A method for constructing a gastrula-like stem cell line and its application

Abstract

The present invention discloses a method for constructing and applying a gastrula-like stem cell line. This study establishes a gastrula-like stem cell line that can be stably passaged. The cell retains the potential of stem cells to a certain extent and expresses genes and proteins of the inner, middle and outer germ layers of cells in the gastrula development stage. Its characteristics are consistent with those of cells in the gastrula stage, and it is possible to better reproduce the key characteristics of cells in the gastrula stage. Using this stem cell line, a three-dimensional gastrula-like body model that can simulate gastrula development can be constructed in mice in vivo or in vitro, and key biological events such as the separation of the inner and outer germ lineages, the formation of the anterior amniotic cavity, the appearance of the primitive streak, and the specialization of the mesoderm lineage during embryonic development in vivo can be partially reproduced. It is possible to better reproduce the key characteristics of the gastrula-stage embryo after implantation. The application of this model can establish a drug screening platform that affects early embryonic development in vitro, providing a 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 a gastrula-like stem cell line and its application, and specifically relates to a method for constructing a gastrula-like stem cell line, a gastrula-like stem cell line obtained by the method, and the construction and application of a model derived from the cell line. Background Art

[0002] During human embryogenesis, the fertilized egg forms tissues and organs through cell division, proliferation, and specialization, forming a highly complex individual. Embryonic development during the first three weeks after fertilization is a critical stage in human embryogenesis, particularly from the periimplantation stage to the gastrula stage, during which cells undergo lineage specification, rearrange themselves, and form a rudimentary embryonic form. Developmental problems during this stage can lead to miscarriage or birth defects. Understanding the mechanisms of early human development is crucial for developmental biology and regenerative medicine. However, due to technical and ethical limitations and limited sample sizes, our understanding of early human embryonic development is limited. Our current understanding of early human embryonic development is largely based on histological and anatomical studies of Carnegie embryos, and much remains to be explored.

[0003] Currently, breakthroughs have been made in the study of human pre-implantation and peri-implantation embryonic development. Researchers are now able to culture human embryos in vitro to embryonic day 14 before gastrulation, or induce human pluripotent stem cells (hPSCs) into pre-implantation blastocysts. Combined with single-cell multi-omics sequencing and fluorescence imaging technologies, this has opened up new avenues for studying human embryonic development and greatly expanded our understanding of the characteristics and mechanisms of human embryonic development from implantation to gastrulation.

[0004] Although some studies have attempted to use hPSCs to establish a three-dimensional gastrula-like body model to simulate the mutually exclusive separation of cells of the three germ layers during the gastrula development stage, this model lacks key embryonic structures (two germinal discs, amniotic cavity, and yolk sac), and no neural cell lineage differentiation was observed. Therefore, an ideal gastrula-like body research model has not yet been established.

[0005] Therefore, there is an urgent need for a method to construct gastrula-like stem cells that can be stably passaged, to obtain gastrula-like stem cells that can retain the pluripotency of stem cells, express genes and proteins of the three germ layers of cells in the inner, middle and outer germ layers during the gastrula development stage, and have the same characteristics as the cells in the gastrula stage, so as to better reproduce the key characteristics of cells in the gastrula stage and provide an ideal model for human embryo research. Summary of the Invention

[0006] In order to solve the above-mentioned technical problems in the existing technology, this study established gastrula-like stem cells that can be stably passaged. These gastrula-like stem cells retain the pluripotency of stem cells to a certain extent, and express genes and proteins of the inner, middle and outer germ layers of cells in the gastrula development stage. Their characteristics are consistent with those of embryonic cells in the gastrula stage, and can better reproduce the key cell characteristics of the gastrula stage.

[0007] By inducing the differentiation of such gastrula-like stem cells, a three-dimensional gastrula-like body model capable of simulating gastrula development can be constructed in vitro or in mice, and can partially reproduce key biological events during in vivo embryonic development, such as the separation of the ectoderm and endoderm lineages, the formation of the anterior amniotic cavity, the appearance of the primitive streak, and the specialization of the mesoderm lineage. Combined with single-cell multi-omics sequencing and fluorescence imaging technology, the above key biological events have been verified at both the protein and transcriptome levels, and can well reproduce the key characteristics of post-implantation gastrula-stage embryos. The application of this model can establish an in vitro drug screening platform that affects early embryonic development, providing a reference for clinical drug use.

[0008] Furthermore, inducing differentiation of this type of gastrula stem cells can form prototype models of tissue organs such as neural epithelium, smooth muscle, and intestine, which are derived from the three germ layers of the outer, middle, and endoderm.

[0009] The first object of the present invention is to provide a method for constructing the aforementioned gastrula-like stem cell line.

[0010] The construction method comprises the following steps:

[0011] (1) Induction of stem cells towards mesoderm:

[0012] (1-1) Digesting stem cells into single cells, centrifuging, and resuspending the cells to obtain 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 embryos within 14 days of fertilization that have not undergone in vivo development;

[0013] (1-2) Cell suspension 1 was centrifuged and the supernatant was discarded. GK15-1 culture medium containing ROCK inhibitor was added to further resuspend the cells to obtain cell suspension 2;

[0014] Preferably, the ratio of the cell suspension 2 to the GK15-1 culture medium containing ROCK inhibitor in step (1-2) is: 1×10 6 cells;

[0015] (1-3) The cell suspension 2 obtained in (1-2) was inoculated into a well plate coated with Matrigel in advance and cultured;

[0016] Preferably, the inoculation in step (1-3) is to inoculate the cell suspension 2 at a concentration of 0.6 to 1×10 5 Density inoculation per square centimeter;

[0017] Preferably, the culture conditions in steps (1-3) are 37° C. and a carbon dioxide concentration of 5.0-5.2% by volume, more preferably, a carbon dioxide concentration of 5.0% by volume;

[0018] (1-4) On the second day of culture, remove the old culture medium and replace it with GK15-1 culture medium. Change the medium every day until newborn mesoderm-like cells are obtained.

[0019] (2) Induction of nascent mesoderm-like cells into primordial germ cell-like cells:

[0020] (2-1) When the neonatal mesoderm-like cells obtained in (1-4) grow to 60-90% confluence, the cells are digested into single cells, centrifuged, and the cells are resuspended to obtain cell suspension 3;

[0021] (2-2) Cell suspension 3 was centrifuged and the supernatant was discarded. GK15-2 culture medium containing ROCK inhibitor was added to further resuspend the cells to obtain cell suspension 4;

[0022] Preferably, the ratio of the cell suspension 3 to the GK15-2 culture medium containing ROCK inhibitor in step (2-2) is: 1×10 5 cells;

[0023] (2-3) Inoculate the cell suspension 4 obtained in (2-2) into a low-viscosity plate for spheroid culture; starting from the second day of culture, remove the old culture medium and replace it with GK15-2 culture medium. Change the medium every day until spheroids containing primordial germ cells are obtained;

[0024] Preferably, the initial cell amount per well of the spheroid culture in step (2-3) is 0.5 to 1×10 4 cells;

[0025] (3) Cell purification:

[0026] (3-1) Digest the cell spheres obtained in (2-3) into single cells and resuspend the cells in GK15-2 culture medium to obtain cell suspension 5;

[0027] (3-2) sorting CD326 and CD49f double-positive cells from the cell suspension 5 obtained in (3-1); adding GK10 culture medium containing ROCK inhibitor to resuspend the cells to obtain cell suspension 6;

[0028] Preferably, in step (3-2), each 1 mL of GK10 culture medium containing ROCK inhibitor contains 1x10 5 Double-positive cells;

[0029] (4) Cell expansion:

[0030] The cell suspension 6 obtained in (3-2) was inoculated into a well plate pre-plated with mitomycin C-treated mouse embryonic fibroblasts as feeder cells; after culturing for 24 hours, fresh GK10 culture medium was replaced, and the medium was changed every day to obtain the gastrula-like stem cell line;

[0031] Preferably, the inoculation in step (4) is to inoculate the cell suspension 6 at a rate of 0.4 to 2×10 4 The cell seeding density was 100 cells;

[0032] Preferably, the culture conditions in step (4) are 37° C. and the volume concentration of carbon dioxide is 5.0%.

[0033] Furthermore, the components of the GK15-1 culture medium containing ROCK inhibitor described in (1-2) include:

[0034] 80-85% by volume of basal medium GMEM, 10-15% by volume of serum substitute 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 supplement, 1% by volume of 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, as well as 25-200 ng / mL of recombinant human activin A factor, 1-10 μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021, and 5-20 μM ROCK inhibitor;

[0035] In a specific embodiment, the components of the GK15-1 culture medium containing ROCK inhibitor in (1-2) include:

[0036] The basal culture medium is 81% by volume GMEM, the serum substitute KOSR is 15% by volume, the penicillin-streptomycin double antibody is 1% by volume, the 10mM non-essential amino acids are 1% by volume, the 200mM GlutaMAX additive is 1% by volume, the 100mM sodium pyruvate additive is 1% by volume, 0.1mM β-mercaptoethanol, as well as 50ng / mL recombinant human activin A factor, 3μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021, and 10μM ROCK inhibitor.

[0037] Furthermore, the components of the GK15-1 culture medium described in (1-4) include:

[0038] 80-85% by volume of basal medium GMEM, 10-15% by volume of serum replacement KOSR, 1% by volume of penicillin-streptomycin, 1% by volume of 10 mM non-essential amino acids, 1% by volume of 200 mM GlutaMAX supplement, 1% by volume of 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, as well as 25-200 ng / mL of recombinant human activin A factor, and 1-10 μM of the glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021;

[0039] In a specific embodiment, the components of the GK15-1 culture medium in (1-4) include:

[0040] The basal culture medium is 81% by volume of GMEM, the serum substitute KOSR is 15% by volume, the penicillin-streptomycin double antibody is 1% by volume, the 10mM non-essential amino acids are 1% by volume, the 200mM GlutaMAX supplement is 1% by volume, the 100mM sodium pyruvate supplement is 1% by volume, 0.1mM β-mercaptoethanol, as well as 50ng / mL of recombinant human activin A factor and 3μM of glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021.

[0041] Furthermore, the components of the GK15-2 culture medium containing the ROCK inhibitor described in (2-2) include:

[0042] The medium includes 80-85% GMEM (basal medium), 10-15% KOSR (serum substitute), 1% penicillin-streptomycin dual antibody, 1% 10mM non-essential amino acids, 1% 200mM GlutaMAX supplement, 1% 100mM sodium pyruvate supplement, 0.1mM β-mercaptoethanol, 100-500ng / mL recombinant human bone morphogenetic protein 4, 50-200ng / mL recombinant human stem cell factor, 1000-5000U / mL recombinant human leukemia inhibitory factor, 50-250ng / mL recombinant human epidermal growth factor, and 5-20μM ROCK inhibitor.

[0043] In a specific embodiment, the components of the GK15-2 culture medium containing ROCK inhibitor in (2-2) include:

[0044] The basal culture medium consists of 81% by volume of GMEM, 15% by volume of serum substitute 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, 200 ng / mL of recombinant human bone morphogenetic protein 4, 100 ng / mL of recombinant human stem cell factor, 1000 U / mL of recombinant human leukemia inhibitory factor, 50 ng / mL of recombinant human epidermal growth factor, and 10 μM ROCK inhibitor.

[0045] Furthermore, the components of the GK15-2 culture medium described in (2-3) and (3-1) include:

[0046] The basal culture medium comprises 80-85% by volume of GMEM, 10-15% by volume of the serum substitute KOSR, 1% by volume of penicillin-streptomycin, 1% by volume of 10 mM non-essential amino acids, 1% by volume of 200 mM GlutaMAX supplement, 1% by volume of 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, 100-500 ng / mL of recombinant human bone morphogenetic protein 4, 50-200 ng / mL of recombinant human stem cell factor, 1000-5000 U / mL of recombinant human leukemia inhibitory factor, and 50-250 ng / mL of recombinant human epidermal growth factor.

[0047] In a specific embodiment, the components of the GK15-2 culture medium described in (2-3) and (3-1) include:

[0048] The basal culture medium consists of 81% by volume of GMEM, 15% by volume of serum substitute 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 supplement, 1% by volume of 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, 200 ng / mL of recombinant human bone morphogenetic protein 4, 100 ng / mL of recombinant human stem cell factor, 1000 U / mL of recombinant human leukemia inhibitory factor, and 50 ng / mL of recombinant human epidermal growth factor.

[0049] Furthermore, the components of the GK10 culture medium containing ROCK inhibitor described in (3-2) include:

[0050] The basal culture medium consists of 80-85% by volume of 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 supplement, 1% by volume of 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, 50-200 ng / mL of recombinant human stem cell factor, and 5-20 μM ROCK inhibitor.

[0051] In a specific embodiment, the components of the GK10 culture medium containing ROCK inhibitor in (3-2) include:

[0052] The basal culture medium consists of 83.5% by volume of 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 supplement, 1% by volume of 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, 100 ng / mL of recombinant human stem cell factor, and 10 μM ROCK inhibitor.

[0053] Furthermore, the components of the GK10 culture medium in (4) include:

[0054] The basal culture medium consists of 80-85% by volume of 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 supplement, 1% by volume of 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, and 50-200 ng / mL of recombinant human stem cell factor.

[0055] In a specific embodiment, the components of the GK10 culture medium in (4) include:

[0056] The basal culture medium consists of 83.5% GMEM by volume, 10% serum substitute KOSR by volume, 2.5% fetal bovine serum FBS by volume, 1% penicillin-streptomycin double antibody by volume, 1% 10mM non-essential amino acids by volume, 1% 200mM GlutaMAX supplement by volume, 1% 100mM sodium pyruvate supplement by volume, 0.1mM β-mercaptoethanol, 10μM forskolin, 10μM rolipram, and 100ng / mL recombinant human stem cell factor.

[0057] The second object of the present invention is to provide a gastrula-like stem cell line, which is constructed using the above method.

