes.

[0179] The tissue blocks were transferred to an embedding frame and infiltrated with paraffin, and then infiltrated with paraffin (1) and paraffin (2) for 45 minutes each.

[0180] After tissue embedding, 5 μM serial sections were prepared and placed in a water bath to flatten the paraffin slides, after which intact sections free of tissue were selected and scooped up, and the sections were attached.

[0181] Sections were dried overnight at 65°C, and slides were equilibrated at 37°C for 30 min before being transferred to room temperature for long-term storage.

[0182] 2.2 HE staining (hematoxylin and eosin) Paraffin sections were dewaxed and incubated in xylene (1) and xylene (2) at 37°C for 15 minutes each.

[0183] The tissue was hydrated at room temperature in an alcohol gradient (100% alcohol (1), 100% alcohol (2), 90% alcohol, 80% alcohol, and 70% alcohol). Each step lasted 2 minutes, and the tissue was finally transferred to tap water for 10 minutes.

[0184] The sections were placed in a hematoxylin staining tank for 40 seconds and then rinsed in running water for 5 minutes.

[0185] The sections were rinsed once in 1% HCl and rinsed with running water for 10 minutes.

[0186] The sections were placed in an eosin staining tank and stained for 3 minutes.

[0187] After staining, the tissue sections were dehydrated in gradient alcohol (70% alcohol, 80% alcohol, 90% alcohol, 100% alcohol (I), 100% alcohol (II)) for 2 minutes each, and then in xylene (I) and xylene (II) for 15 minutes each.

[0188] The tissue was dripped with resin, covered with a coverslip, placed in a 37°C oven for 2 hours, removed, stored at room temperature, and photographed under an upright microscope.

[0189] 2.3 Immunofluorescence staining (IF) The tissue sections were dewaxed, hydrated, and transferred to tap water using the same procedure as for HE staining.

[0190] Antigen repair: 200 mL of acidic antigen repair solution was prepared and added to the antigen repair box. The sections were transferred to the repair box and heated in a microwave oven on high heat for 3 minutes and on low heat for 7 minutes to repair the antigens, and then allowed to cool to room temperature.

[0191] Blocking: The slides were washed with PBS, a circle was drawn around the tissue using an immunohistochemistry pen, 100 μL of 5% BSA solution was added to the circle, and the slides were incubated at room temperature for 2 hours.

[0192] Primary antibody incubation: Aspirate as much liquid as possible from the tissue, then re-cover the tissue with antibody diluted in 5% BSA and incubate overnight at 4°C.

[0193] The antibody on the tissue was removed and washed three times with PBS, for 5 minutes each time.

[0194] Fluorescent secondary antibodies and live cell staining solution Hoechst 33342 were diluted 1:1000 with 5% BSA and added to the tissue slides and incubated at room temperature for 2 hours. The slides were then washed three times with PBS for 5 minutes each time.

[0195] Slides were mounted with glycerol and photographed under a confocal fluorescence microscope.

[0196] 2.4 HE and IF results of three germ layer organ primordia After injecting the gastruloid stem cell line CCRM-hGOSC-1 described in this invention into the testes of nude mice, 30 to 40 days later, the amniotic cavity further proliferated and expanded, and neuroectoderm began to develop, resulting in a neuroectoderm model. By 90 days, CCRM-hGOSC-1 differentiated into tissues derived from the three germ layers: ectoderm, mesoderm, and endoderm, such as neuroepithelium, smooth muscle, and intestine. organ It can be observed that this occurs.

[0197] Endoderm: Immunofluorescent anti-CDX2 & GATA6 antibody labeling and HE staining morphological analysis showed that CCRM-hGOSC-1 gradually formed an intestinal structure over time after injection, and by day 90, intestinal tubes surrounded by a muscle layer had formed. organ The formation of these organ primordia was observed, and a model of intestinal organ primordium was obtained.

[0198] Mesoderm: Immunofluorescence revealed that SOX9 labeled cartilage and ACTA2 labeled muscle. Morphological analysis using HE staining revealed that CCRM-hGOSC-1 cells developed muscle and cartilage 90 days after injection, resulting in muscle and cartilage primordium models.

[0199] Ectoderm: Antibody labeling with SOX17, BLIMP1, and TFAP2C revealed the presence of a small number of primordial germ cells 30 days after CCRM-hGOSC-1 intraductal injection, forming a primordial germ cell model. Antibody labeling with GATA2, GATA3, and KRT7 revealed amniotic epithelial cells near the primordial germ cells, forming a model of the amniotic cavity structure. After 70-90 days of culture, KER15, which indicates keratinocytes, ACTA2, which indicates muscle, and HE staining revealed the development of skin morphology, forming a model of the skin primordium. OTX2 and SOX2 labeled neuroepithelial or radial glial cells, while TUJ1 and DCX labeled neuronal cells. Immunofluorescence staining revealed that by 40-50 days, the majority of the cells were stem-like neuroepithelial cells, forming a model of the neuroepithelial cells. By 70-90 days, the neuroepithelium began to differentiate, forming neurons, forming a model of the neural primordium (Figures 8 and 9).

[0200] That is, after culturing for 30 to 40 days, a neuroectoderm model, and / or a primordial germ cell model, and / or an amniotic epithelial cell model was obtained. After culturing for 40-50 days, a neuroepithelial cell model was obtained. After culturing for 70 to 90 days, an intestinal organ primordium model, and / or a muscle primordium model, and / or a cartilage primordium model, and / or a nerve primordium model, and / or a skin primordium model was obtained.

[0201] In conclusion, this study established a stable gastruloid stem cell line, CCRM-hGOSC-1, which retains a certain degree of stem cell pluripotency and expresses genes and proteins of the three germ layers of endoderm, mesoderm, and ectoderm during gastrula development, with characteristics consistent with those of gastrula-stage cells, closely reproducing the important characteristics of gastrula-stage cells. This stem cell line can be used to construct a gastruloid model in vivo in mice, and tissues derived from the three germ layers, ectoderm, mesoderm, and endoderm, such as neuroepithelium, smooth muscle, and intestine, can be generated. organ It can form a primordium model of the embryo, and establish an in vitro drug screening platform that affects early embryonic development, providing reference for clinical drug use.

