Embryo-like model, and construction method therefor and use thereof

Abstract

Provided is an embryo-like model construction method. The method comprises: culturing primordial stem cells in vitro or injecting primordial stem cells into an immunodeficient animal for differentiation, so as to obtain an embryo-like model. By means of the method, during the preparation of the embryo-like model, there is no need to add cytokines and small molecule pathway interference factors, and there is no need to perform gene editing and only a single initiating cell type is used. The method has the advantages of simplicity and high repeatability, and is beneficial to large-scale preparation and application. Moreover, the prepared embryo-like model has all extraembryonic cell lineages and all embryonic cell lineages other than trophectoderm, can simulate the development process of a post-implantation embryo of Carnegie stage 3 to Carnegie stage 9 neurula, and then can be used for preparation of a variety of progenitor cell lineages, disease modeling, drug screening, and scientific researches.

Description

Embryo-like model and its construction method and use Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an embryo-like model and a construction method and use thereof. More particularly, the present invention relates to a method for constructing an embryo-like model using stem cells in vivo and in vitro. Background Art

[0002] Early human embryonic development progresses primarily through the stages of fertilization, morula, blastocyst, gastrulation, neurulation, and organogenesis. Due to ethical concerns and the limited availability of human tissue materials, many developmental stages, such as gastrulation, remain largely mysterious. Much of our understanding of development comes from model organisms such as mice, but mice and humans differ significantly in their early embryos. For example, morphologically, after implantation, the epiblast of the mouse embryo forms a cup-shaped structure, while the human embryo forms a double-layered disc-like structure. This makes it difficult to directly extrapolate research findings from other species to humans. In addition to model animals, human stem cell-based embryo models are also important tools for studying early human embryonic development. Compared to human embryos, human stem cell-based embryo models also allow for gene editing when studying embryonic development.

[0003] Selecting appropriate in vitro embryo models for research is of great help in understanding the mechanisms of human embryonic development and related diseases. However, current in vitro embryo models, especially those simulating the gastrula stage (such as gastrula-like embryos), can only simulate key events in embryonic development due to their limited cell types, but cannot truly reflect other events.

[0004] Therefore, it is crucial to construct a model that can efficiently simulate the complexity of the embryo.

[0005] Summary of the Invention

[0006] The present invention aims to develop a scalable, cytokine-free, small molecule pathway-interfering factor-free method for generating three-dimensional cell aggregates including intra- and extra-embryonic tissues from a single starting cell type.

[0007] To this end, the present invention defines a three-dimensional cell aggregate comprising both intra-embryonic and extra-embryonic tissues derived from the differentiation of human pluripotent stem cells / totipotent cells. The inventors discovered that these embryonic tissues include endoderm cells and their derivatives, mesoderm cells and their derivatives, and ectoderm cells and their derivatives; and that extra-embryonic tissues include yolk sac endoderm cells and their derivatives, yolk sac mesothelial cells and their derivatives, extra-embryonic mesoderm cells and their derivatives, and amniotic cells and their derivatives. The cell types in this model are highly similar to those of human gastrulation cells and can be used as a model for studying diseases related to embryonic development.

[0008] Therefore, in one aspect of the present invention, a method for constructing an embryo-like model is proposed. According to an embodiment of the present invention, the method comprises: culturing naive stem cells in vitro or injecting the naive stem cells into an immunodeficient animal for differentiation to obtain the embryo-like model. The inventors found that in the process of preparing the embryo-like model using this method, there is no need to add cytokines and small molecule pathway inhibitors or activators, and no gene editing is required and only a single starting cell type is used. The method has the advantages of being simple, highly reproducible, and more conducive to large-scale application, and the embryo-like model prepared therefrom is a 3D embryo-like model with multi-lineage cell types, that is, it has all extraembryonic cell lineages and all intraembryonic cell lineages except the trophectoderm, and can simulate the entire developmental process from the post-implantation embryo of Carnegie stage 3 to the neural embryo of Carnegie stage 9, thereby being used for preparing a variety of progenitor cell lineages, disease modeling, drug screening, and scientific research.

[0009] It should be noted that the "primitive stem cells ( PSCs) refers to a cell state of pluripotent stem cells. In vitro culture, pluripotent stem cells exist in at least two completely different states: the original state and primed, both of which can differentiate into the three germ layers within the embryo, namely, endoderm, mesoderm, and ectoderm. According to the embodiments of the present application, the starting cells used in the present application are The primordial stem cells (human primordial cells) according to the embodiment of the present application are closest to the inner cell mass and have the potential to differentiate into the epiblast (which then develops into the endoderm, mesoderm, and ectoderm) and the hypoblast (which then develops into a complete yolk sac).