[0058] Furthermore, the gastrula-like stem cell line was deposited with the China Center for Type Culture Collection, with the culture name being Human Gastrula-like Stem Cell Line CCRM-hGOSC-1, the deposit number being CCTCC NO. C2022114, and the deposit date being April 27, 2022. The gastrula-like stem cell line was constructed using human embryonic stem cells.

[0059] Furthermore, the gastrula-like stem cell line was deposited with the China Center for Type Culture Collection, with the culture name being human gastrula-like stem cell line DYR0100-hGOSC-1, the deposit number being CCTCC NO. C2022115, and the deposit date being April 27, 2022. The gastrula-like stem cell line was constructed using human induced pluripotent stem cells.

[0060] The third object of the present invention is to provide the use of the aforementioned gastrula-like stem cell line in constructing a gastrula-like model.

[0061] The fourth object of the present invention is to provide a gastrula-like embryo model, wherein the gastrula-like embryo model is obtained by inducing differentiation of the aforementioned gastrula-like embryo stem cell line.

[0062] The fifth object of the present invention is to provide a method for constructing a gastrula-like embryo model, wherein the construction method comprises inducing differentiation of the aforementioned gastrula-like embryo stem cell line, wherein the induced differentiation is in vivo induced differentiation or in vitro induced differentiation.

[0063] Furthermore, the in vivo differentiation induction comprises the following steps:

[0064] The aforementioned gastrula-like stem cell line was resuspended in GK10 culture medium, and the cell suspension was injected into the mouse testis and cultured for 10 to 20 days to obtain the gastrula-like embryo model;

[0065] Preferably, the animal is an immunodeficient mouse;

[0066] More preferably, the animal is a BALB / c nude mouse.

[0067] Furthermore, the injection is to inject the cell suspension into the mouse testicle through the vas deferens of the mouse.

[0068] Furthermore, the injection volume is 2 to 8 × 10 4 cell.

[0069] Furthermore, the in vitro differentiation induction comprises the following steps:

[0070] (1) When the gastrula-like embryonic stem cells grow to 60-90% confluence, the cells are 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 are sorted; centrifuged, and mTR culture medium is used to culture the cells at a rate of 1.5-1.75×10 4 Resuspend the cells at a concentration of 10 cells / mL and inoculate into low-viscosity well plates at a concentration of 6.0-7.0 x 10 cells per well. 3 cells; culture until the gastrula-like stem cells assemble into a three-dimensional structure;

[0071] (2) Without discarding the mTR culture medium in (1), E6BIN culture medium is added; culture is carried out until the amniotic cavity is formed and the mesendoderm lineage is fully specified, thereby obtaining a gastrula-like embryo model.

[0072] Preferably, the amount of the E6BIN culture medium added is 50-200 μL / well.

[0073] Preferably, the culture conditions in steps (1) and (2) are 37° C. and a volume concentration of carbon dioxide of 5.0%.

[0074] Furthermore, the mTR culture medium in step (1) comprises: 99% by volume of mTeSR TM 1 Complete culture medium, 1% penicillin-streptomycin double antibody by volume, 5-20 μM ROCK inhibitor.

[0075] In a specific embodiment, the mTR culture medium composition of step (1) comprises: mTeSR with a volume ratio of 99% TM 1 complete culture medium, 1% penicillin-streptomycin double antibody by volume, 10 μM ROCK inhibitor.

[0076] Furthermore, the E6BIN culture medium in step (2) comprises: 100% by volume of Essential 6 culture medium, 20 ng / mL recombinant human fibroblast growth factor 2, 50 ng / mL recombinant human noggin protein, and 5 μM IWP-2.

[0077] The sixth object of the present invention is to provide an organ prototype model, wherein the gastrula-like embryo model is obtained by injecting the aforementioned gastrula-like embryo stem cell line into animal testicles for culture, and the organ is a tissue organ derived from the ectodermal, mesodermal, and endoderm layers;

[0078] Preferably, the culture days are 30 to 90 days.

[0079] A seventh object of the present invention is to provide a method for constructing an organ prototype model, the method comprising the following steps: resuspending the aforementioned gastrula-like stem cell line in GK10 culture medium, injecting the cell suspension into mouse testicles, and culturing for 30 to 90 days to obtain the organ prototype model;

[0080] Preferably, the animal is an immunodeficient mouse;

[0081] More preferably, the animal is a BALB / c nude mouse.

[0082] Furthermore, the injection is to inject the cell suspension into the mouse testicle through the vas deferens of the mouse.

[0083] Furthermore, 2 to 8 × 10 4 cell;

[0084] Furthermore, a neural ectoderm model, and / or a primordial germ cell model, and / or an amniotic epithelial cell model is obtained after 30 to 40 days of culture; a neural epithelial cell model is obtained after 40 to 50 days of culture; and an intestinal organoid prototype model, and / or a muscle prototype model, and / or a cartilage prototype model, and / or a neuron prototype model, and / or a skin prototype model is obtained after 70 to 90 days of culture.

[0085] The eighth purpose of this patent is to provide the aforementioned gastrula-like stem cell line, or the aforementioned gastrula-like model, or the aforementioned organ prototype model, or the tissue or organ derived from the cell line, gastrula-like model, organ prototype model or its culture in the study of the mechanism of human early embryonic development.

[0086] The ninth objective of this patent is to provide the aforementioned gastrula-like stem cell line, or the aforementioned gastrula-like model, or the aforementioned organ prototype model, or the tissue or organ derived from the cell line, gastrula-like model, organ prototype model, or their culture in diagnostic strategies and / or therapeutic strategies for human early embryonic developmental diseases.

[0087] The tenth objective of this patent is to provide the aforementioned gastrula-like stem cell line, or the aforementioned gastrula-like model, or the aforementioned organ prototype model, or the tissue or organ derived from the cell line, gastrula-like model, organ prototype model, or its culture in screening, verifying, evaluating, assessing or studying the efficacy of drugs for preventing and / or treating human early embryonic development diseases.

[0088] The penicillin-streptomycin dual antibiotic solution of the present invention contains 10,000 units / mL of penicillin and 10,000 μg / mL of streptomycin.

[0089] The ROCK inhibitors of the present invention include but are not limited to Y-27632, ROCK-IN-1, and Chroman.

[0090] The gastrula-like stem cell line CCRM-hGOSC-1 described herein is a novel cell line derived in vitro from human embryonic stem cells, and its cellular properties resemble those of human gastrula stem cells. In a specific embodiment, a gastrula-like embryo model obtained by injecting the gastrula-like stem cell line CCRM-hGOSC-1 into nude mouse testes for 10-20 days exhibits characteristics of a human gastrula-stage embryo and can simulate gastrula formation. Furthermore, injection of CCRM-hGOSC-1 into nude mouse testes for 30-90 days can simulate the formation of organ embryos, resulting in a model similar to human organ embryos. The gastrula-like stem cell line CCRM-hGOSC-1, gastrula-like embryo model, organ embryo model, or tissues or organs derived from this cell line or gastrula-like embryo model, or their cultures, cannot develop into a human or animal individual due to the lack of cell types such as the trophoblast.

[0091] The CCRM-hGOSC-1 cells provided by the present invention have at least the following characteristics:

[0092] Feature 1: CCRM-hGOSC-1 cells proliferate rapidly, and clone formation can be seen 3 days after single-cell passaging. The cells are relatively uniform in size and morphology, appearing round or oval, with clear boundaries of cell clones.

[0093] Feature 2: CCRM-hGOSC-1 cells grow actively, have good cell activity, high cell culture stability, and have stable cell growth characteristics in vitro.

[0094] Feature 3: CCRM-hGOSC-1 cells were immunostained by cell slides. The cell population in the same clone simultaneously expressed three germ layer proteins: pluripotency genes OCT4 and SOX2, mesoderm genes EOMES and TBXT, and endoderm genes GATA4 and GATA6.

[0095] Feature 4: The chromosome structure and number of CCRM-hGOSC-1 cells are normal, with 44+XY chromosomes, indicating a diploid male cell line.

[0096] Feature 5: RNA sequencing revealed that CCRM-hGOSC-1 cells express markers for three germ layers, including the mesoderm genes MIXL1, EOMES, MESP1, WNT3, TBXT, and GSC; the endoderm genes ELF3, FOXA2, CXCR4, GATA4, GATA6, and SOX17; and the pluripotency markers POU5F1 (OCT4), NANOG, KLF4, and TFCP2L1. While developing multilineage specialization, the proliferating cell line retains stem cell pluripotency to a certain extent, exhibiting gene expression for multiple germ layer lineages with characteristics similar to those of gastrula-stage cells.

[0097] Feature 6: CCRM-hGOSC-1 cells injected into nude mouse testes can mimic human gastrulation for 10-20 days. In the 10-day testis, a blastocyst-like double blastoderm structure, as well as amniotic and yolk sac structures, were observed forming within the testicular lumen. OCT4- and SOX2-positive epiblast-like cell clusters formed an amniotic cavity. GATA6 / GATA4 / EOMES-positive cells indicated the migration and assembly of primitive endoderm-like cells to form a primary yolk sac-like structure. The epiblast and hypoblast were orderly arranged between the amniotic cavity and yolk sac, forming an embryo-like structure similar to CS5b and CS5c embryos. Furthermore, some embryo-like cells were observed to begin developing into gastrulation-like structures: epiblast cells underwent epithelial-mesenchymal transition (EMT), generating EOMES / T-positive, OCT4-reduced, and SOX2-negative gastrulating motor cells. At 20 days, gastrulation cells appeared, forming a gastrula-like structure: an amniotic cavity was further formed around OCT4-positive cells, which gradually differentiated into KRT7 / GATA2 / GATA3-positive amniotic-like epithelial cells on the top side of the amniotic cavity, and EOMES / T-positive gastrulation cells appeared. The yolk sac of some embryos was gradually covered by proliferating and migrating mesendoderm cells.

[0098] Feature 7: CCRM-hGOSC-1 injected into the nude mouse testes can simulate organoid formation over a 30-90-day period. The amniotic cavity proliferates and expands, and the neural ectoderm appears over a 30-40-day period. After 50-90 days, hGOSCs differentiate into neural epithelium, smooth muscle, and intestine, forming tissues and organs derived from the ectoderm, mesoderm, and endoderm. Endoderm: Immunofluorescence analysis of CDX2 and GATA6 labeling and morphological analysis with HE staining revealed that hGOSCs gradually formed intestinal structures over time after injection, forming intestinal organoids encased in a muscle layer by 90 days. Mesoderm: Immunofluorescence analysis of SOX9 labeling cartilage and ACTA2 labeling muscle, combined with HE staining, revealed that hGOSCs generated muscle and cartilage 90 days after injection. Ectoderm: A small number of primordial germ cells were found 30 days after hGOSCs were injected into the testicular lumen, marked by SOX17, BLIMP1, and TFAP2C. At the same time, amniotic epithelial cells were found near the primordial germ cells by labeling with GATA2, GATA3, and KRT7. Combining the morphology of HE staining and KER15 indicating keratinocytes and ACTA2 indicating muscles, the morphological structure of the skin appeared. HE combined with immunofluorescence staining showed that stem-like neuroepithelial cells appeared 40-50 days later, and the neuroepithelium differentiated into neurons 70-90 days later: OTX2 and SOX2 marked neuroepithelial or radioglial cells, and TUJ1 and DCX marked neuronal cells.

[0099] The gastrula-like stem cell line DYR0100-hGOSC-1 described herein is a novel cell line derived in vitro from pluripotent stem cells, and its cellular properties resemble those of gastrula stem cells. In a specific embodiment, a gastrula-like embryo model differentiated from the gastrula-like stem cell line DYR0100-hGOSC-1 exhibits characteristics of a human gastrula-stage embryo, can simulate gastrula formation, and is used to study morphological developmental characteristics and gene function during the gastrula stage. The gastrula-like stem cell line DYR0100-hGOSC-1, gastrula-like embryo model, organ prototype model, or tissues, organs, or cultures thereof derived from such cell line, gastrula-like embryo model, or organ prototype model cannot develop into individuals due to the lack of cell types such as the trophoblast.

[0100] The DYR0100-hGOSC-1 provided by the present invention has at least the following characteristics:

[0101] Feature 1: DYR0100-hGOSC-1 proliferates rapidly, with colony formation visible 3 days after single-cell passaging. The cells are relatively uniform in size and morphology, appearing round or oval, with clear colony boundaries.

[0102] Feature 2: DYR0100-hGOSC-1 grows actively, has good cell activity, high cell culture stability, and has stable cell growth characteristics in vitro culture.

[0103] Feature 3: Immunofluorescence staining of DYR0100-hGOSC-1 cells on cell slides revealed that the cell population within the same clone simultaneously expressed three germ layer proteins: the pluripotency genes OCT4 and SOX2; the mesoderm genes EOMES, TBXT, CDX2, and MIXL1; and the endoderm genes GATA4, GATA6, SOX17, and OTX2.

[0104] Feature 4: The chromosome structure and number of DYR0100-hGOSC-1 are normal, with 44+XY chromosomes, indicating a diploid male cell line.

[0105] Feature 5: DYR0100-hGOSC-1 cells retain a certain degree of stem cell pluripotency while undergoing multi-lineage cell specialization. They also exhibit gene expression patterns characteristic of cells from multiple germ-layer lineages, similar to those of gastrula-stage cells. RNA sequencing revealed that DYR0100-hGOSC-1 cells express markers for three germ-layer proteins, including the mesoderm genes MIXL1, EOMES, MESP1, WNT3, TBXT, and GSC; the endoderm genes ELF3, FOXA2, CXCR4, GATA4, GATA6, and SOX17; and the pluripotency markers POU5F1 (OCT4), NANOG, KLF4, TFCP2L1, and SOX2.

[0106] Feature 6: DYR0100-hGOSC-1 can complete spheroidization within 12 hours, with good cell activity and high induction stability, and has stable cell growth characteristics during the continuous 4-day gastruloid embryo culture.