[0202] 2. Method for inducing human induced pluripotent stem cells into gastruloid stem cell lines The present invention further discloses a method for inducing human induced pluripotent stem cells into a gastruloid stem cell line, and constructs a human in vitro-like post-implantation gastruloid model derived from the cell line. The reagents, instruments, cell lines, etc. used in the present invention are all commercially available. Human induced pluripotent stem cells were purchased from the Cell Bank / Stem Cell Bank of the Committee for the Depositary of Type Cultures of the Chinese Academy of Sciences, with the cell name DYR0100 and catalog number SCSP-1301.

[0203] (1) The sources of the components of the culture medium used to induce human pluripotent stem cells to human gastruloid stem cell lines in vitro are as follows: GMEM (Glasgow's MEM) medium: 11710035, purchased from Gibco, USA. Serum substitute (Thermo Fisher Scientific confidential formulation): 10828028, purchased from Gibco, USA. Fetal bovine serum: 12483020, purchased from Gibco, USA. Gibco® MEM Non-Essential Amino Acids: 11140076, purchased from Gibco, USA. GlutaMAX TM Additive: 35050061, purchased from Gibco, USA. Sodium pyruvate additive: 11360070, purchased from Gibco, USA. Penicillin-streptomycin double antibody: 15140122, 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 stem cell factor: 7734-LF, purchased from R&D Systems, USA. Recombinant human leukemia inhibitory factor: 225-SC, purchased from R&D Systems, USA. Recombinant human epidermal growth factor: 236-EG, purchased from R&D Systems, USA. Recombinant human activin A protein: 338-AC, purchased from R&D Systems, USA. ROCK inhibitors: Specific types are Y-27632 and HY-10071, and are purchased from MCE, USA. CHIR 99021: The specific type is Laduviglusib trihydrochloride, HY-10182B, purchased from MCE, USA. Adenylate cyclase activator: A specific type is Forskolin 1099, purchased from R&D Systems, USA. PDE4 inhibitors: specific types are Rolipram, 0905, purchased from R&D Systems, USA.

[0204] (2) The sources of the components of the culture medium used to induce human pluripotent stem cells in vitro into post-implantation blastocyst-like embryos are as follows: mTeSR TM 1 Medium: #85850, purchased from STEMCELL Technologies, Canada. Essential 6 medium: A1516401, purchased from Gibco, USA. Recombinant human fibroblast growth factor 2:3718-FB, purchased from R&D Systems, USA. Recombinant human Noggin protein: HY-P7051A, purchased from MCE, USA. ROCK inhibitors: Specific types are Y-27632 and HY-10071, and are purchased from MCE, USA. WNT inhibitors: specific types are IWP-2 and S7085, purchased from Selleck, USA.

[0205] (3) Preparation of GK15-1 culture medium containing Y-27632 for the first stage of mesoderm induction of pluripotent stem cells: The medium consisted of 81% basal medium GMEM (GMEM), 15% serum replacement KOSR, 1% penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10 mM non-essential amino acids, 1% 200 mM GlutaMAX supplement, 1% 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, 50 ng / mL recombinant human activin A protein, 3 μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021, and 10 μM ROCK inhibitor Y-27632.

[0206] (4) Preparation of GK15-1 culture medium for mesoderm induction from pluripotent stem cells (stage 1): The medium consisted of 81% basal medium GMEM (GMEM), 15% serum replacement KOSR, 1% penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10 mM non-essential amino acids, 1% 200 mM GlutaMAX supplement, 1% 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, 50 ng / mL recombinant human activin A protein, and 3 μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021.

[0207] (5) Preparation of GK15-2 culture medium containing Y-27632 for the induction of primordial germ cell-like cells from neonatal mesoderm-like cells (stage 2) and cell purification: The medium consisted of 81% GMEM basal medium (volume percentage), 15% KOSR serum replacement (volume percentage), 1% penicillin-streptomycin double antibody (10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX additive (volume percentage), 1% 100 mM sodium pyruvate additive (volume percentage), 0.1 mM β-mercaptoethanol, 100 ng / mL recombinant human stem cell factor, 200 ng / mL recombinant human bone morphogenetic protein 4, 1000 U / mL recombinant human leukemia inhibitory factor, 50 ng / mL recombinant human epidermal growth factor, and 10 μM ROCK inhibitor Y-27632.

[0208] (6) Preparation of GK15-2 culture medium for inducing primordial germ cell-like cells from neonatal mesoderm-like cells (stage 2) and cell purification: The medium consisted of 81% GMEM basal medium (volume percentage), 15% KOSR serum replacement (volume percentage), 1% penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10 mM non-essential amino acids (volume percentage), 1% 200 mM GlutaMAX additive (volume percentage), 1% 100 mM sodium pyruvate additive (volume percentage), 0.1 mM β-mercaptoethanol, 200 ng / mL recombinant human bone morphogenetic protein 4, 100 ng / mL recombinant human stem cell factor, 1000 U / mL recombinant human leukemia inhibitory factor, and 50 ng / mL recombinant human epidermal growth factor.

[0209] (7) Preparation of GK10 culture medium containing Y-27632: The medium consisted of 83.5% basal medium GMEM (by volume), 10% serum replacement KOSR (by volume), 2.5% fetal bovine serum (FBS), 1% penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10 mM non-essential amino acids, 1% 200 mM GlutaMAX supplement, 1% 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, 100 ng / mL recombinant human stem cell factor, and 10 μM ROCK inhibitor Y-27632.

[0210] (8) Preparation of GK10 medium for in vitro expansion of gastruloid stem cell lines: The medium consisted of 83.5% basal medium GMEM (by volume), 10% serum replacement KOSR (by volume), 2.5% fetal bovine serum (FBS), 1% penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10 mM non-essential amino acids, 1% 200 mM GlutaMAX additive, 1% 100 mM sodium pyruvate additive, 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, and 100 ng / mL recombinant human stem cell factor.

[0211] (9) Preparation of mTR culture medium for the first stage of post-implantation gastruloid induction: 99% mTeSR by volume TM 1 complete medium, 1% by volume of double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), and a small molecule compound: 10 μM ROCK inhibitor Y-27632.

[0212] (10) Preparation of E6BIN culture medium for the second stage of post-implantation gastruloid induction: The medium was 100% Essential 6 medium by volume and several cytokines: 20 ng / mL recombinant human fibroblast growth factor 2, 50 ng / mL recombinant human noggin protein, and 5 μM small molecule compound IWP-2. [Example]

[0213] Construction of gastruloid stem cell line DYR0100-hGOSC-1 An embodiment of the present invention provides a gastruloid stem cell named human gastruloid-like stem cell line DYR0100-hGOSC-1, which has been deposited in the China Center for Type Cultures Depositary under accession number CCTCC NO. C2022115.

[0214] (1) Induction of mesodermal development from human induced pluripotent stem cells (Step 1): (1-1) Human induced pluripotent stem cells DYR0100 were purchased from the Cell Bank / Stem Cell Bank of the Committee for the Deposit of Typical Cultures of the Chinese Academy of Sciences, catalog number SCSP-1301.

[0215] When human induced pluripotent stem cells grew to 80-90% confluence, the cells were digested into single cells using the digestion enzyme TrypLE Select, centrifuged at 1300 rpm for 3 minutes using a horizontal centrifuge, and then resuspended in PBS to obtain cell suspension 1.

[0216] (1-2) Cell suspension 1 was taken and centrifuged, the supernatant was discarded, and the cells were further resuspended in GK15-1 culture medium containing Y-27632 to obtain cell suspension 2. 1 × 10 cells were collected per mL of GK15-1 culture medium containing Y-27632. 6 It contains cells.

[0217] (1-3) The cell suspension 2 obtained in step (1-2) was seeded into a 6-well plate pre-coated with Matrigel, at a density of 1 × 10 cells per square centimeter. 6 The cells were inoculated at a cell density of 1000 cells / well, shaken evenly, and placed in an incubator at 37°C with a volume concentration of 5.0% carbon dioxide.

[0218] (1-4) On the second day of culture, the old medium was removed and replaced with GK15-1 medium, which was then replaced every day until new mesoderm-like cells were obtained.

[0219] The above-mentioned step of inducing pluripotent stem cells toward the mesoderm occurs between days 0 and 2 of the gastruloid stem cell line construction method.

[0220] (2) Induction of newborn mesoderm-like cells toward primordial germ cell-like cells (stage 2): (2-1) When the nascent mesoderm-like cells obtained in step (1-4) grew to 80-90% confluence, the cells were digested into single cells using the digestion enzyme TrypLE Select, centrifuged at 1300 rpm for 3 minutes using a horizontal centrifuge, and resuspended in PBS to obtain cell suspension 3.

[0221] (2-2) Cell suspension 3 was taken and centrifuged, and the supernatant was discarded. Then, GK15-2 culture medium containing Y-27632 was added to further resuspend the cells, and the cells were diluted to 1 × 10 per mL of GK15-2 culture medium containing Y-27632. 5 A cell suspension containing 4 cells was obtained.

[0222] (2-3) The cell suspension 4 obtained in step (2-2) was inoculated into a round-bottom, transparent, low-adhesion 96-well plate for spheroid culture, and the initial cell amount of the cell spheroids in each well was 1 × 10 4 On the second day of culture, the old medium was removed and replaced with GK15-2 medium, and half of the GK15-2 medium was replaced every day until cell spheroids containing primordial germ cell-like cells were obtained through culture.

[0223] The above-mentioned step of inducing the newborn mesoderm-like cells to form primordial germ cell-like cells occurs on days 3 to 6 of the gastruloid stem cell line construction method.

[0224] (3) Cell purification: (3-1) The cell spheroids obtained in step (2-3) were digested into single cells using collagenase IV and 0.25% Trypsin-EDTA trypsin, centrifuged at 1300 rpm for 3 minutes using a horizontal centrifuge, and the cells were resuspended in GK15-2 culture medium to obtain cell suspension 5.

[0225] (3-2) Using a flow cell sorter, CD326 and CD49f double-positive cells were selected from the cell suspension 5 obtained in step (3-1), centrifuged at 1300 rpm for 3 minutes, and 1.0 × 10 5 The cells were seeded into a 12-well plate at a density of 1 / well, and the cells in each well were resuspended in 1 mL of GK10 culture medium containing Y-27632 to obtain cell suspension 6.

[0226] (4) Cell amplification: The cell suspension obtained in step (3-2) was diluted to 2 x 10 cells per square centimeter. 4 The feeder layer cells were seeded at a density of 100 cells per well onto 12-well plates pre-plated with mitomycin C-treated mouse embryonic fibroblasts. The plates were shaken uniformly and placed in an incubator at 37°C with 5.0% CO2. After 24 hours, the medium was replaced with fresh GK10 medium, which was then replaced daily to monitor clone formation. The gastruloid stem cell line DYR0100-hGOSC-1 was obtained and deposited at the China Center for Genetic Cell Cultures (CCTCC) under accession number C2022115. [Example]

[0227] Explanation of each biological property of a cell 1.1 Cell morphology observation Gastruloid stem cells DYR0100-hGOSC-1 were observed under an upright microscope. The cells were round or oval in shape, had a rapid proliferation rate, and clone formation was observed within 2-3 days of single cell subculture. The boundaries of the cell clones were clearly defined. The results of the morphological observation are shown in Figure 10.

[0228] 1.2 Growth curve measurement 1.2.1 Growth curve measurement procedure Except for replacing the cells with gastruloid stem cells DYR0100-hGOSC-1, the other procedures were the same as in 1.2.1 Growth curve measurement procedure in Example 2.

[0229] 1.2.2 Growth curve measurement results Measurements were taken over five consecutive days, and the growth curve data shown in Table 2 below was obtained.

[0230] Table 2 shows the growth curve data obtained over five consecutive days.

[0231] [Table 2]

[0232] Table 2 Based on the cell growth curve data, a schematic diagram of the cell growth curve shown in Figure 11 was obtained. In Figure 11, the horizontal axis represents the culture time (days), and the vertical axis represents the number of cells (×10 5 (pieces).

[0233] Table 2 Referring to the growth curve data and the schematic diagram of the growth curve shown in Figure 11, it can be seen that gastruloid stem cells DYR0100-hGOSC-1 grew well for 5 consecutive days, with the logarithmic growth phase being 3 days.

[0234] From the above, it can be seen that DYR0100-hGOSC-1 cells have a fast proliferation rate, active cell growth, good cell activity, high cell culture stability, and stable cell growth characteristics in vitro.

[0235] 1.3 Immunofluorescence identification 1.3.1 Immunofluorescence identification procedure Small circular glass slides were disinfected with 75% ethanol and sterilized with ultraviolet light, then transferred to cell culture dishes and coated with fibronectin to enhance the adhesion of the slides. DYR0100-hGOSC-1 cells were then seeded and cultured according to the usual cell subculture procedure.

[0236] DYR0100-hGOSC-1 cells were cultured until passage, and the small circular glass slide was removed and placed on a clean new culture dish. The DYR0100-hGOSC-1 cells on the small circular glass slide were fixed with 4% PFA for 30 minutes at room temperature.