[0107] Feature 7: At 0-1 days of gastrula induction, gastrula stem cells begin to assemble and form a stable three-dimensional structure, inside which there are cells of the epiblast (expressing OCT4, SOX2), primitive streak (expressing TBXT, MIXL1), endoderm (expressing SOX17, OTX2, FOXA2), as well as a small amount of amniotic epithelial cells (expressing CDX2) and extraembryonic mesoderm cells (expressing LUM).

[0108] Feature 8: When the gastrula is induced for 2-4 days, the lineage separation of the epiblast (expressing OCT4 and SOX2), the mesoderm developed from the primitive streak (expressing EOMES and MESP1), and the endoderm (expressing SOX17, OTX2, and FOXA2) begins to appear inside the three-dimensional model formed by the assembly of gastrula stem cells. The OCT4 and SOX2-positive epiblast cells form a cavity similar to the amniotic cavity.

[0109] Feature 9: The gastrula-like state after 4 days of induction culture is similar to that of a Carnegie stage 7 embryo.

[0110] Beneficial effects:

[0111] The method for constructing a gastrula-like stem cell line of the present invention, the gastrula-like stem cell line obtained by the method, and the model constructed from the cell line provide a platform for in vitro research on human gastrula development, aiming to understand the complexity of human early embryonic development and provide a research platform for the development of clinical treatments for early embryonic peri-implantation diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0112] Figure 1 is a cell morphology diagram provided in Example 2 of the present invention, wherein: Figure 1 A is human embryonic stem cell, Figure 1 B is a newborn mesoderm-like cell. Figure 1 C is a primordial germ cell ball. Figure 1 D is gastrula-like stem cells;

[0113] Figure 2 This is a growth curve diagram provided in Example 2 of the present invention;

[0114] Figure 3 This is a graph showing the immunofluorescence identification results provided in Example 2 of the present invention, wherein OCT4 and SOX2 are pluripotency genes, EOMES and TBXT are mesoderm genes, and GATA4 and GATA6 are endoderm genes; scale bar, 100 μm;

[0115] Figure 4 This is a chromosome karyotype analysis diagram provided in Example 2 of the present invention;

[0116] Figure 5 This is an RNA sequencing analysis diagram provided in Example 2 of the present invention;

[0117] Figure 6 This is the in vivo gastrula HE provided in Example 3 of the present invention, where the asterisk * indicates the amniotic cavity, and the broad arrow Points to amniotic epithelial cells, square ■ position points to yolk sac, narrow arrow ↑ points to epiblast cells, triangle ▲ points to primitive endoderm cells, and five-pointed star ★ position points to gastrulation cells; ruler, 100 μm;

[0118] Figure 7 This is the in vivo gastrula-like IF provided in Example 3 of the present invention, wherein OCT4 / SOX2 marks epiblast cells, GATA6 / GATA4 marks primitive endoderm cells, T+EOMES marks gastrulating motor cells, and KRT7 / GATA2 / GATA3 is used to mark amniotic epithelial cells; scale bar, 20 μm;

[0119] Figure 8The HE of the embryonic form of organ development in vivo provided in Example 4 of the present invention, wherein Figure 8 A is the prototype of the digestive tract organs derived from the endoderm. Figure 8 Figure 8B shows the prototype of the skin organ derived from the ectoderm (Ecdoderm), and Figure 8C shows the prototype of the cartilage organ derived from the mesoderm (Mesoderm);

[0120] Figure 9 This is the HE of the in vivo organ development prototype provided in Example 4 of the present invention, wherein GATA6 and CDX2 mark the digestive tract; KRT15 and ACTA2 mark the skin; and SOX9 marks the cartilage.

[0121] Figure 10 The different growth generations P1 ( Figure 10 A),P7( Figure 10 B),P20( Figure 10 C),P30 ( Figure 10 D) Cell morphology, scale bar, 100 μm;

[0122] Figure 11 This is a growth curve diagram provided in Example 6 of the present invention;

[0123] Figure 12 This is the immunofluorescence identification result provided in Example 6 of the present invention, scale: 100 μm;

[0124] Figure 13 This is a chromosome karyotype analysis diagram provided in Example 6 of the present invention;

[0125] Figure 14 This is an RNA sequencing analysis diagram of a gastrula-like stem cell line provided in Example 6 of the present invention;

[0126] Figure 15 This is a white light image of the gastrula-like embryo induction culture process from day 0 to day 4 provided in Example 9 of the present invention. Scale: 100 μm.

[0127] Figure 16 This is a white light image of the sampling process of gastrula-like embryos from day 0 to day 4 of induction culture provided in Example 9 of the present invention. Scale: 100 μm.

[0128] Figure 17 This is a growth curve of gastruloid embryos induced on day 1-4, as provided in Example 9 of the present invention;

[0129] Figure 18 This is the process of assembling three-dimensional structures of gastrula-like stem cells induced by one-day gastrula-like embryos provided in Example 9 of the present invention. IF, scale, 50 μm;

[0130] Figure 19This is the process of the gastrula model provided in Example 9 of the present invention being completed after 2-4 days of induction, during which the anterior amniotic cavity is formed and the mesendoderm lineage is specified. IF, scale bar, 50 μm;

[0131] Figure 20 This is an RNA sequencing analysis diagram of the gastrula-like embryo model provided in Example 9 of the present invention;

[0132] Biomaterial deposit information

[0133] The gastrula-like stem cell line with accession number CCTCC NO.C2022114 has been deposited in the China Center for Type Culture Collection on April 27, 2020, at Wuhan University, Wuhan, China. The classification name is human gastrula-like stem cell line CCRM-hGOSC-1.

[0134] The gastrula-like stem cell line with accession number CCTCC NO.C2022115 has been deposited in the China Center for Type Culture Collection on April 27, 2020, at Wuhan University, Wuhan, China. The classification name is human gastrula-like stem cell line DYR0100-hGOSC-1. DETAILED DESCRIPTION

[0135] 1. Method for constructing a gastrula-like stem cell line from human embryonic stem cells

[0136] The present invention discloses a method for converting human embryonic stem cells into a gastrula-like stem cell line and constructing a human in vitro post-implantation gastrula-like model derived from the cell line. The reagents, instruments, cell lines, etc. used in the present invention can all be purchased commercially.

[0137] The human embryonic stem cells were obtained from the Reproductive Medicine Center of Jiangsu Provincial People's Hospital, and the cell name was CCRM-hESCs-22 (46, XY).

[0138] (1) The sources of the culture medium components used for inducing human embryonic stem cells into human gastrula stem cell lines in vitro:

[0139] GMEM (Glasgow's MEM) medium: 11710035, purchased from Gibco, USA;

[0140] Serum replacement (confidential formula of Thermo Fisher Scientific): 10828028, purchased from Gibco, USA;

[0141] Fetal bovine serum: 12483020, purchased from Gibco, USA;

[0142] MEM non-essential amino acids: 11140076, purchased from Gibco, USA;

[0143] GlutaMAX TM Additive: 35050061, purchased from Gibco, USA;

[0144] Sodium pyruvate additive: 11360070, purchased from Gibco, USA;

[0145] Penicillin-streptomycin dual antibody: 15140122, Gibco, USA;

[0146] β-Mercaptoethanol: 21985023, purchased from Gibco, USA;

[0147] Recombinant human bone morphogenetic protein 4:314-BP was purchased from R&D Systems, USA;

[0148] Recombinant human stem cell factor: 7734-LF, purchased from R&D Systems, USA;

[0149] Recombinant human leukemia inhibitory factor: 225-SC, purchased from R&D Systems, USA;

[0150] Recombinant human epidermal growth factor: 236-EG, purchased from R&D Systems, USA;

[0151] Recombinant human activin A protein: 338-AC, purchased from R&D Systems, USA;

[0152] Glycogen synthase kinase-3 (GSK-3) inhibitors: CHIR 99021;

[0153] ROCK inhibitors: Y-27632 and HY-10071, purchased from MCE, USA;

[0154] Adenylate cyclase activator: Forskolin 1099, purchased from R&D Systems, USA;

[0155] PDE4 inhibitors: Rolipram, 0905, purchased from R&D Systems, USA;

[0156] (2) Sources of culture medium components used for inducing human pluripotent stem cells into post-implantation blastocysts:

[0157] mTeSR TM 1. Culture medium: #85850, purchased from STEMCELL Technologies, Canada;

[0158] Essential 6 medium: A1516401, purchased from Gibco, USA;

[0159] Recombinant human fibroblast growth factor 2: 3718-FB, purchased from R&D Systems, USA;

[0160] Recombinant human noggin protein: HY-P7051A, purchased from MCE, USA;

[0161] ROCK inhibitors: Y-27632 and HY-10071, purchased from MCE, USA;

[0162] WNT inhibitors: specific types include IWP-2 and S7085, purchased from Selleck, USA.

[0163] (3) Preparation of GK15-1 culture medium containing ROCK inhibitor Y-27632 in embryonic stem cell mesoderm orientation induction (stage 1):

[0164] The volume percentage of the basal culture medium is 81% GMEM, the volume percentage of the serum substitute KOSR is 15%, the volume percentage of the penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin) is 1%, the volume percentage of 10 mM non-essential amino acids is 1%, the volume percentage of 200 mM GlutaMAX supplement is 1%, the volume percentage of 100 mM sodium pyruvate supplement is 1%, 0.1 mM β-mercaptoethanol, as well as 50 ng / mL recombinant human activin A factor, 3 μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021, and 10 μM ROCK inhibitor Y-27632.

[0165] (4) Preparation of GK15-1 culture medium for mesoderm orientation induction of embryonic stem cells (stage 1):

[0166] The medium consists of 81% GMEM (basal medium), 15% KOSR (serum replacement), 1% penicillin-streptomycin (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 multiple cytokines: 50 ng / mL recombinant human activin A factor, 3 μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021.

[0167] (5) Preparation of GK15-2 culture medium containing ROCK inhibitor Y-27632 for induction of nascent mesoderm-like cells into primordial germ cell-like cells (second stage) and cell purification:

[0168] The basal culture medium consists of 81% GMEM by volume, 10% KOSR serum substitute, 1% penicillin-streptomycin dual antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10mM non-essential amino acids by volume, 1% 200mM GlutaMAX supplement by volume, 1% 100mM sodium pyruvate supplement by volume, 0.1mM β-mercaptoethanol, 100ng / mL recombinant human stem cell factor, 200ng / mL recombinant human bone morphogenetic protein 4, 1000U / mL recombinant human leukemia inhibitory factor, 50ng / mL recombinant human epidermal growth factor, and 10μM ROCK inhibitor Y-27632.

[0169] (6) Preparation of GK15-2 culture medium for induction of nascent mesoderm-like cells into primordial germ cell-like cells (second stage) and cell purification:

[0170] The basal culture medium consists of 81% GMEM by volume, 15% KOSR serum substitute, 1% penicillin-streptomycin dual antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% 10mM non-essential amino acids by volume, 1% 200mM GlutaMAX supplement by volume, 1% 100mM sodium pyruvate supplement by volume, 0.1mM β-mercaptoethanol, 200ng / mL recombinant human bone morphogenetic protein 4, 100ng / mL recombinant human stem cell factor, 1000U / mL recombinant human leukemia inhibitory factor, and 50ng / mL recombinant human epidermal growth factor.

[0171] (7) Preparation of GK10 culture medium containing ROCK inhibitor Y-27632 for in vitro expansion of gastrula-like stem cell lines:

[0172] The basal culture medium consists of 83.5% GMEM by volume, 10% serum substitute KOSR by volume, 2.5% fetal bovine serum FBS by volume, 1% penicillin-streptomycin dual antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin by volume), 1% 10 mM non-essential amino acids by volume, 1% 200 mM GlutaMAX supplement by volume, 1% 100 mM sodium pyruvate supplement by volume, 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.

[0173] (8) Preparation of GK10 culture medium for in vitro expansion of gastrula-like stem cell lines:

[0174] The basal culture medium consists of 83.5% by volume of GMEM, 10% by volume of serum substitute KOSR, 2.5% by volume of fetal bovine serum FBS, 1% by volume of penicillin-streptomycin dual antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), 1% by volume of 10 mM non-essential amino acids, 1% by volume of 200 mM GlutaMAX supplement, 1% by volume of 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, and 100 ng / mL of recombinant human stem cell factor.

[0175] Example 1 Construction of gastrula-like stem cell line CCRM-hGOSC-1

[0176] An embodiment of the present invention provides a gastrula-like stem cell line, named human gastrula-like stem cell line CCRM-hGOSC-1, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO.C2022114.

[0177] (1) Induction of embryonic stem cells into mesoderm (stage 1):

[0178] (1-1) The human embryonic stem cells were CCRM-hESCs-22 (46, XY) human embryonic stem cells provided by the Reproductive Medicine Center of Jiangsu Provincial People's Hospital; when the human embryonic stem cells grew to 80-90% confluence, the cells were digested into single cells using TrypLE Select, centrifuged at 1300 rpm for 3 minutes, and then resuspended in PBS to obtain cell suspension 1;

[0179] (1-2) Centrifuge cell suspension 1 and discard the supernatant. Add GK15-1 culture medium containing Y-27632 to resuspend the cells to obtain cell suspension 2. Each 1 mL of GK15-1 culture medium containing Y-27632 contains 1x10 6 cells;

[0180] (1-3) Cell suspension 2 was seeded into a six-well plate coated with matrigel in advance, with 8×10 cells per square centimeter. 5 Inoculate cells at a cell density of 100 cells, shake the cells in the six-well plate evenly and transfer to a 37°C, 5% CO2 incubator for culture;

[0181] (1-4) On the second day of culture, remove the old culture medium and replace it with GK15-1 culture medium. Change the medium every day until newborn mesoderm-like cells are obtained.

[0182] The above embryonic stem cell mesoderm direction induction step is on days 0 to 2 of the gastrula-like stem cell line construction method.