[0237] The 4% PFA fixative was discarded, and PBS was added at room temperature to wash out the fixative three times for 5 minutes each time. The PBS was then discarded, and 5% BSA, a blocking solution, was added, followed by blocking at room temperature for 2 hours.

[0238] The 5% BSA blocking solution was discarded, and the corresponding primary antibody diluted in 5% BSA was added and incubated with DYR0100-hGOSC-1 cells on small circular glass slides overnight at 4°C.

[0239] After discarding the primary antibody incubation solution, PBS was added at room temperature to wash out the remaining primary antibody incubation solution three times for 5 minutes each time. Secondary antibody diluted with 5% BSA and live cell staining solution Hoechst 33342 were added to the dish and incubated at room temperature for 2 hours in the dark.

[0240] After discarding the secondary antibody incubation solution, PBS was added to wash out the remaining secondary antibody incubation solution three times for 5 minutes each time. Glycerol was then added to the slide. The small circular slide was removed from the culture dish and placed upside down on the glycerol-added slide. The position of the small circular slide was fixed using nail polish, and the slide was then imaged using a confocal fluorescence microscope.

[0241] 1.3.2 Results of immunofluorescence identification The results of immunofluorescence identification are shown in Figure 12. Immunofluorescence staining of gastruloid stem cells (DYR0100-hGOSC-1 cells) was performed using cell slides to identify the gene expression profiles of each germ layer in DYR0100-hGOSC-1 cells. We found that the pluripotency genes OCT4 and SOX2, the mesoderm genes EOMES, TBXT, MIXL1, and CDX2, and the endoderm genes GATA4, GATA6, SOX17, FOXA2, and OTX2 were expressed in the cell clones. Overall, pluripotency genes were expressed in the center of the cell clones, mesoderm genes were expressed in a scattered manner within the cell clones, and endoderm genes were expressed at the edge of the cell clones without co-localizing with pluripotency genes.

[0242] 1.4 Chromosome karyotype analysis and identification 1.4.1 Chromosome karyotyping and identification procedures 20 μg / mL colchicine was added to the gastruloid stem cell GK10 culture medium at a ratio of 1:200 to a final concentration of 0.1 μg / mL, and the mixture was then incubated in an incubator at 37°C with a volumetric carbon dioxide concentration of 5.0% for 3 hours.

[0243] A hypotonic 0.56% KCl solution was preheated to 37°C. The culture dish was removed, and the colchicine-treated gastruloid stem cells were washed at least three times with PBS (0.01M, pH 7.4) to remove old medium and any unhealthy cells. DYR0100-hGOSC-1 gastruloid stem cells were then digested into a single-cell suspension using TrypLE Select. The cell suspension was transferred to a 15 mL centrifuge tube and centrifuged at 2000 rpm for 10 minutes in a horizontal centrifuge. The supernatant was discarded, and 9 mL of hypotonic 0.56% KCl solution preheated to 37°C was added to the cell pellet. The tube was gently squirted 50 times with a rubber-tipped glass dropper until a single-cell suspension was obtained. The tube was then placed in a 37°C water bath for 25 minutes.

[0244] A fixative solution was prepared using a 3:1 methanol / glacial acetic acid solution and mixed uniformly at room temperature. 1 mL of fixative was added to the hypotonic gastruloid stem cell suspension, gently bubbled 20 times with a rubber-tipped glass dropper, and centrifuged at 2000 rpm for 10 minutes. The supernatant was discarded, and 10 mL of fresh fixative was added. A single-cell suspension was obtained by gently bubbled 10 times with a rubber-tipped glass dropper. The fixative was then allowed to settle for 2 hours at room temperature. The suspension was centrifuged at 2000 rpm for 10 minutes. The supernatant was discarded, and 10 mL of fresh fixative was added. A single-cell suspension was obtained by gently bubbled 10 times with a rubber-tipped glass dropper. The fixative was then allowed to settle for 30 minutes at room temperature. The suspension was then centrifuged at 2000 rpm for 10 minutes. The supernatant was discarded, and 0.2–0.6 mL of fixative was added depending on the amount of cell pellet. The gastruloid stem cells were then resuspended.

[0245] Light the alcohol lamp and remove a clean glass slide from the distilled water. No draining is necessary. Place the undrained glass slide tilted over a waste tank. Using a pipette gun, aspirate the cell suspension and drop it onto the slide from 50 cm above. Place one drop at each of three or four different locations on each slide. Immediately afterwards, bake the backside of the slide five times over the alcohol lamp. Then, transfer the slide to a 37°C oven and bake overnight.

[0246] A glass slide of human gastroblastoid stem cell chromosomes from a 37°C dryer was placed in an 80°C oven for 2.5 hours. 0.25% Trypsin-EDTA was preheated to 37°C. The slide was immersed in 0.25% Trypsin-EDTA trypsin preheated to 37°C and treated for 30–40 seconds depending on the condition of the slide. The slide was then removed and rinsed three times on both the front and back of the slide under a thin stream of tap water. The slide was then immersed in Giemsa stain preheated to 37°C for approximately 10 minutes. The front and back of the slide were then rinsed three times on both sides of the slide with tap water, and the slide was then dried by absorbing the moisture from the surface with lens paper.

[0247] Under a low-magnification microscope, medium-length, well-dispersed mitotic phases were selected, and then the objective lens was switched to an oil-based objective to observe and photograph them, and the results of chromosome karyotype analysis and identification were obtained.

[0248] 1.4.2 Results of chromosome karyotype analysis and identification The results of karyotype analysis and identification are shown in Figure 13. Referring to Figure 13, the karyotype analysis and identification showed that the chromosome structure and number of the cells were normal, and the chromosome number of the cells was 44+XY, which means that the cells belonged to a male cell line with a diploid karyotype.

[0249] 1.5 RNA sequencing and identification 1.5.1 RNA sequencing and identification procedures The procedure was the same as that in 1.5.1 of Example 1, except that the cells were replaced with gastruloid stem cells DYR0100-hGOSC-1 cells.

[0250] 1.5.2 RNA sequencing and identification results The results of RNA sequencing and identification are shown in Figure 14. Comparison of the transcriptome sequences of gastruloid stem cells DYR0100-hGOSC-1 and human induced pluripotent stem cells DYR0100-hiPSCs revealed that the expression of mesodermal genes MIXL1, EOMES, MESP1, WNT3, TBXT, and GSC, as well as endodermal genes ELF3, FOXA2, CXCR4, GATA4, GATA6, and SOX17, was upregulated in gastruloid stem cells DYR0100-hGOSC-1 compared to human induced pluripotent stem cells DYR0100-hiPSCs.