[0183] (2) Induction of nascent mesoderm-like cells into primordial germ cell-like cells (second stage):

[0184] (2-1) When the neonatal mesoderm-like cells obtained in (1-4) grew to 80-90% confluence, the cells were digested into single cells using TrypLESelect, centrifuged at 1300 rpm for 3 minutes using a horizontal centrifuge, and the cells were resuspended in PBS to obtain cell suspension 3;

[0185] (2-2) Centrifuge the cell suspension for 3 minutes and discard the supernatant. Add GK15-2 culture medium containing Y-27632 to further resuspend the cells. Each 1 mL of GK15-2 culture medium containing Y-27632 contains 1x10 5 cells, and obtain cell suspension 4;

[0186] (2-3) The cell suspension 4 obtained in (2-2) was re-inoculated into a round-bottomed transparent low-adhesion U-shaped 96-well plate for spherical culture, with 5x10 cells per well. 3 On the second day of culture, remove the old culture medium and replace it with GK15-2 culture medium. Change half of the medium every day until cell spheres containing primordial germ cells are obtained.

[0187] The above steps of inducing mesoderm-like cells to endoderm are on days 3 to 6 of the method for constructing a gastrula-like stem cell line.

[0188] (3) Cell purification:

[0189] (3-1) The cell spheres 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 resuspended in GK15-2 culture medium to obtain cell suspension 5;

[0190] (3-2) Use flow cytometry to separate CD326 and CD49f double-positive cells from the cell suspension 5 obtained in (3-1); centrifuge at 200 g for 3 minutes, add GK10 culture medium containing Y-27632 to resuspend the cells. Each 1 mL of GK10 culture medium containing Y-27632 contains 1x10 5 cells, and the resulting cell suspension was 6;

[0191] (4) Cell expansion:

[0192] Mouse embryonic fibroblasts treated with mitomycin C were revived one day in advance and plated as feeder cells; the cell suspension obtained in (3-2) was plated at 2x10 per square centimeter. 4 The cells were inoculated at a density of 100 cells per well onto the prepared feeder layer cells, shaken and placed in a 37°C, 5% CO2 incubator. Fresh GK10 culture medium was replaced after 24 hours. The culture medium was changed daily until colony formation was observed. The gastrula-like stem cell line CCRM-hGOSC-1 was obtained and deposited with the China Center for Type Culture Collection, with the deposit number CCTCC NO. C2022114.

[0193] Example 2 The biological characteristics of cells are described.

[0194] 1.1 Cell morphology observation

[0195] Observation of human gastrula stem cells CCRM-hGOSC-1 under an inverted microscope showed that the cells proliferated rapidly and clones were formed 2 days after single cell passage. The cells were relatively uniform in size and morphology, with round or oval shapes and clear boundaries of cell clones. Figure 1 shown.

[0196] 1.2 Growth curve determination

[0197] 1.2.1 Growth curve determination steps

[0198] When the confluence of human gastrula stem cell CCRM-hGOSC-1 reached 70-90%, the culture medium was removed and the cells were washed at least twice with PBS (0.01M, pH 7.4) to remove the old culture medium and the cells in poor condition. The cells were digested with TrypLESelect and observed under a microscope. The digested cells were collected and digested with GK10 medium until all cells were digested. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and GK10 medium was added. Each 1 mL of GK10 medium contained 1x10 5 cells.

[0199] Press 1x10 5 Seed the cells at a density of 10 cells per well (12-well plate) onto the prepared feeder layer, shake well, and incubate at 37°C, 5% CO2. After 24 hours, replace with fresh GK10 medium daily. Discard the medium from three wells every 24 hours, add TrypLE Select for digestion, resuspend the cells, and count the total number of cells in the three wells. Count each well three times and take the average value. Continue culturing the cells in the remaining wells until day 5. Change the medium daily.

[0200] 1.2.2 Growth curve measurement results

[0201] The measurement was continued for 5 days, and the growth curve data shown in Table 1 below were obtained.

[0202] Table 1 shows the growth curve data obtained from 5 consecutive days of measurement.

[0203] Table 1

[0204] Cultivation time (days) Average total number of cells 1 <![CDATA[1.35×10 5 ]]> 2 <![CDATA[2.23×10 5 ]]> 3 <![CDATA[3.73×10 5 ]]> 4 <![CDATA[5.28×10 5 ]]> 5 <![CDATA[7.91×10 5 ]]>

[0205] Based on the cell growth curve data in Table 1, we can get Figure 2 Schematic diagram of cell growth curve shown. Figure 2 The horizontal axis is the culture time (days), and the vertical axis is the number of cells (×10 5 indivual).

[0206] Refer to the growth curve data in Table 1 and Figure 2 As shown in the growth curve diagram, it can be seen that CCRM-hGOSC-1 cells grew well for 5 consecutive days.

[0207] In summary, CCRM-hGOSC-1 cells proliferate rapidly, have active cell growth, good cell activity, high cell culture stability, and have stable cell growth characteristics in vitro.

[0208] 1.3 Immunofluorescence identification

[0209] 1.3.1 Immunofluorescence identification steps

[0210] The small round glass slides were disinfected with 75% ethanol and sterilized by UV, then transferred to a cell culture dish. Fibronectin was used to increase the adhesion of the glass slides, and CCRM-hGOSC-1 cells were then inoculated and cultured according to normal cell subculture procedures.

[0211] CCRM-hGOSC-1 cells were cultured until passage, and small round slides were removed and placed in a dish for fixation with 4% PFA for 40 minutes;

[0212] Discard the 4% PFA fixative and wash three times with PBS, then add 5% BSA and block at room temperature for 2 hours;

[0213] Discard the 5% BSA blocking solution, add the diluted antibody in proportion, and incubate at 4°C overnight;

[0214] After discarding the primary antibody, wash three times with PBS, add the diluted secondary antibody and live cell staining solution Hoechst 33342 to the dish, and incubate at room temperature for 2 hours;

[0215] After discarding the secondary antibody, wash with PBS three times, add glycerol on the slide, take out the small round glass slide from the dish and turn it upside down on the slide with glycerol, and use nail polish to fix the position of the small glass slide, and take pictures with a confocal fluorescence microscope.

[0216] 1.3.2 Immunofluorescence identification results

[0217] Immunofluorescence identification results Figure 3 The proliferating cell lines were immunofluorescently stained on cell slides to identify genes of each germ layer: pluripotency genes OCT4 and SOX2, mesoderm genes EOMES and TBXT, and endoderm genes GATA4 and GATA6 were partially expressed in the cell clones.

[0218] 1.4 Chromosome karyotype analysis and identification

[0219] 1.4.1 Chromosome karyotype analysis and identification steps

[0220] Cell culture

[0221] When the cell confluence reached 70-90%, the culture medium was removed and the cells were washed at least twice with PBS (0.01M, pH 7.4) to remove old culture medium and detached cells in poor condition. The cells were then digested with 1-2 mL of TrypLE Select and observed under a microscope. The digested cells were collected and digested with GK10 medium until all cells were digested. The cells were centrifuged at 1000 rpm for 5 minutes and the supernatant was removed.

[0222] Colchicine treatment

[0223] 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 at 37°C for 3 hours to obtain colchicine-treated gastrula-like stem cells CCRM-hGOSC-1.

[0224] Hypotonic treatment

[0225] Preheat 0.56% KCl hypotonic solution to 37°C. Remove the culture dish and digest the colchicine-treated gastrulation-like stem cells CCRM-hGOSC-1 into a single-cell suspension. Transfer the cell suspension to a 15mL centrifuge tube and centrifuge at 2000 rpm for 10 minutes. Discard the supernatant. Add 9mL of 0.56% KCl hypotonic solution, preheated to 37°C, to the cell pellet. Gently pipette approximately 50 times with a rubber-tipped glass pipette. Incubate at 37°C for 40 minutes.

[0226] fixed

[0227] Prepare fixative (methanol: glacial acetic acid = 4:1) and mix thoroughly at room temperature. Add 1 mL of fixative to the cells, gently pipette to mix, and centrifuge at 300 g for 10 minutes. Discard the supernatant, add 10 mL of fresh fixative, gently pipette to a single-cell suspension, and fix at room temperature for 1 hour. Centrifuge at 300 g for 10 minutes. Discard the supernatant, add 10 mL of fresh fixative, gently pipette to resuspend the cells, and fix at room temperature for 30 minutes. Centrifuge at 300 g for 10 minutes. Discard the supernatant and resuspend the cells in a small amount of fixative (0.2-0.6 mL) depending on the size of the cell pellet.

[0228] Producer

[0229] Light an alcohol lamp and take a clean glass slide from distilled water without draining it. With one hand, tilt the water-containing slide and place it on a waste liquid tank. With the other hand, use a pipette to draw up the cell suspension and drop it 30-60 cm above the slide. Place one drop of cell suspension on three different positions of each slide. Immediately, burn the back of the slide five times back and forth on the alcohol lamp. Mark the slide and transfer it to a 37°C oven for baking overnight.

[0230] dyeing

[0231] Place the human gastrula stem cell slides in an 80°C oven at 37°C for 2.5 hours and then develop the bands. Preheat 0.25% Trypsin-EDTA in advance to ensure it reaches 37°C before use. Immerse the slides in 0.25% Trypsin-EDTA for 30-40 seconds. Remove the slides and rinse both sides of the slide under a gentle stream of water 2-3 times. Stain the slides in Giemsa stain preheated at 37°C for approximately 10 minutes. Rinse both sides of the slide 2-3 times with tap water and then blot dry with lens paper.

[0232] Microscopic examination

[0233] Under a low-power microscope, well-dispersed mitotic phases of moderate length were selected, and then observed and photographed with an oil immersion lens to obtain the results of chromosome karyotype analysis.

[0234] 1.4.2 Chromosome karyotype analysis results

[0235] The results of chromosome karyotype analysis were as follows Figure 4 Please refer to Figure 4 Chromosome karyotype analysis showed that the chromosome structure and number of the cells were normal, with the number of chromosomes being 44+XY, belonging to a diploid male cell line.

[0236] 1.5 RNA Sequencing Identification

[0237] 1.5.1 RNA Sequencing Identification Steps

[0238] When CCRM-hGOSC-1 cells reached 70-90% confluence, the culture medium was removed and the cells were washed at least three times with PBS (0.01M, pH 7.4) to remove old culture medium and detached cells in poor condition. DYR0100-hGOSC-1 cells were digested into single cells using TrypLE Select, harvested, and digestion terminated with GK10 medium. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant removed, and 1 mL of Trizol was added and stored in a low-temperature refrigerator (-80°C).

[0239] RNA was extracted from DYR0100-hGOSC-1 cell samples using the phenol-chloroform method.

[0240] The cell sample was taken out of the low-temperature refrigerator, melted on ice, and 200 μl of chloroform was added. After vigorous shaking, the sample was placed at room temperature for 3 minutes and centrifuged at 12,000 rpm in a desktop high-speed centrifuge precooled to 4°C for 15 minutes.

[0241] After centrifugation, the sample was transferred to ice, and the supernatant was taken. According to the liquid volume of the supernatant, isopropanol was added at a ratio of 1:1, and glycogen was added at a ratio of 200:1. After shaking, the sample was stored in a low-temperature (-80°C) refrigerator for 30 minutes. The sample was taken out, thawed on ice, and centrifuged at 12000 rpm in a desktop high-speed centrifuge precooled to 4°C for 15 minutes.

[0242] The supernatant was discarded, and the cell pellet was washed once with enzyme-free 75% alcohol. The cell pellet was centrifuged at 12,000 rpm for 10 minutes in a tabletop high-speed centrifuge precooled to 4°C. The supernatant was discarded, and the remaining 75% alcohol was dried in a fume hood.

[0243] After drying, the cell samples were redissolved by adding 10-20 μl of enzyme-free water according to the amount of RNA, and then transferred to enzyme-free EP tubes for sequencing.

[0244] 1.5.2 RNA Sequencing Identification Results

[0245] RNA sequencing identification results Figure 5 As shown. Transcriptome sequencing comparison of CCRM-hGOSC-1 proliferating cells and hESCs (human embryonic stem cells, hESCs) revealed that compared with hESCs, proliferating cells had increased expression of multi-lineage cell (ectoderm, endoderm, mesoderm, amnion, primordial germ cell) marker genes. Specifically, the expression of mesoderm genes MIXL1, EOMES, MESP1, WNT3, TBXT, GSC and endoderm genes ELF3, FOXA2, CXCR4, GATA4, GATA6, SOX17 increased significantly. Although the expression of SOX2 decreased, POU5F1 (OCT4) and NANOG were still expressed. Increased expression of the pluripotency factors KLF4 and TFCP2L1 in the proliferative cell lineages indicates that the proliferative cell lineages remain pluripotent. While developing multi-lineage cell specialization, the proliferative cell lineages retain a certain degree of stem cell pluripotency and exhibit gene expression of cells of multiple germ layer lineages, with characteristics similar to those of cells at the gastrula stage.

[0246] Example 3: The cell line was injected into mice to form a gastrula-like embryo model

[0247] CCRM-hGOSC-1 cells have characteristics of the gastrula stage and can simulate gastrula formation, allowing for the study of morphological development and gene function during the gastrula stage. Injecting these cells into the mouse testis results in the formation of gastrula-like embryos within 10-20 days.

[0248] 1. Nude Mouse Testicular Injection

[0249] Preparation: Use a needle puller to make suitable capillary glass needles. The parameters of the needle puller are: Heat 515, Pull 100, Trip 75, Delay 75.

[0250] When CCRM-hGOSC-1 cells reached 70-90% confluence, the culture medium was removed and the cells were washed at least twice with PBS (0.01M, pH 7.4) to remove old culture medium and detached cells in poor condition. The cells were then digested with 1-2 mL of TrypLE and observed under a microscope. The digested cells were collected and digested with GK10 medium until all cells were digested. The cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was removed.

[0251] Resuspend in GK10 culture medium and adjust the cell density to 2x10 6 / mL; 5-6x10 5 cells.

[0252] After injection, the mice were fed normally.

[0253] 2. Verification of Gastrulation

[0254] 2.1 Testicular tissue sampling and preparation

[0255] Testicular tissues of the injected mice were collected on the 10th and 20th days after injection.

[0256] 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;

[0257] After 42 hours, the fixative was discarded and the testes were dehydrated at room temperature: 70% alcohol for 24 hours, then 80% alcohol for 2 hours, 90% alcohol for 2 hours, and 100% alcohol for 1 hour. The solution was then dehydrated in an alcohol:xylene ratio of 1:1 for 25 minutes, and the tissue was permeabilized with xylene for 25 minutes.

[0258] The tissue blocks were transferred to an embedding frame and immersed in paraffin wax (I) and paraffin wax (II) for 45 min each;

[0259] After embedding, the tissue was sliced into 5 μm sections, placed in a slide spreader to flatten the wax slide, and then the complete tissue-free sections were selected and mounted;

[0260] Bake the slides at 65°C overnight, equilibrate the slides at 37°C for 30 minutes, and then transfer to room temperature for long-term storage.

[0261] 2.2 HE staining (hematoxylin and eosin)

[0262] Paraffin sections were dewaxed and incubated in xylene (I) and xylene (II) at 37°C for 15 min each;

[0263] Hydrate the tissue using a gradient of alcohol at room temperature: 100% alcohol (I), 100% alcohol (II), 90% alcohol, 80% alcohol, and 70% alcohol. Each step takes 2 minutes, and the tissue is then transferred to tap water and soaked for 10 minutes.

[0264] Place the sections in a hematoxylin staining jar for 40 seconds and rinse with running water for 5 minutes;

[0265] Wash once in 1% HCl and rinse with running water for 10 minutes;

[0266] Place the sections in an eosin staining jar for 3 minutes;

[0267] After staining, the tissue sections were dehydrated in graded alcohols: 70% alcohol, 80% alcohol, 90% alcohol, 100% alcohol (I), 100% alcohol (II) for 2 minutes each, and xylene (I) and xylene (II) for 15 minutes each.

[0268] Drop resin on the tissue, cover with a coverslip, place in a 37°C oven for 2 hours, take out and store at room temperature, and photograph using an upright microscope.

[0269] 2.3 Immunofluorescence staining (IF)

[0270] The tissue sections were dewaxed, hydrated and transferred to tap water, and the steps were the same as those for HE staining;

[0271] Antigen retrieval: Prepare 200 mL of acidic antigen retrieval solution and add it to the antigen retrieval box. Transfer the sections to the retrieval box and microwave the antigens for 3 minutes on high heat and 7 minutes on low heat. Allow to cool naturally to room temperature.

[0272] Blocking: Wash the slides with PBS, draw a circle around the tissue using an immunohistochemistry pen, add 100 μl of 5% BSA solution inside the circle and incubate at room temperature for 2 hours;

[0273] Primary antibody incubation: remove as much liquid as possible from the tissue, re-cover the tissue with antibody diluted proportionally with 5% BSA, and incubate overnight at 4°C;

[0274] The antibodies on the tissue were removed and washed with PBS three times for 5 minutes each time;

[0275] Use 5% BSA 1:1000 dilution of fluorescent secondary antibody and Hoechst 33342, add to tissue slides, and incubate at room temperature for 2 hours; wash the slides 3 times with PBS, each time for 5 minutes;

[0276] The slides were mounted with glycerol and photographed using a confocal fluorescence microscope.

[0277] 2.4 HE and IF verification results of gastrula

[0278] After injection into mouse testes, the gastrula-like stem cell line CCRM-hGOSC-1 described herein formed a blastocyst-like double blastoderm structure, as well as amniotic and yolk sac structures, within the lumen of the testes at day 10. A pre-amniotic cavity-like cavity emerged within the OCT4- and SOX2-positive epiblast-like cell cluster; primitive endoderm-like cells, indicated by GATA6 / GATA4 / EOMES-positive cells, migrated and assembled to form a primary yolk sac-like structure; and the epiblast and hypoblast arranged themselves in an orderly fashion between the amniotic cavity and the yolk sac, forming an embryo-like structure similar to CS5b and CS5c embryos. Furthermore, within the 10-day testicular lumen, some embryo-like cells were observed to begin developing into gastrula-like structures: Epi-like cells underwent an epithelial-mesenchymal transition (EMT), generating EOMES / T-positive, OCT4-reduced, and SOX2-negative gastrulating motor cells.

[0279] At 20 days, gastrulation cells appeared, forming a gastrula-like structure, and a gastrula-like model was obtained: OCT4-positive cells further formed an amniotic cavity, and gradually differentiated into KRT7 / GATA2 / GATA3-positive amniotic epithelial cells on the top side of the amniotic cavity, and there were EOMES / T-positive gastrulation cells. The yolk sac of some embryos was gradually covered by proliferating and migrating mesendoderm cells ( Figure 6 、 Figure 7 ).

[0280] Example 4: The cell line was injected into mice to form an organ prototype model

[0281] The gastrula-like stem cell line CCRM-hGOSC-1 cells of the present invention can simulate the development of organ prototypes when injected into mouse testicles for 30-90 days.

[0282] 1. Nude Mouse Testicular Injection

[0283] Preparation: Use a needle puller to make suitable capillary glass needles. The parameters of the needle puller are: Heat 515, Pull 100, Trip 75, Delay 75.

[0284] When CCRM-hGOSC-1 cells reached 70-90% confluence, remove the culture medium and wash the cells at least twice with PBS (0.01M, pH 7.4) to remove old culture medium and detached cells in poor condition. Digest the cells with 1-2 mL of EDTA-Trypsin and observe them under a microscope. Continue digestion and continue until all cells are digested. Centrifuge at 1000 rpm for 5 minutes, and remove the supernatant.

[0285] Resuspend in GK10 culture medium and adjust the cell density to 2x10 6 / mL; 5-6x10 5 cells.

[0286] After injection, the mice were fed normally.

[0287] 2. Verification of Organ Prototype Formation

[0288] 2.1 Testicular tissue sampling and preparation

[0289] Testicular tissues were collected from the injected mice every 10 days from the 30th to the 90th day after injection.

[0290] 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;

[0291] After 42 hours, the fixative was discarded and the testes were dehydrated at room temperature: 70% alcohol for 24 hours, then 80% alcohol for 2 hours, 90% alcohol for 2 hours, and 100% alcohol for 1 hour. The solution was then dehydrated in an alcohol:xylene ratio of 1:1 for 25 minutes, and the tissue was permeabilized with xylene for 25 minutes.

[0292] The tissue blocks were transferred to an embedding frame and immersed in paraffin wax (I) and paraffin wax (II) for 45 min each;

[0293] After embedding, the tissue was sliced into 5 μm sections, placed in a slide spreader to flatten the wax slide, and then the complete tissue-free sections were selected and mounted;

[0294] Bake the slides at 65°C overnight, equilibrate the slides at 37°C for 30 minutes, and then transfer to room temperature for long-term storage.

[0295] 2.2 HE staining (hematoxylin and eosin)

[0296] Paraffin sections were dewaxed and incubated in xylene (I) and xylene (II) at 37°C for 15 min each;

[0297] Hydrate the tissue using a gradient of alcohol at room temperature: 100% alcohol (I), 100% alcohol (II), 90% alcohol, 80% alcohol, and 70% alcohol. Each step takes 2 minutes, and the tissue is then transferred to tap water and soaked for 10 minutes.

[0298] Place the sections in a hematoxylin staining jar for 40 seconds and rinse with running water for 5 minutes;

[0299] Wash once in 1% HCl and rinse with running water for 10 minutes;

[0300] Place the sections in an eosin staining jar for 3 minutes;

[0301] After staining, the tissue sections were dehydrated in graded alcohols: 70% alcohol, 80% alcohol, 90% alcohol, 100% alcohol (I), 100% alcohol (II) for 2 minutes each, and xylene (I) and xylene (II) for 15 minutes each.

[0302] Drop resin on the tissue, cover with a coverslip, place in a 37°C oven for 2 hours, take out and store at room temperature, and photograph using an upright microscope.

[0303] 2.3 Immunofluorescence staining (IF)

[0304] The tissue sections were dewaxed, hydrated and transferred to tap water, and the steps were the same as those for HE staining;

[0305] Antigen retrieval: Prepare 200 mL of acidic antigen retrieval solution and add it to the antigen retrieval box. Transfer the sections to the retrieval box and microwave the antigens for 3 minutes on high heat and 7 minutes on low heat. Allow to cool naturally to room temperature.

[0306] Blocking: Wash the slides with PBS, draw a circle around the tissue using an immunohistochemistry pen, add 100 μl of 5% BSA solution inside the circle and incubate at room temperature for 2 hours;

[0307] Primary antibody incubation: remove as much liquid as possible from the tissue, re-cover the tissue with antibody diluted proportionally with 5% BSA, and incubate overnight at 4°C;

[0308] The antibodies on the tissue were removed and washed with PBS three times for 5 minutes each time;

[0309] Fluorescent secondary antibodies and live cell staining solution Hoechst 33342 were diluted 1:1000 in 5% BSA and added to tissue slides for incubation at room temperature for 2 hours; the slides were washed with PBS three times for 5 minutes each time;

[0310] The slides were mounted with glycerol and photographed using a confocal fluorescence microscope.

[0311] 2.4 HE and IF results of three germ layer organ rudiments

[0312] After the gastrula-like stem cell line CCRM-hGOSC-1 of the present invention is injected into the mouse testis, the amniotic cavity further proliferates and expands at 30-40 days, and the neuroectoderm begins to appear, thus obtaining a neuroectoderm model. By 90 days, CCRM-hGOSC-1 can be seen to differentiate into neural epithelium, smooth muscle, intestine, and other tissues and organs derived from the ectoderm, mesoderm, and endoderm.

[0313] Endoderm: Immunofluorescence anti-CDX2 & GATA6 antibody labeling and HE staining morphological analysis showed that CCRM-hGOSC-1 gradually formed intestinal structures over time after injection. At 90 days, intestinal organoids wrapped in a muscle layer were formed, and an intestinal organoid prototype model was obtained.

[0314] Mesoderm: Immunofluorescence SOX9 marked cartilage and ACTA2 marked muscle. Combined with HE staining morphological analysis, it can be seen that CCRM-hGOSC-1 produced muscle and cartilage 90 days after injection, obtaining muscle prototype models and cartilage prototype models.

[0315] Ectoderm: SOX17, BLIMP1, and TFAP2C antibody labeling revealed a small number of primordial germ cells 30 days after CCRM-hGOSC-1 was injected into the testicular lumen, thus obtaining a primordial germ cell model; at the same time, amniotic epithelial cells were found near the primordial germ cells by labeling with GATA2, GATA3, and KRT7 antibodies, thus obtaining an amniotic cavity structure model; KER15 indicated keratinocytes, and ACTA2 indicated muscles. Combined with the morphology of HE staining, the morphological structure of the skin was found at 70 to 90 days of culture, thus obtaining a skin prototype model; OTX2 and SOX2 labeled neuroepithelium or radioactive glial cells, and TUJ1 and DCX labeled neuronal cells. Immunofluorescence staining revealed that the main stem-like neuroepithelial cells were found at 40 to 50 days, thus obtaining a neuroepithelial cell model. At 70 to 90 days, the neuroepithelium began to differentiate into neurons, thus obtaining a neural prototype model ( Figure 8 、 Figure 9 ).

[0316] That is, a neuroectoderm model, and / or a primordial germ cell model, and / or an amniotic epithelial cell model is obtained at 30 to 40 days of culture;

[0317] The neuroepithelial cell model was obtained after 40 to 50 days of culture;

[0318] After 70 to 90 days of culture, an intestinal organoid prototype model, and / or a muscle prototype model, and / or a cartilage prototype model, and / or a nerve prototype model, and / or a skin prototype model are obtained.

[0319] In summary, this study established a stably passaged gastrula-like stem cell line, CCRM-hGOSC-1. These cells retain stem cell pluripotency to a certain extent and express genes and proteins characteristic of cells from the inner, middle, and outer germ layers during gastrula development. Their characteristics are consistent with those of cells from the gastrula stage, and they can well reproduce the key characteristics of cells from the gastrula stage. This stem cell line can be used to construct a gastrula-like model in mice and form prototype models of tissues and organs derived from the outer, middle, and inner germ layers, such as the neural epithelium, smooth muscle, and intestine. This model can be used to establish an in vitro drug screening platform for drugs that affect early embryonic development, providing a reference for clinical drug use.

[0320] II. Methods for constructing a gastrula-like stem cell line from human induced pluripotent stem cells

[0321] The present invention also discloses a method for inducing human induced pluripotent stem cells into a gastrula-like stem cell line, and constructs a human in vitro post-implantation gastrula-like model derived from this cell line. The reagents, instruments, and cell lines used in the present invention are all commercially available. The human induced pluripotent stem cells were purchased from the Cell Bank / Stem Cell Bank of the Committee for Type Culture Collection of the Chinese Academy of Sciences, with the cell name DYR0100 and catalog number SCSP-1301.

[0322] (1) The sources of the culture medium components used for inducing human pluripotent stem cells into human gastrula stem cell lines in vitro:

[0323] GMEM (Glasgow's MEM) medium: 11710035, purchased from Gibco, USA;

[0324] Serum replacement (confidential formula of Thermo Fisher Scientific): 10828028, purchased from Gibco, USA;

[0325] Fetal bovine serum: 12483020, purchased from Gibco, USA;

[0326] MEM non-essential amino acids: 11140076, purchased from Gibco, USA;

[0327] GlutaMAX TM Additive: 35050061, purchased from Gibco, USA;

[0328] Sodium pyruvate additive: 11360070, purchased from Gibco, USA;

[0329] Penicillin-streptomycin dual antibody: 15140122, Gibco, USA;

[0330] β-Mercaptoethanol: 21985023, purchased from Gibco, USA;

[0331] Recombinant human bone morphogenetic protein 4:314-BP was purchased from R&D Systems, USA;

[0332] Recombinant human stem cell factor: 7734-LF, purchased from R&D Systems, USA;

[0333] Recombinant human leukemia inhibitory factor: 225-SC, purchased from R&D Systems, USA;

[0334] Recombinant human epidermal growth factor: 236-EG, purchased from R&D Systems, USA;

[0335] Recombinant human activin A protein: 338-AC, purchased from R&D Systems, USA;

[0336] ROCK inhibitors: Y-27632 and HY-10071, purchased from MCE, USA;

[0337] CHIR 99021: The specific type is Laduviglusib trihydrochloride, HY-10182B, purchased from MCE Company in the United States.