[0251] The expression level of the pluripotency gene SOX2 in gastruloid stem cells DYR0100-hGOSC-1 was slightly reduced compared to human induced pluripotent stem cells DYR0100-hiPSCs, but the remaining pluripotency genes POU5F1 (OCT4) and NANOG were still expressed. Furthermore, the expression of pluripotency factors KLF4 and TFCP2L1, which are upregulated in naive human induced pluripotent stem cells, was elevated in gastruloid stem cells DYR0100-hGOSC-1, indicating that the gastruloid stem cell line DYR0100-hGOSC-1 still retains stem cell pluripotency.

[0252] Gastruloid stem cells DYR0100-hGOSC-1 retain a certain degree of stem cell pluripotency and exhibit gene expression of multi-dermal lineage cells, characteristics of which are similar to those of embryonic cells at the gastrula stage of human embryonic development. [Example]

[0253] Induction of a post-implantation gastruloid three-dimensional model using gastruloid stem cells DYR0100-hGOSC-1 (Stage 1, Days 0-1): The gastruloid stem cell DYR0100-hGOSC-1 possesses the characteristics of human gastrula stage embryos, can simulate the formation of gastrula embryos, and can be used to study the morphological developmental characteristics and gene functions of the gastrula stage. Under in vitro culture conditions, this cell line can reconstruct structures similar to those of in vivo gastrula embryos and partially reproduce the biological events in gastrula development.

[0254] When the gastruloid stem cells obtained in Example 5 grew to 70-80% confluence, they were digested into single cells using TrypLE Select and resuspended in GK10 to obtain cell suspension 7. CD326 and CD49f double-positive cells were selected using a flow cell sorter. After centrifugation at 1300 rpm for 3 minutes, 6.0-7.0 × 10 4 Take 10 cells and add 4 mL of mTR medium to resuspend the cells, resulting in a density of 6.0–7.0 × 10 cells per well. 3The cells were seeded into a round-bottom, transparent, low-adhesion 96-well plate, centrifuged at 800 rpm for 3 minutes in a horizontal centrifuge, and then placed in an incubator at 37°C with 5.0% CO2 until the gastruloid stem cells organized into three-dimensional structures. [Example]

[0255] Induction of post-implantation gastruloid three-dimensional models using gastruloid stem cells DYR0100-hGOSC-1 (Phase 2, Days 2-4): Instead of discarding the mTR culture medium in the well plate at the end of Example 7, E6BIN culture medium was added so that the final culture medium contained 20 ng / mL recombinant human fibroblast growth factor 2, 50 ng / mL recombinant human noggin protein, and 5 μM small molecule compound IWP-2. The well plate was placed in an incubator at 37°C with a volume concentration of 5.0% carbon dioxide until the formation of the pre-amniotic cavity and the completion of mesendoderm lineage specification, yielding a gastruloid model. [Example]

[0256] Biological characteristics of the gastruloid model induced by gastruloid stem cells: 1.1 Morphological observation of induced gastruloids Postimplantation gastruloid models cultured in 96-well plates for 0-96 hours were observed under an upright microscope, revealing rapid cell aggregation into spheroids. After 12 hours of induction, cells aggregated to form three-dimensional embryonic spheroids. The volume of the spheroids then slowly increased. After 48-60 hours, two distinct compartments, dense and sparse, developed within the spheroids, forming mutually exclusive double spheroids, followed by an amniotic cavity-like structure. After 96 hours, the maximum diameter of the gastruloids was approximately 150-250 μm. Morphological observations are shown in Figures 15 and 16.

[0257] 1.2 Measurement of the growth curve of induced post-implantation gastruloids 1.2.1 Growth curve measurement procedure During the in vitro construction of the post-implantation gastruloid model using gastruloid stem cells, DYR0100-hGOSC-1, samples were taken every 24 hours for days 1 to 4. The gastruloids were washed at least twice with PBS (0.01M, pH 7.4) to remove the original medium and any unhealthy cell fragments. The post-implantation gastruloids were fixed with 4% PFA at room temperature for 30 minutes, after which the fixative was discarded and the gastruloids were washed at least three times with PBST (0.01M, pH 7.4, containing 1% tTriton) for 5 minutes each time.

[0258] After blocking the gastruloids in 5% BSA (containing 1% tTriton) for 4 hours at room temperature, the DAPI staining solution was diluted 1:200 in 5% BSA, and the gastruloids were then incubated at room temperature for 4 hours.

[0259] After discarding the DAPI dilution solution, the sample was washed three times with PBS for 5 minutes each time, then transferred to the staining chamber and cleared by adding a certain amount of prepared iohexol solution. The chamber was then lightly covered with a glass slide and photographed using a confocal microscope.

[0260] The cell number within each cell spheroid was estimated using Imaris software (Bitplane) on Dapi-stained confocal images. Counting spots were drawn using an internal algorithm, with an estimated x-y size of 6–10 µM, a quality threshold of 2.5, and background noise was removed (Figure 17).

[0261] 1.2.2 Growth curve measurement results Measurements were carried out continuously for four days, and the cell count data within the cell spheroids at each time point was obtained as shown in Table 3 below.

[0262] [Table 3]

[0263] Table 3 shows the growth curve data of DYR0100-hGOSC-1-induced gastruloids obtained by measuring for 4 consecutive days, and the number of samples (n) counted at each counting time point was 20–30.

[0264] Table 3 Based on the cell growth curve data, a schematic diagram of the cell growth curve shown in FIG. 17 was obtained. Figure 17 The horizontal axis represents the culture time (h) and the vertical axis represents the number of cells (cells).

[0265] Referring to the growth curve data in Table 3 and the schematic diagram of the growth curve shown in Figure 17, it can be seen that gastruloids induced by gastruloid stem cells DYR0100-hGOSC-1 have a slow cell proliferation rate, high cell induction stability, and in vitro induction has stable embryo-like growth characteristics.

[0266] 1.3 Immunofluorescence identification 1.3.1 Immunofluorescence identification procedure During days 1 to 4 of gastruloid model induction using gastruloid stem cells DYR0100-hGOSC-1, cells were sampled every 24 hours.

[0267] Gastruloids were fixed with 4% PFA for 30 minutes, and after discarding the fixative, they were washed three times with PBST (0.01 M, pH 7.4, containing 1% tTriton) for 5 minutes each time.

[0268] Gastruloids were blocked with 5% BSA (containing 1% Triton) for 4 hours at room temperature.

[0269] The blocking solution was discarded, and the diluted antibodies were added and incubated at 4°C for 24 to 48 hours.

[0270] After discarding the primary antibody incubation solution, the sections were washed three times with PBS (0.01 M, pH 7.4, containing 1% tTriton) for 5 minutes each time, and then diluted secondary antibody incubation solution and DAPI staining solution were added and incubated at room temperature for 4 hours.

[0271] After discarding the secondary antibody incubation solution, the samples were washed three times with PBS (0.01 M, pH 7.4, containing 1% tTriton) for 5 minutes each time. The samples were then transferred to a 0.2 mM or 0.75 mM silica gel staining chamber and cleared with a certain amount of prepared iohexol solution. The chamber was then lightly covered with a glass slide, and the immunofluorescent staining results were photographed using a confocal microscope.

[0272] 1.3.2 Results of immunofluorescence identification The results of immunofluorescence identification are shown in Figures 18-19.

[0273] On day 1 of gastruloid stem cell induction, gastruloid stem cells began to organize into stable three-dimensional cellular spheroid structures within the gastruloid. At this point, epidermal-like cells (expressing OCT4 and SOX2), primitive streak-like cells (expressing NCAD, TBXT, and MIXL1), mesendodermal-like cells (expressing EOMES, GATA6, and NCAD), and endoderm-like cells (expressing NCAD, SOX17, OTX2, and FOXA2) coexisted within the gastruloid. A small number of cells suspected to be extraembryonic mesodermal-like cells (expressing LUM) were also present. At this point, there was no clear spatial distribution pattern for each type of cell.

[0274] On day 2 of induction, OCT4- and SOX2-positive epidermal-like cells began to migrate to one side of the gastruloid, while endoderm-like cells, directed by SOX17, FOXA2, and OTX2, simultaneously migrated in the opposite direction. Concurrently, MIXL1- and TBXT-positive primitive streak-like cells also migrated in the opposite direction to the epidermal cells. However, the number of MIXL1- and TBXT-positive primitive streak cells decreased compared with gastruloids on day 1 of induction, suggesting that at this time point, primitive streak-like cells may have begun to specialize toward mesoderm- or definitive endoderm-like cell fates.

[0275] On days 3-4 of induction, OCT4- and SOX2-positive epidermal-like cells formed a cavity similar to the amniotic cavity. Compared to day 2 of gastruloid induction, at this time point, SOX17-, FOXA2-, and OTX2-positive endoderm-like cells completely separated from OCT4- and SOX2-positive epidermal-like cells, forming a well-ordered double scutellum structure between the two cell types. The nuclei of OCT4- and SOX2-positive epidermal-like cells displayed a columnar 3D cell morphology. Mutually exclusive SOX17-, FOXA2-, and OTX2-positive endoderm-like cells and EOMES-positive, FOXA2-negative mesodermal-like cells simultaneously expressed the epithelial-mesenchymal transition marker NCAD.

[0276] 1.5 RNA sequencing and identification 1.5.1 RNA sequencing and identification procedures After 96 hours of induction of the gastruloid model using the gastruloid stem cells DYR0100-hGOSC-1, the gastruloids were sampled, the medium removed, and the gastruloids were washed at least twice with PBS (0.01 M, pH 7.4) to remove old medium and shed dead cell fragments. The gastruloids were placed in a 37°C environment and digested with 1-2 mL of 0.25% Trypsin-EDTA trypsin for 3 minutes. During digestion, the gastruloids were gently blown with a pipette gun to break up the gastruloids into single cells. The digestion was then terminated with serum-containing medium. After centrifugation at 1300 rpm for 5 minutes, the supernatant was removed. The cells were resuspended in PBS and transferred to enzyme-free EP tubes for measurement.

[0277] 1.5.2 RNA sequencing and identification results The results of single-cell RNA sequencing and identification are shown in Figure 20.

[0278] In single-cell RNA sequencing data from a gastruloid model cultured for 96 hours, uniform manifold approximation and projection (UMAP) analysis of 4563 cells divided the cells into 12 cell clusters.

[0279] Based on the gene expression of each cell group, nine cell types were finally identified: amnion cells, epiblast cells, somitic mesoderm cells, vascular endothelial cells, fibroblast cells, mesoderm cells, primordial germ cell-like cells (PGCs), endoderm cells, and unknown cells. Consistent with the immunofluorescence images in Figure 19, only 10 primitive streak cells expressing MIXL1 and TBXT were identified at this time point.

[0280] In this dataset, we were unable to detect any cell types that simultaneously expressed two or more neuroectodermal markers.

[0281] These data indicate that in the 96-hour embryo-like model, epiblast cells undergo transient gastrulation, and the resulting primitive streak cells complete the differentiation process into definitive endoderm and mesoderm, but neural differentiation has not yet begun at this stage, suggesting that the embryo-like structure at this stage may resemble an embryo at Carnegie stage 7 of embryogenesis.

[0282] This study established stably passaged gastruloid stem cells that retain a certain degree of stem cell pluripotency and express genes and proteins representative of the three germ layers of endoderm, mesoderm, and ectoderm during gastrula development. These genes and proteins are consistent with those of gastrula-stage embryonic cells, closely recapitulating key features of gastrula-stage cells. Using these cells, a three-dimensional gastruloid model can be constructed in vitro to simulate gastrula development, partially recapitulating key biological events during in vivo embryonic development, such as separation of the endodermal and ectoderm lineages, formation of the pre-amniotic cavity, development of the primitive streak, and mesoderm lineage specification. Using single-cell multi-omics sequencing and fluorescence imaging techniques, these key biological events were verified at both the protein and transcriptome levels, closely recapitulating key features of the postimplantation gastrula-stage embryo. This model can be used to establish an in vitro drug screening platform that influences early embryonic development, providing reference for clinical drug use.