[0338] Adenylate cyclase activator: Forskolin 1099, purchased from R&D Systems, USA;

[0339] PDE4 inhibitors: Rolipram, 0905, purchased from R&D Systems, USA;

[0340] (2) Sources of culture medium components used for inducing human pluripotent stem cells into post-implantation blastocysts:

[0341] mTeSR TM 1. Culture medium: #85850, purchased from STEMCELL Technologies, Canada;

[0342] Essential 6 medium: A1516401, purchased from Gibco, USA;

[0343] Recombinant human fibroblast growth factor 2: 3718-FB, purchased from R&D Systems, USA;

[0344] Recombinant human noggin protein: HY-P7051A, purchased from MCE, USA;

[0345] ROCK inhibitors: Y-27632 and HY-10071, purchased from MCE, USA;

[0346] WNT inhibitors: specific types include IWP-2 and S7085, purchased from Selleck, USA.

[0347] (3) Preparation of GK15-1 culture medium containing Y-27632 for the first stage of induction of pluripotent stem cells into mesoderm:

[0348] The volume percentage is 81% basal culture medium GMEM, the volume percentage is 15% serum replacement KOSR, the volume percentage is 1% penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), the volume percentage is 1% 10mM non-essential amino acids, the volume percentage is 1% 200mM GlutaMAX additive, the volume percentage is 100mM sodium pyruvate additive, 0.1mM β-mercaptoethanol, as well as 50ng / mL recombinant human activin A protein, 3μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021, and 10μM ROCK inhibitor Y-27632.

[0349] (4) Preparation of GK15-1 culture medium for induction of pluripotent stem cells into mesoderm (stage 1):

[0350] The volume percentage is 81% basal culture medium GMEM, the volume percentage is 15% serum replacement KOSR, the volume percentage is 1% penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), the volume percentage is 1% 10mM non-essential amino acids, the volume percentage is 1% 200mM GlutaMAX additive, the volume percentage is 100mM sodium pyruvate additive, 0.1mM β-mercaptoethanol, as well as 50ng / mL recombinant human activin A protein, and 3μM glycogen synthase kinase-3 (GSK-3) inhibitor CHIR 99021.

[0351] (5) Preparation of GK15-2 culture medium containing Y-27632 for induction of nascent mesoderm-like cells into primordial germ cell-like cells (second stage) and cell purification:

[0352] The volume percentage is 81% basal culture medium GMEM, the volume percentage is 15% serum substitute KOSR, the volume percentage is 1% penicillin-streptomycin dual antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), the volume percentage is 1% 10mM non-essential amino acids, the volume percentage is 1% 200mM GlutaMAX additive, the volume percentage is 100mM sodium pyruvate additive, 0.1mM β-mercaptoethanol, 100ng / mL recombinant human stem cell factor, 200ng / mL recombinant human bone morphogenetic protein 4, 1000U / mL recombinant human leukemia inhibitory factor, 50ng / mL recombinant human epidermal growth factor, and 10μM ROCK inhibitor Y-27632.

[0353] (6) Preparation of GK15-2 culture medium for induction of nascent mesoderm-like cells into primordial germ cell-like cells (second stage) and cell purification:

[0354] The volume percentage is 81% basal culture medium GMEM, the volume percentage is 15% serum substitute KOSR, the volume percentage is 1% penicillin-streptomycin dual antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin), the volume percentage is 1% 10mM non-essential amino acids, the volume percentage is 1% 200mM GlutaMAX additive, the volume percentage is 1% 100mM sodium pyruvate additive, 0.1mM β-mercaptoethanol, 200ng / mL recombinant human bone morphogenetic protein 4, 100ng / mL recombinant human stem cell factor, 1000U / mL recombinant human leukemia inhibitory factor, and 50ng / mL recombinant human epidermal growth factor.

[0355] (7) Preparation of GK10 culture medium containing Y-27632:

[0356] The basal culture medium consists of 83.5% GMEM by volume, 10% serum substitute KOSR by volume, 2.5% fetal bovine serum FBS by volume, 1% penicillin-streptomycin dual antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin by volume), 1% 10 mM non-essential amino acids by volume, 1% 200 mM GlutaMAX supplement by volume, 1% 100 mM sodium pyruvate supplement by volume, 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.

[0357] (8) Preparation of GK10 culture medium for in vitro expansion of gastrula-like stem cell lines:

[0358] The volume percentage of the basal culture medium is 83.5% GMEM, the volume percentage of the serum substitute KOSR is 10%, the volume percentage of the fetal bovine serum FBS is 2.5%, the volume percentage of the penicillin-streptomycin double antibody (containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin) is 1%, the volume percentage of the 10mM non-essential amino acids is 1%, the volume percentage of the 200mM GlutaMAX supplement is 1%, the volume percentage of the 100mM sodium pyruvate supplement is 1%, 0.1mM β-mercaptoethanol, 10μM forskolin, 10μM rolipram, and 100ng / mL recombinant human stem cell factor.

[0359] (9) Preparation of mTR culture medium for the first stage of gastruloid induction after implantation:

[0360] 99% mTeSR by volume TM 1Complete culture medium, 1% by volume containing 10,000 units / mL penicillin and 10,000 μg / mL streptomycin, and a small molecule compound: 10 μM ROCK inhibitor Y-27632.

[0361] (10) Preparation of E6BIN culture medium for the second stage of post-implantation gastruloid induction:

[0362] Essential 6 medium with a volume ratio of 100% and multiple cytokines: 20 ng / mL recombinant human fibroblast growth factor 2 and 50 ng / mL recombinant human noggin protein, 5 μM small molecule compound IWP-2.

[0363] Example 5 Construction of gastrula-like stem cell line DYR0100-hGOSC-1

[0364] An embodiment of the present invention provides a gastrula-like stem cell line, named human gastrula-like stem cell line DYR0100-hGOSC-1, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO. C2022115.

[0365] (1) Induction of human induced pluripotent stem cells towards mesoderm (stage 1):

[0366] (1-1) Human induced pluripotent stem cells DYR0100 were purchased from the Cell Bank / Stem Cell Bank of the Committee of Type Culture Collection of the Chinese Academy of Sciences with the catalog number SCSP-1301.

[0367] When human induced pluripotent stem cells grow to 80-90% confluence, the cells are digested into single cells using the digestion enzyme TrypLE Select, centrifuged at 1300 rpm for 3 minutes using a swing-out centrifuge, and then resuspended in PBS to obtain cell suspension 1;

[0368] (1-2) Cell suspension 1 was centrifuged and the supernatant was discarded. GK15-1 culture medium containing Y-27632 was added to further resuspend the cells to obtain cell suspension 2. Each 1 mL of GK15-1 culture medium containing Y-27632 contained 1x10 6 cells;

[0369] (1-3) The cell suspension 2 obtained in (1-2) was inoculated into a six-well plate coated with Matrigel in advance, with 1x10 cells per square centimeter. 6 Inoculate cells at a cell density of 100 cells; shake well and place in an incubator at 37°C with a carbon dioxide concentration of 5.0%;

[0370] (1-4) On the second day of culture, remove the old culture medium and replace it with GK15-1 culture medium. Change the medium every day until newborn mesoderm-like cells are obtained.

[0371] The above steps of inducing the mesoderm direction of pluripotent stem cells are on days 0 to 2 of the method for constructing a gastrula-like stem cell line.

[0372] (2) Induction of nascent mesoderm-like cells into primordial germ cell-like cells (second stage):

[0373] (2-1) When the neonatal mesoderm-like cells obtained in (1-4) grow to 80-90% confluence, the cells are digested into single cells using the digestion enzyme TrypLESelect, centrifuged at 1300 rpm for 3 minutes using a horizontal centrifuge, and the cells are resuspended in PBS to obtain cell suspension 3;

[0374] (2-2) After centrifugation of the cell suspension for 3 times and discarding the supernatant, add GK15-2 culture medium containing Y-27632 to further resuspend the cells. Each 1 mL of GK15-2 culture medium containing Y-27632 contains 1x10 5 cells, and obtain cell suspension 4;

[0375] (2-3) The cell suspension 4 obtained in (2-2) was inoculated into a round-bottomed transparent low-adhesion 96-well plate for spheroid culture. The initial cell number of each well was 1x10 4 On the second day of culture, remove the old culture medium and replace it with GK15-2 culture medium. Change half of the GK15-2 culture medium every day until cell spheres containing primordial germ cells are obtained.

[0376] The above steps of inducing the neonatal mesoderm-like cells to the primordial germ cell-like direction are on days 3 to 6 of the method for constructing the gastrula-like stem cell line.

[0377] (3) Cell purification:

[0378] (3-1) The cell spheres 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, and resuspended in GK15-2 culture medium to obtain cell suspension 5;

[0379] (3-2) Using a flow cytometer, the CD326 and CD49f double-positive cells in the cell suspension 5 obtained in (3-1) were sorted; centrifuged at 1300 rpm for 3 minutes, and the cells were centrifuged at 1.0×10 5 The cells were seeded into a twelve-well plate at a density of 1 mL per well, and 1 mL of GK10 culture medium containing Y-27632 was added to each well to resuspend the cells, resulting in a cell suspension of 6;

[0380] (4) Cell expansion:

[0381] The cell suspension obtained in (3-2) was inoculated at a rate of 2 x 10 cells per square centimeter. 4 The cells were seeded at a density of 100 cells / well into a twelve-well plate pre-plated with mitomycin C-treated mouse embryonic fibroblasts as feeder cells; the cells were shaken and placed in an incubator at 37°C with a carbon dioxide concentration of 5.0% by volume; fresh GK10 culture medium was replaced after 24 hours, and the medium was changed daily until colony formation was observed, thereby obtaining the aforementioned gastrula-like stem cell line DYR0100-hGOSC-1, which was deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO. C2022115.

[0382] Example 6 The biological characteristics of the cells are described.

[0383] 1.1 Cell morphology observation

[0384] Observation of DYR0100-hGOSC-1 gastrula-like stem cells under an upright microscope revealed round or oval cell morphology and rapid cell proliferation. Colony formation was observed 2-3 days after single-cell passaging, with clear cell colony boundaries. The morphological observation results are shown in Figure 10.

[0385] 1.2 Growth curve determination

[0386] 1.2.1 Growth curve determination steps

[0387] Except that the cells were replaced with gastrula-like stem cells DYR0100-hGOSC-1, other operations were the same as the growth curve determination step 1.2.1 in Example 2.

[0388] 1.2.2 Growth curve measurement results

[0389] The measurement was continued for 5 days, and the growth curve data shown in Table 2 below were obtained.

[0390] Table 2 shows the growth curve data obtained from 5 consecutive days of measurement.

[0391] Table 2

[0392] Cultivation time (days) <![CDATA[Average number of cells (×10 5 cells)]]> 1 day <![CDATA[1.24×10 5 ]]> 2 days <![CDATA[1.32×10 5 ]]> 3 days <![CDATA[2.56×10 5 ]]> 4 days <![CDATA[4.00×10 5 ]]> 5 days <![CDATA[6.04×10 5 ]]>

[0393] Based on the cell growth curve data in Table 1, we can get Figure 11 Schematic diagram of cell growth curve shown. Figure 11 The horizontal axis is the culture time (days), and the vertical axis is the number of cells (×10 5 indivual).

[0394] Refer to the growth curve data in Table 1 and Figure 11 As shown in the growth curve diagram, it can be seen that the gastrula-like stem cells DYR0100-hGOSC-1 grew well for 5 consecutive days, and its logarithmic growth phase was 3 days.

[0395] In summary, DYR0100-hGOSC-1 cells proliferate rapidly, have active cell growth, good cell activity, high cell culture stability, and have stable cell growth characteristics in vitro.

[0396] 1.3 Immunofluorescence identification

[0397] 1.3.1 Immunofluorescence identification steps

[0398] The small round glass slides were disinfected with 75% ethanol and sterilized by UV, then transferred to a cell culture dish. They were coated with Fibronectin to increase the adhesion of the glass slides. DYR0100-hGOSC-1 cells were then inoculated and cultured according to normal cell subculture procedures.

[0399] DYR0100-hGOSC-1 cells were cultured until passage, and then the small round glass slide was removed and placed in a clean new culture dish. The DYR0100-hGOSC-1 cells on the small round glass slide were fixed with 4% PFA at room temperature for 30 minutes.

[0400] Discard the 4% PFA fixative, add PBS to wash away the fixative three times at room temperature, 5 minutes each time, discard the PBS, add 5% BSA blocking solution, and block at room temperature for 2 hours;

[0401] Discard the 5% BSA blocking solution, add the corresponding primary antibody diluted in 5% BSA, and incubate the DYR0100-hGOSC-1 cells on the small round glass slides at 4°C overnight;

[0402] After discarding the primary antibody incubation solution, add PBS to wash away the residual primary antibody incubation solution three times at room temperature, 5 minutes each time, add the secondary antibody diluted with 5% BSA and the live cell staining solution Hoechst 33342 to the dish, and incubate at room temperature for 2 hours in the dark;

[0403] After discarding the secondary antibody incubation solution, add PBS to wash away the residual secondary antibody incubation solution three times, 5 minutes each time, add glycerol to the slide, take out the small round glass slide from the culture dish and place it upside down on the slide with glycerol, and use nail polish to fix the position of the small round glass slide, and use a confocal fluorescence microscope for imaging.