[0283] Although the present invention has been described to some extent above, it is clear that appropriate 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 embodiments, but rather falls within the scope of the claims, including equivalent replacements of each described element. The specification and drawings of the present invention are for illustrative purposes only and do not limit 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, and for example, "preferably," "according to one preferred embodiment," or "optionally" all indicate that each paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. In the entire text, features described as "preferably" are optional and should not be construed as essential, and the applicant reserves the right to waive or delete related preferred features at any time. [Accession number]

[0284] Human gastruloid-like stem cell line CCRM-hGOSC-1 CCTCC NO.C2022114 Human gastruloid-like stem cell line DYR0100-hGOSC-1 CCTCC NO.C2022115

Claims

1. A method for constructing a gastruloid stem cell line, comprising the steps of: (1) Induction of stem cells toward mesoderm: (1-1) Digesting stem cells into single cells and resuspending the cells to obtain a cell suspension 1; the stem cells are human embryonic stem cells or human induced pluripotent stem cells; The human embryonic stem cells are established human embryonic stem cells derived from an embryo within 14 days of fertilization that has not undergone in vivo development; (1-2) A step of taking cell suspension 1, centrifuging it, discarding the supernatant, adding a GK15-1 culture medium containing a ROCK inhibitor, and further resuspending the cells to obtain cell suspension 2, Preferably, the ratio of the cell suspension 2 to the GK15-1 culture medium containing a ROCK inhibitor in step (1-2) is 1 x 10 per mL of the GK15-1 culture medium containing a ROCK inhibitor. ６ cells; (1-3) A step of inoculating the cell suspension 2 obtained in (1-2) into a well plate pre-coated with Matrigel and culturing the cells, Preferably, the inoculation in step (1-3) is carried out by inoculating the cell suspension 2 at a density of 0.6 to 1 × 10 per square centimeter. ５ cells were seeded at a density of Preferably, the culture conditions in step (1-3) are 37°C and a volume concentration of carbon dioxide of 5.0 to 5.2%; (1-4) On the second day of culture, the old medium is removed and replaced with GK15-1 culture medium, and the medium is replaced every day until newborn mesoderm-like cells are obtained; (2) Induction of newborn mesoderm-like cells toward primordial germ cell-like cells: (2-1) When the newborn mesoderm-like cells obtained in (1-4) grow to 60 to 90% confluence, the cells are digested into single cells and resuspended to obtain a cell suspension 3; (2-2) A step of taking the cell suspension 3, centrifuging it, discarding the supernatant, and then adding a GK15-2 culture medium containing a ROCK inhibitor to further resuspend the cells to obtain a cell suspension 4; Preferably, the ratio of the cell suspension 3 to the GK15-2 culture medium containing a ROCK inhibitor in step (2-2) is 1 × 10 per mL of the GK15-2 culture medium containing a ROCK inhibitor. ５ cells; (2-3) a step of inoculating the cell suspension 4 obtained in (2-2) into a low-adhesion well plate to culture the cells as spheroids, and from the second day of culture, removing the old medium and replacing it with GK15-2 culture medium every day until cell spheroids containing primordial germ cell-like cells are obtained by culture, Preferably, in step (2-3), the initial cell amount per well is 0.5 to 1 × 10 ４ a step of generating cells; (3) Cell purification: (3-1) digesting the cell spheroids obtained in (2-3) into single cells and resuspending the cells in GK15-2 culture medium to obtain a cell suspension 5; (3-2) A step of selecting CD326 and CD49f double-positive cells from the cell suspension 5 obtained in (3-1), and resuspending the cells by adding a GK10 culture medium containing a ROCK inhibitor to obtain a cell suspension 6, Preferably, the GK10 culture medium containing a ROCK inhibitor in step (3-2) contains 1 x 10 per mL. ５ a step including cells; (4) Cell amplification: a step of inoculating the cell suspension 6 obtained in (3-2) into a well plate on which mitomycin C-treated MEFs have been previously laid as feeder layer cells, culturing the cells, and replacing the culture medium with fresh GK10 culture medium after 24 hours to obtain the gastruloid stem cell line, Preferably, the inoculation in step (4) is carried out by inoculating the cell suspension 6 at a density of 0.4 to 2 × 10 per square centimeter. ４ cells at a density of Preferably, the culture conditions in step (4) are 37°C and a volume concentration of carbon dioxide of 5.0%. A method comprising:

2. The components of the GK15-1 culture medium containing the ROCK inhibitor in (1-2) above are: the medium comprises 80-85% by volume of basal medium GMEM, 10-15% by volume of serum replacement KOSR, 1% by volume of penicillin-streptomycin double antibody, 1% by volume of 10 mM non-essential amino acids, 1% by volume of 200 mM GlutaMAX additive, 1% by volume of 100 mM sodium pyruvate additive, 0.1 mM β-mercaptoethanol, 25-200 ng / mL recombinant human activin A factor, 1-10 μM of glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021, and 5-20 μM of ROCK inhibitor; The components of the GK15-1 culture medium in (1-4) are: the medium comprises 80-85% by volume of basal medium GMEM, 10-15% by volume of serum replacement KOSR, 1% by volume of penicillin-streptomycin double antibody, 1% by volume of 10 mM non-essential amino acids, 1% by volume of 200 mM GlutaMAX additive, 1% by volume of 100 mM sodium pyruvate additive, 0.1 mM β-mercaptoethanol, 25-200 ng / mL recombinant human activin A factor, and 1-10 μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021; The components of the GK15-2 culture medium containing the ROCK inhibitor in (2-2) above are: the medium contains 80-85% by volume of basal medium GMEM, 10-15% by volume of serum replacement KOSR, 1% by volume of penicillin-streptomycin double antibody, 1% by volume of 10 mM non-essential amino acids, 1% by volume of 200 mM GlutaMAX additive, 1% by volume of 100 mM sodium pyruvate additive, 0.1 mM β-mercaptoethanol, 100-500 ng / mL recombinant human bone morphogenetic protein 4, 50-200 ng / mL recombinant human stem cell factor, 1000-5000 U / mL recombinant human leukemia inhibitory factor, 50-250 ng / mL recombinant human epidermal growth factor, and 5-20 μM ROCK inhibitor; The components of the GK15-2 culture medium in (2-3) and (3-1) are: The medium contains 80-85% by volume of basal medium GMEM, 10-15% by volume of serum replacement KOSR, 1% by volume of penicillin-streptomycin double antibody, 1% by volume of 10 mM non-essential amino acids, 1% by volume of 200 mM GlutaMAX additive, 1% by volume of 100 mM sodium pyruvate additive, 0.1 mM β-mercaptoethanol, 100-500 ng / mL recombinant human bone morphogenetic protein 4, 50-200 ng / mL recombinant human stem cell factor, 1000-5000 U / mL recombinant human leukemia inhibitory factor, and 50-250 ng / mL recombinant human epidermal growth factor; The components of the GK10 culture medium containing the ROCK inhibitor in (3-2) are: The medium contains 80 to 85% by volume of basal medium GMEM, 10% by volume of serum substitute KOSR, 2.5% by volume of fetal bovine serum FBS, 1% by volume of penicillin-streptomycin double antibody, 1% by volume of 10 mM non-essential amino acids, 1% by volume of 200 mM GlutaMAX additive, 1% by volume of 100 mM sodium pyruvate additive, 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, 50 to 200 ng / mL recombinant human stem cell factor, and 5 to 20 μM ROCK inhibitor; The components of the GK10 culture medium in (4) are:

2. The method for constructing a gastruloid stem cell line according to claim 1, characterized in that the medium contains 80-85% by volume of basal medium GMEM, 10% by volume of serum substitute KOSR, 2.5% by volume of fetal bovine serum (FBS), 1% by volume of penicillin-streptomycin double antibody, 1% by volume of 10 mM non-essential amino acids, 1% by volume of 200 mM GlutaMAX additive, 1% by volume of 100 mM sodium pyruvate additive, 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, and 50-200 ng / mL recombinant human stem cell factor.

3. A gastruloid stem cell line constructed using the method of claim 1 or 2.

4. 4. The gastruloid stem cell line of claim 3, which has been deposited at the China Center for Typical Cultures Depositary, with the name of the culture being human gastruloid-like stem cell line CCRM-hGOSC-1, the accession number being CCTCC NO. C2022114, and the date of deposit being April 27, 2022.

5. The gastruloid stem cell line of claim 3, which has been deposited at the China Center for Typical Cultures Depositary, has the name of the culture as human gastruloid-like stem cell line DYR0100-hGOSC-1, has the accession number CCTCC NO. C2022115, and was deposited on April 27, 2022.

6. Use of the gastruloid stem cell line of claim 3 in constructing a gastruloid model.

7. A gastruloid model obtained by inducing differentiation using the gastruloid stem cell line according to claim 3.

8. A method for constructing a gastruloid model, comprising inducing differentiation of the gastruloid stem cell line described in claim 3, characterized in that the differentiation induction is in vivo or in vitro.

9. The in vivo differentiation induction is The method comprises the steps of: obtaining the gastruloid stem cell line according to claim 3, resuspending it in GK10 culture medium, injecting the cell suspension into the testis of a mouse, and culturing it for 10 to 20 days to obtain the gastruloid model; Preferably, the animal is an immunodeficient mouse; More preferably, the animal is a BALB / c nude mouse.

10. The injection dose was 2 to 8 x 10 ４ The method of constructing a tumor cell according to claim 9, characterized in that the cells are injected.

11. The in vitro differentiation induction is (1) When gastruloid stem cells grew to 60-90% confluence, the cells were digested into single cells and resuspended in GK10 culture medium to obtain cell suspension 7. CD326 and CD49f double-positive cells in cell suspension 7 were selected and centrifuged, and 1.5-1.75 × 10 cells were cultured in mTR culture medium. ４ Resuspend the cells at a concentration of 6.0-7.0 x 10 cells / mL and plate them in a low-adhesion well plate at 6.0-7.0 x 10 cells / mL. ３ seeding the cells and culturing them until the gastruloid stem cells are organized into a three-dimensional structure; (2) adding E6BIN medium to the mTR culture medium in (1) without discarding it, and culturing until the amniotic cavity is formed and mesendoderm lineage specification is complete, thereby obtaining a gastruloid model; The method for constructing a cell culture medium according to claim 8, wherein the culture conditions in steps (1) and (2) are preferably 37°C and a volume concentration of carbon dioxide of 5.0%.

12. The components of the mTR culture solution in step (1) are 99% by volume of mTeSR ＴＭ 12. The method for constructing a gastruloid model according to claim 11, characterized in that it contains a complete culture medium, 1% by volume of penicillin-streptomycin double antibody, and 5 to 20 μM of a ROCK inhibitor.

13. The method for constructing a gastruloid model according to claim 11, characterized in that the components of the E6BIN medium in step (2) include, by volume, 100% Essential 6 medium, 20 ng / mL recombinant human fibroblast growth factor 2, 50 ng / mL recombinant human noggin protein, and 5 μM IWP-2.

14. The gastruloid model is obtained by injecting the gastruloid stem cell line described in claim 3 into the testes of an animal and culturing it, and the organ is a tissue organ derived from three germ layers: ectoderm, mesoderm, and endoderm, and preferably the culture period is 30 to 90 days.

15. A method for constructing an organ primordium model, comprising the steps of: taking the gastruloid stem cell line according to claim 3, resuspending it in GK10 culture medium, injecting the cell suspension into the testis of a mouse, and culturing it for 30 to 90 days to obtain the organ primordium model; Preferably, the animal is an immunodeficient mouse; More preferably, the method for constructing a primordium model is characterized in that the animal is a BALB / c nude mouse.

16. 2-8 x 10 in one testicle ４ The method of claim 16, characterized in that the cells are injected.

17. After culturing for 30 to 40 days, a neuroectoderm model, and / or a primordial germ cell model, and / or an amniotic epithelial cell model is obtained; After culturing for 40-50 days, a neuroepithelial cell model was obtained. The method for constructing an organ primordium model according to claim 16, characterized in that an intestinal organ primordium model, and / or a muscle primordium model, and / or a cartilage primordium model, and / or a nerve primordium model, and / or a skin primordium model is obtained when cultured for 70 to 90 days.

18. Use of a gastruloid stem cell line described in any one of claims 3 to 5, or a gastruloid model described in claim 6, or an organ primordium model described in claim 15, or a tissue or organ derived from said cell line, gastruloid model, or organ primordium model, or a culture thereof, in researching the developmental mechanisms of early human embryos.

19. Use of a gastruloid stem cell line described in any one of claims 3 to 5, or a gastruloid model described in claim 6, or an organ primordium model described in claim 15, or a tissue or organ derived from said cell line, gastruloid model, or organ primordium model, or a culture thereof, in diagnostic and / or therapeutic strategies for human early embryonic development disorders.

20. Use of a gastruloid stem cell line described in any one of claims 3 to 5, or a gastruloid model described in claim 6, or an organ primordium model described in claim 15, or a tissue or organ derived from said cell line, gastruloid model, or organ primordium model, or a culture thereof, in screening, verifying, evaluating, assessing or researching the efficacy of drugs for preventing and / or treating human early embryonic developmental disorders.

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