[0404] 1.3.2 Immunofluorescence identification results

[0405] Immunofluorescence identification results Figure 12 As shown. Immunofluorescence staining of gastrula-like stem cells (DYR0100-hGOSC-1) was performed on cell slides to identify the expression of genes from each germ layer in DYR0100-hGOSC-1 cells. The results showed that pluripotency genes OCT4 and SOX2, mesoderm genes EOMES, TBXT, MIXL1, and CDX2, and 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 scattered within the cell clones, and endoderm genes were expressed at the edges of the cell clones without co-localization with pluripotency genes.

[0406] 1.4 Chromosome karyotype analysis and identification

[0407] 1.4.1 Chromosome karyotype analysis and identification steps

[0408] 20 μg / mL colchicine was added to the GK10 culture medium of gastrula-like embryonic stem cells at a ratio of 1:200 to a final concentration of 0.1 μg / mL, and then placed in an incubator at 37°C and a carbon dioxide concentration of 5.0% for 3 hours;

[0409] Preheat 0.56% KCl hypotonic solution to 37°C; remove the culture dish, wash the colchicine-treated gastrula-like stem cells at least three times with PBS (0.01M, pH 7.4), remove the old culture medium and detached cells in poor condition, and then digest the gastrula-like stem cells DYR0100-hGOSC-1 cells into a single-cell suspension using TrypLESelect; transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 2000 rpm in a horizontal centrifuge for 10 minutes. After discarding the supernatant, add 9 mL of 0.56% KCl hypotonic solution preheated to 37°C to the cell pellet, gently pipette approximately 50 times with a rubber-tipped glass dropper until a single-cell suspension is obtained, and place in a 37°C water bath for hypotonic treatment for 25 minutes;

[0410] Prepare a fixative using methanol and glacial acetic acid solution in a ratio of 3:1 and mix well at room temperature. Add 1 mL of fixative to the hypotonic treated gastrula-like stem cell suspension, gently blow bubbles 20 times from top to bottom with a rubber-tipped glass pipette, and centrifuge at 2000 rpm for 10 minutes; discard the supernatant, add 10 mL of fresh fixative, and gently blow bubbles 10 times from top to bottom with a rubber-tipped glass pipette until it becomes a single-cell suspension, and fix at room temperature for 2 hours; centrifuge at 2000 rpm for 10 minutes, discard the supernatant, add 10 mL of fresh fixative, and gently blow bubbles 10 times from top to bottom with a rubber-tipped glass pipette until it becomes a single-cell suspension, and fix at room temperature for 30 minutes; centrifuge at 2000 rpm for 10 minutes. Discard the supernatant and resuspend the gastrula-like stem cells in 0.2-0.6 mL of fixative depending on the amount of cell pellet;

[0411] Light an alcohol lamp and take a clean glass slide from distilled water. Without draining the water, place the water-containing slide at an angle on a waste liquid tank. Use a pipette to draw up the cell suspension and drop it 50 cm above the slide. Drop one drop of cell suspension on 3-4 different positions of each slide. Immediately afterwards, burn the back of the slide back and forth over the alcohol lamp 5 times, and then transfer the slide to a 37°C oven for baking overnight.

[0412] Place the human gastrula stem cell chromosome slide specimen in a 37°C oven in an 80°C oven for 2.5 hours; preheat 0.25% Trypsin-EDTA to 37°C; immerse the slide specimen in 37°C preheated 0.25% Trypsin-EDTA for 30-40 seconds, depending on the specimen; remove the slide and rinse the front and back of the slide three times with tap water under a thin stream; immerse the specimen in 37°C preheated Giemsa stain for approximately 10 minutes; rinse the front and back of the slide three times with tap water, and then dry the surface of the slide with lens paper.

[0413] Under a low-power microscope, select the mitotic phases with good dispersion and moderate length, then switch to the oil immersion lens for observation and photography to obtain the results of chromosome karyotype analysis and identification.

[0414] 1.4.2 Chromosome karyotype analysis results

[0415] The results of chromosome karyotype analysis were as follows Figure 13 Please refer to Figure 13 Chromosome karyotype analysis showed that the chromosome structure and number of the cells were normal, with the number of chromosomes being 44+XY, belonging to a diploid male cell line.

[0416] 1.5 RNA Sequencing Identification

[0417] 1.5.1 RNA Sequencing Identification Steps

[0418] Except that the cells were replaced with gastrula-like stem cells DYR0100-hGOSC-1 cells, the rest of the operations were the same as those in 1.5.1 of Example 1.

[0419] 1.5.2 RNA Sequencing Identification Results

[0420] RNA sequencing identification results Figure 14 Transcriptome sequencing of gastrula-like stem cells (DYR0100-hGOSC-1) and human induced pluripotent stem cells (DYR0100-hiPSCs) revealed that mesoderm genes MIXL1, EOMES, MESP1, WNT3, TBXT, GSC, and endoderm genes ELF3, FOXA2, CXCR4, GATA4, GATA6, and SOX17 were all upregulated in gastrula-like stem cells (DYR0100-hGOSC-1) compared to human induced pluripotent stem cells (DYR0100-hiPSCs).

[0421] The expression of the pluripotency gene SOX2 in gastrula-like stem cells DYR0100-hGOSC-1 was slightly downregulated compared with that in human induced pluripotent stem cells DYR0100-hiPSCs, while the other pluripotency genes POU5F1 (OCT4) and NANOG were still expressed. The pluripotency factors KLF4 and TFCP2L1, which are upregulated in human induced pluripotent stem cells in the induced pluripotent state, are expressed at elevated levels in the gastrula-like embryonic stem cells DYR0100-hGOSC-1, indicating that the gastrula-like embryonic stem cell line DYR0100-hGOSC-1 still has the pluripotency of stem cells.

[0422] The gastrula-like stem cells DYR0100-hGOSC-1 retain the pluripotency of stem cells to a certain extent, and show gene expression of cells of multiple germ layer lineages, whose characteristics are similar to those of embryonic cells in the gastrula stage of human embryonic development.

[0423] Example 7 Three-dimensional model of gastrula-like embryos after induction and implantation of gastrula-like stem cells DYR0100-hGOSC-1 (first stage, day 0-1):

[0424] The gastrula-like stem cell line DYR0100-hGOSC-1 possesses characteristics of human gastrula-stage embryos, mimicking gastrula formation and enabling research into morphological development and gene function during this stage. Under in vitro culture conditions, this cell line can reconstruct in vivo gastrula-like structures and partially reproduce the biological events of gastrula development.

[0425] When the gastrula-like stem cells obtained in Example 5 grew to 70-80% confluence, the cells were digested into single cells using TrypLE Select and resuspended using GK10 to obtain cell suspension 7; CD326 and CD49f double-positive cells were sorted using a flow cytometer; centrifuged at 1300 rpm for 3 minutes, and 6.0-7.0 x 10 4 4 mL of mTR culture medium was added to resuspend the cells and re-seeded into round-bottomed transparent low-adhesion 96-well plates at 6.0-7.0 x 10 cells per well. 3 The cells were centrifuged at 800 rpm for 3 minutes using a horizontal centrifuge and placed in an incubator at 37° C. with a carbon dioxide concentration of 5.0% until the gastrula-like stem cells assembled into a three-dimensional structure.

[0426] Example 8 Three-dimensional model of gastrula-like embryos after induction and implantation of gastrula-like stem cells DYR0100-hGOSC-1 (second stage, day 2-4):

[0427] Without 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 and 50 ng / mL recombinant human noggin protein, as well as the small molecule compound: 5 μM IWP-2; and the cells were placed in an incubator at 37°C with a carbon dioxide concentration of 5.0% by volume until the anterior amniotic cavity was formed and the mesendoderm lineage was specified, thereby obtaining a gastrula-like embryo model.

[0428] Example 9 describes the biological characteristics of the gastrula-like embryo model induced by gastrula-like stem cells:

[0429] 1.1 Morphological observation of induced gastrulae

[0430] Under an upright microscope, the post-implantation gastrula-like embryo model induced and cultured in a 96-well plate for 0-96 hours was observed. It was observed that the cells aggregated into balls quickly. After 12 hours of induction, the cells aggregated into three-dimensional embryo-like cell balls. Then the volume of the cell balls slowly increased. After 48-60 hours, two partitions of dense and loose cells appeared inside the cell balls, forming mutually exclusive double balls, and then an amniotic cavity was formed. At 96 hours, the maximum diameter of the gastrula-like embryo was about 150-250μm. The morphological observation results are as follows. Figure 15 Figure 16 shown.

[0431] 1.2 Growth curve of gastruloid embryos after induction and implantation

[0432] 1.2.1 Growth curve determination steps

[0433] During the 1-4 days of in vitro construction of the post-implantation gastruloid model using the gastruloid stem cell DYR0100-hGOSC-1, samples were collected every 24 hours. The gastruloids were washed at least twice with PBS (0.01M, pH 7.4) to remove the original culture medium and detached cell debris in poor condition. The post-implantation gastruloids were fixed with 4% PFA at room temperature for 30 minutes, 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.

[0434] After blocking the gastruloids with 5% BSA (containing 1% tTriton) at room temperature for 4 hours, DAPI staining solution was diluted in 5% BSA at a ratio of 1:200 and the gastruloids were incubated at room temperature for 4 hours.

[0435] After discarding the DAPI dilution solution, wash with PBS three times, 5 minutes each time. Move the sample into the chamber staining chamber and add a certain amount of prepared iohexol solution for transparency. Lightly cover the chamber with a slide and use a confocal microscope to photograph.

[0436] The number of cells within each sphere was estimated on confocal images stained with Dapi using Imaris software (Bitplane). Count points were drawn using an in-house algorithm using an estimated xy size of 6-10 μm, a quality threshold of 2.5, and background noise removal (Figure 17).

[0437] 1.2.2 Growth curve measurement results

[0438] The measurement was continued for 4 days, and the cell count data inside the cell spheres at each time point were obtained as shown in Table 3 below.

[0439] Table 3

[0440]

[0441]

[0442] Table 3 shows the growth curve data of DYR0100-hGOSC-1-induced gastruloids obtained by continuous measurement for 4 days. The number of samples (n) involved in the counting at each counting time point ranged from 20 to 30.

[0443] Based on the cell growth curve data in Table 2, we obtained Figure 8 Schematic diagram of cell growth curve shown. Figure 8 The horizontal axis is the culture time (hours), and the vertical axis is the number of cells (cells).

[0444] Refer to the growth curve data in Table 3 and Figure 17 As shown in the growth curve diagram, it can be seen that in the gastrula-like embryos induced by the gastrula-like stem cells DYR0100-hGOSC-1, the cell proliferation rate is slow, the cell induction stability is high, and the in vitro induction has stable embryo-like growth characteristics.

[0445] 1.3 Immunofluorescence identification

[0446] 1.3.1 Immunofluorescence identification steps

[0447] During the 1-4 days of induction of the gastrula model by DYR0100-hGOSC-1, samples were collected every 24 hours;

[0448] The gastruloids were fixed with 4% PFA for 30 minutes, the fixative was discarded, and PBST (0.01 M, pH 7.4, containing 1% t-Triton) was added for washing three times, 5 minutes each time;

[0449] Gastruloid embryos were blocked with 5% BSA (containing 1% Triton) for 4 h at room temperature;

[0450] Discard the blocking solution, add the diluted antibody in proportion, and incubate at 4°C for 24-48 hours;

[0451] After discarding the primary antibody incubation solution, add PBS (0.01M, pH 7.4, containing 1% tTriton) and wash three times, 5 minutes each time, then add diluted secondary antibody incubation solution and DAPI staining solution, and incubate at room temperature for 4 hours;

[0452] After discarding the secondary antibody incubation solution, add PBS (0.01M, pH 7.4, containing 1% tTriton) and wash three times, 5 minutes each time. Move the sample into a silica gel staining chamber with a depth of 0.2 mm or 0.75 mm, and add a certain amount of prepared iohexol solution for transparency. After lightly covering the chamber with a slide, use a confocal microscope to capture the results of immunofluorescence staining.

[0453] 1.3.2 Immunofluorescence identification results

[0454] Immunofluorescence identification results Figure 18-19 shown.

[0455] One day after induction of gastrula stem cells, gastrula stem cells began to assemble in the gastrula and formed a stable three-dimensional cell ball structure. Inside the gastrula at this time, there were epiblast-like (expressing OCT4, SOX2), primitive streak-like (expressing NCAD, TBXT, MIXL1), mesendoderm-like (expressing EOMES, GATA6, NCAD), and endoderm-like (expressing NCAD, SOX17, OTX2, FOXA2) cells. There were also a small number of cells suspected to be extraembryonic mesoderm-like (expressing LUM). At this time, there was no obvious spatial distribution pattern of the various cell types.

[0456] On day 2 of induction, OCT4- and SOX2-positive epiblast-like cells began to migrate toward one side of the gastrula-like embryo, while endoderm-like cells indicated by SOX17, FOXA2, and OTX2 also migrated in the opposite direction. At the same time, MIXL1- and TBXT-positive primitive streak-like cells also migrated in the opposite direction of the epiblast-like cells. However, the number of MIXL1- and TBXT-positive primitive streak cells decreased compared with that of the gastrula-like embryo induced for one day, suggesting that the primitive streak-like cells may have begun to specialize into a mesoderm-like or definitive endoderm-like cell fate at this time.

[0457] On days 3-4 of induction, OCT4- and SOX2-positive epiblast-like cells formed a cavity resembling the amniotic cavity. Compared to day 2 of gastrulation, SOX17-, FOXA2-, and OTX2-positive endoderm-like cells completely separated from OCT4- and SOX2-positive epiblast-like cells, forming a neatly arranged two-germ disc structure between the two cell types. The nuclei of the OCT4- and SOX2-positive epiblast-like cells exhibited a columnar, three-dimensional morphology. The mutually exclusive SOX17-, FOXA2-, and OTX2-positive endoderm-like cells and EOMES- and FOXA2-negative mesoderm-like cells simultaneously expressed signals for the epithelial-mesenchymal transition marker NCAD.

[0458] 1.5 RNA Sequencing Identification

[0459] 1.5.1 RNA Sequencing Identification Steps

[0460] At 96 hours into the DYR0100-hGOSC-1 induction model, gastrulae were harvested. The culture medium was removed and the embryos were washed at least twice with PBS (0.01M, pH 7.4) to remove old culture medium and detached dead cell debris. The embryos were then digested with 1-2 mL of 0.25% Trypsin-EDTA at 37°C for 3 minutes. During digestion, the embryos were gently pipetted to single cells. Digestion was terminated with serum-supplemented medium. The cells were centrifuged at 1300 rpm for 5 minutes, and the supernatant was removed. The cells were resuspended in PBS and transferred to enzyme-free EP tubes for testing.

[0461] 1.5.2 RNA Sequencing Identification Results

[0462] Single-cell RNA sequencing identification results Figure 20 shown.

[0463] In the single-cell RNA sequencing data of the gastrula model cultured for 96 hours, uniform manifold approximation and projection (UMAP) analysis of 4563 cells divided the cells into 12 cell clusters.

[0464] Based on the gene expression of each cell group, 9 types of cells were finally identified, namely amnion cells, epiblast cells, somite mesoderm cells, vascular endothelial cells, fibroblasts, mesoderm cells, primordial germ cell-like cells (PGC), endoderm cells, and some cells of unknown type (Unknown). Figure 19 Consistent with the immunofluorescence images, only 10 primitive streak cells expressing MIXL1 and TBXT were identified at this time point.

[0465] In this dataset, we were unable to detect any cell types that simultaneously expressed two or more neuroectodermal markers.

[0466] These data indicate that in the 96-hour embryo-like model, epiblast cells have transiently undergone gastrulation, and the primitive streak cells that give rise to them have completed their differentiation into definitive endoderm and mesoderm, while neural differentiation has not yet begun. This suggests that the embryo-like model at this time may be close to Carnegie stage 7 embryos.

[0467] In summary, this study established stably passaged gastrula-like stem cells that retain stem cell pluripotency to a certain extent and express genes and proteins characteristic of cells from the inner, middle, and outer germ layers of the gastrula developmental stage. These characteristics are consistent with those of embryonic cells at the gastrula stage, and they can well reproduce the key cellular features of the gastrula stage. Using these cells, a three-dimensional gastrula-like body model capable of mimicking gastrula development was constructed in vitro, partially reproducing key biological events during in vivo embryonic development, such as the separation of the inner and outer germ lineages, the formation of the anterior amniotic cavity, the emergence of the primitive streak, and the specification of the mesoderm lineage. Combined with single-cell multi-omics sequencing and fluorescence imaging, these key biological events were verified at both the protein and transcriptome levels, reproducing the key features of post-implantation gastrula-stage embryos. This model can be used to establish an in vitro platform for drug screening that affects early embryonic development, providing a reference for clinical drug use.

[0468] Although the present invention has been described to a certain extent, it is obvious that appropriate changes in various conditions can be made without departing from the spirit and scope of the present invention. It will be understood that the present invention is not limited to the described embodiments, but falls within the scope of the claims, which include equivalent replacements for each of the described factors. The present specification and its drawings are illustrative and do not constitute a limitation to the claims. The scope of protection of the present invention is defined by the claims and their equivalents. The present specification contains multiple inventive concepts, such as "preferably", "according to a preferred embodiment" or "optionally", all of which indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application based on each inventive concept. Throughout the text, the features guided by "preferably" are only an optional method and should not be understood as having to be set, so the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. A method for constructing a gastrula-like stem cell line, characterized in that: The construction method comprises the following steps: (1) Stem cells are induced to mesoderm: (1-1) Digest the stem cells into single cells and resuspend the cells to obtain 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, which are derived from embryos within 14 days of fertilization that have not undergone in vivo development; (1-2) Cell suspension 1 was centrifuged and the supernatant was discarded. GK15-1 culture medium containing ROCK inhibitor was added to further resuspend the cells to obtain cell suspension 2; The components of the GK15-1 culture medium containing ROCK inhibitor described in (1-2) include: The basal medium consisted of 80-85% GMEM by volume, 10-15% serum replacement KOSR, 1% penicillin-streptomycin, 1% 10 mM non-essential amino acids, 1% 200 mM GlutaMAX supplement, 1% 100 mM sodium pyruvate supplement, 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. (1-3) The cell suspension 2 obtained in (1-2) is inoculated into a well plate coated with Matrigel in advance and cultured; (1-4) On the second day of culture, remove the old culture medium and replace it with GK15-1 culture medium. Change the medium every day until newborn mesoderm-like cells are obtained. The components of the GK15-1 culture medium described in (1-4) include: The basal medium consisted of 80-85% GMEM by volume, 10-15% serum replacement KOSR by volume, 1% penicillin-streptomycin, 1% 10 mM non-essential amino acids by volume, 1% 200 mM GlutaMAX supplement by volume, 1% 100 mM sodium pyruvate supplement by volume, 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. (2) Induction of nascent mesoderm-like cells into primordial germ cell-like cells: (2-1) When the neonatal mesoderm-like cells obtained in (1-4) grow to 60-90% confluence, the cells are digested into single cells and resuspended to obtain cell suspension 3; The components of the GK15-2 culture medium containing ROCK inhibitor described in (2-2) include: The basal medium consists of 80-85% GMEM by volume, 10-15% serum replacement KOSR by volume, 1% penicillin-streptomycin, 1% 10 mM non-essential amino acids by volume, 1% 200 mM GlutaMAX supplement by volume, 1% 100 mM sodium pyruvate supplement by volume, 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. (2-2) Cell suspension 3 was centrifuged and the supernatant was discarded. GK15-2 culture medium containing ROCK inhibitor was added to further resuspend the cells to obtain cell suspension 4; (2-3) Inoculate the cell suspension 4 obtained in (2-2) into a low-viscosity plate for spheroid culture; starting from the second day of culture, remove the old culture medium and replace it with GK15-2 culture medium. Change the medium every day until spheroids containing primordial germ cells are obtained; (3) Cell purification: (3-1) Digest the cell spheres obtained in (2-3) into single cells and resuspend the cells in GK15-2 culture medium to obtain cell suspension 5; The components of the GK15-2 culture medium described in (2-3) and (3-1) include: The basal medium consists of 80-85% GMEM by volume, 10-15% serum substitute KOSR by volume, 1% penicillin-streptomycin dual antibody by volume, 1% 10 mM non-essential amino acids by volume, 1% 200 mM GlutaMAX supplement by volume, 1% 100 mM sodium pyruvate supplement by volume, 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. (3-2) sorting CD326 and CD49f double-positive cells from the cell suspension 5 obtained in (3-1); adding GK10 culture medium containing ROCK inhibitor to resuspend the cells to obtain cell suspension 6; The components of the GK10 culture medium containing ROCK inhibitor described in (3-2) include: 80-85% by volume of basal culture 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 supplement, 1% by volume of 100 mM sodium pyruvate supplement, 0.1 mM β-mercaptoethanol, 10 μM forskolin, 10 μM rolipram, 50-200 ng / mL of recombinant human stem cell factor, and 5-20 μM ROCK inhibitor; (4) Cell expansion: The cell suspension 6 obtained in (3-2) was inoculated into a well plate pre-plated with MEFs treated with mitomycin C as feeder cells; cultured, and after 24 hours, fresh GK10 culture medium was replaced to obtain the gastrula-like stem cell line; The components of the GK10 culture medium described in (4) include: The basal medium consists of 80-85% GMEM by volume, 10% serum substitute KOSR, 2.5% fetal bovine serum (FBS), 1% penicillin-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, and 50-200 ng / mL recombinant human stem cell factor.

2. The method for constructing a gastrula-like stem cell line according to claim 1, wherein: The ratio of cell suspension 2 and GK15-1 culture medium containing ROCK inhibitor in step (1-2) is: 1x10 6 cells.

3. The method for constructing a gastrula-like stem cell line according to claim 1, wherein: The culture conditions in steps (1-3) are 37° C. and a carbon dioxide volume concentration of 5.0-5.2%.

4. The method for constructing a gastrula-like stem cell line according to claim 1, wherein: In step (1-3), the cell suspension 2 was inoculated at a concentration of 0.6~1×10 5 The cells were seeded at a density of 10 cells per square centimeter.

5. The method for constructing a gastrula-like stem cell line according to claim 1, wherein: The ratio of the cell suspension 3 and the GK15-2 culture medium containing ROCK inhibitor in step (2-2) is: 1x10 5 cells.

6. The method for constructing a gastrula-like stem cell line according to claim 1, wherein: The initial cell volume per well in steps (2-3) is 0.5~1×10 4 cells.

7. The method for constructing a gastrula-like stem cell line according to claim 1, wherein: In step (3-2), each 1 mL of GK10 culture medium containing ROCK inhibitor contains 1x10 5 cells.

8. The method for constructing a gastrula-like stem cell line according to claim 1, wherein: The inoculation in step (4) is to inoculate the cell suspension 6 at a rate of 0.4~2×10 4 The cells were seeded at a density of 100 cells.

9. The method for constructing a gastrula-like stem cell line according to claim 1, wherein: The culture conditions in step (4) are 37° C. and a carbon dioxide volume concentration of 5.0%.

10. A gastrula-like stem cell line, characterized in that: The gastrula-like stem cell line is constructed using the method of claim 1 or 2.

11. The gastrula-like stem cell line according to claim 10, characterized in that The gastrula-like stem cell line is deposited in the China Center for Type Culture Collection, the culture name is human gastrula-like stem cell line CCRM-hGOSC-1, the preservation number is CCTCC NO.C2022114, and the preservation date is April 27, 2022.

12. The gastrula-like stem cell line according to claim 10, characterized in that The gastrula-like stem cell line is deposited in the China Center for Type Culture Collection, the culture name is human gastrula-like stem cell line DYR0100-hGOSC-1, the preservation number is CCTCC NO.C2022115, and the preservation date is April 27, 2022.

13. Use of the gastrula-like stem cell line according to claim 10 in constructing a gastrula-like model.

14. A method for constructing a gastrula-like embryo model, characterized in that: The construction method is to induce differentiation of the gastrula-like stem cell line according to claim 10, wherein the induced differentiation is in vivo induced differentiation or in vitro induced differentiation, and the in vivo induced differentiation comprises the following steps: The gastrula-like stem cell line according to claim 10 is resuspended in GK10 culture medium, and the cell suspension is injected into mouse testicles and cultured for 10 to 20 days to obtain the gastrula-like embryo model; The in vitro differentiation induction comprises the following steps: (1) When the gastrula-like embryonic stem cells grow to 60-90% confluence, the cells are 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 are sorted; centrifuged, and mTR culture medium is used to culture the cells at a rate of 1.5-1.75 × 10 4 Resuspend the cells at a concentration of 10 cells / mL and inoculate into low-viscosity well plates at a concentration of 6.0-7.0 x 10 cells per well. 3 cells; cultured until the gastrula-like stem cells assembled into a three-dimensional structure; (2) Without discarding the mTR culture medium from (1), add E6BIN culture medium; culture until the amniotic cavity is formed and the mesendoderm lineage is fully specified, thus obtaining a gastrula-like embryo model; The mTR culture medium in step (1) comprises: 99% by volume of mTeSR TM 1Complete culture medium, 1% penicillin-streptomycin, 5-20 μM ROCK inhibitor; The E6BIN culture medium in step (2) comprises: 100% by volume of Essential 6 culture medium, 20 ng / mL recombinant human fibroblast growth factor 2, 50 ng / mL recombinant human noggin protein, and 5 μM IWP-2.

15. The method for constructing a gastrula-like embryo model according to claim 14, wherein: The mice are immunodeficient mice.

16. The method for constructing a gastrula-like embryo model according to claim 15, wherein: The mice are BALB / cNude nude mice.

17. The method for constructing a gastrula-like embryo model according to claim 14, wherein: The culture conditions in steps (1) and (2) are 37° C. and a carbon dioxide volume concentration of 5.0%.

18. The construction method according to claim 14, characterized in that: The injection volume is 2-8×10 4 cell.

19. A method for constructing an organ prototype model, characterized in that: The construction method comprises the following steps: taking the gastrula-like stem cell line according to claim 10 and resuspending it in GK10 culture medium, injecting the cell suspension into mouse testicles, and culturing for 30 to 90 days to obtain the organ prototype model.

20. The method for constructing an organ prototype model according to claim 19, wherein: The mice are immunodeficient mice.

21. The method for constructing an organ prototype model according to claim 20, characterized in that: The mice are BALB / cNude nude mice.

22. The construction method according to claim 19, characterized in that: Inject 2~8×10 4 cell.

23. The method for constructing an organ prototype model according to claim 19, wherein: Obtain a neuroectoderm model, and / or a primordial germ cell model, and / or an amniotic epithelial cell model on day 30-40 of culture; The neuroepithelial cell model was obtained after 40–50 days of culture; After 70 to 90 days of culture, intestinal organoid prototype models, and / or muscle prototype models, and / or cartilage prototype models, and / or neural prototype models, and / or skin prototype models are obtained.

24. Use of the gastrula-like stem cell line according to any one of claims 10 to 12, or a tissue or organ derived therefrom, or a culture thereof, in the preparation of reagents for studying the mechanism of human early embryonic development.

25. Use of the gastrula-like stem cell line according to any one of claims 10 to 12, or a tissue or organ derived therefrom, or a culture thereof, in the preparation of reagents for diagnosing and / or treating human early embryonic developmental diseases.

26. Use of the gastrula-like stem cell line according to any one of claims 10 to 12, or a tissue or organ derived therefrom, or a culture thereof, in the preparation of a reagent for screening, validating, evaluating, assessing or studying the efficacy of drugs for preventing and / or treating human early embryonic developmental disorders.

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