[0010] According to an embodiment of the present invention, the method further includes at least one of the following additional technical features:

[0011] According to an embodiment of the present invention, the in vitro culture includes: subjecting the naive stem cells to a first culture treatment in a first culture medium and a second culture medium to obtain a first cell group; subjecting the first cell group to a second culture treatment in a third culture medium to obtain a second cell group; subjecting the second cell group to a third culture treatment in a fourth culture medium and a fifth culture medium to obtain the embryo-like model. The different culture media used at different stages do not require the addition of cytokines and small molecule pathway interference factors, and the cells are in a natural differentiation state during the culture process. The naive stem cells can spontaneously differentiate into epiblast cells and hypoblast cells during the culture process without human intervention and induction, and different culture media are used at different stages to prepare an embryo-like model with a relatively complete extraembryonic lineage, which can simulate embryonic development (especially the human gastrulation period), disease modeling, drug screening, and scientific research.

[0012] According to an embodiment of the present invention, the naive stem cells are pre-cultured, and the coverage of the naive stem cells in the culture dish after the pre-culture is 70-80%.

[0013] According to an embodiment of the present invention, the naive stem cells after the pre-culture treatment are subjected to a digestion treatment.

[0014] According to an embodiment of the present invention, the digestion process is carried out in TrypLE TM Express enzyme in the presence of conditions.

[0015] It should be noted that the “TrypLE TM "Express enzyme" is a commercial enzyme that can be used directly after purchase or diluted with EDTA solution. When diluted, TrypLE TM The volume ratio of Express enzyme to 0.3-0.7 mM EDTA solution is 1:(0.8-1.2).

[0016] According to an embodiment of the present invention, the first culture medium comprises complete culture medium for human primed pluripotent stem cells, penicillin-streptomycin and a Rock pathway inhibitor.

[0017] It should be noted that the "complete culture medium for human primed pluripotent stem cells" described in the present invention can be selected from mTeSR or other equivalent culture media, as long as it can be used to culture human primed pluripotent stem cells.

[0018] According to an embodiment of the present invention, the second culture medium comprises complete culture medium for human primed pluripotent stem cells and penicillin-streptomycin. The culture medium components are all basic, without the need for the addition of cytokines or small molecule pathway disruptors. The cells are in a naturally differentiated state during culture, and the naive stem cells can spontaneously differentiate into epiblast and hypoblast cells during culture without the need for human intervention or induction.

[0019] According to an embodiment of the present invention, the mass volume fraction of the penicillin-streptomycin in the first culture medium or the second culture medium is 0.5-1.5%.

[0020] According to an embodiment of the present invention, the Rock pathway inhibitor includes Y27632.

[0021] According to an embodiment of the present invention, the final concentration of Y27632 in the first culture medium is 45-55 μM.

[0022] According to an embodiment of the present invention, the first culture treatment is performed in a low-adsorption round-bottom 96-well plate, a 3D scaffold or a microfluidic device.

[0023] According to an embodiment of the present invention, the duration of the first culturing treatment is 5 to 7 days.

[0024] According to a specific embodiment of the present invention, the first culture treatment lasts for 5 days.

[0025] According to an embodiment of the present invention, the first culturing process includes a first-stage culturing process and a second-stage culturing process, the first-stage culturing process is performed in the first culture medium, and the second-stage culturing process is performed in the second culture medium.

[0026] According to an embodiment of the present invention, the duration of the first stage of culturing is 3 days.

[0027] According to an embodiment of the present invention, after the naive stem cells are cultured in the first culture medium for one day, the first culture medium is replaced and the naive stem cells are cultured in the first culture medium for another two days.

[0028] According to an embodiment of the present invention, the duration of the second stage of culturing treatment is 2 to 4 days.

[0029] According to an embodiment of the present invention, the duration of the second stage of culturing is 2 days.

[0030] According to an embodiment of the present invention, the third culture medium comprises DMEM-F12 basal medium, N2 supplement, glutaminase, non-essential amino acids, heparin, and penicillin-streptomycin. The culture medium components are all basic, and no cytokines or small molecule pathway disruptors are required. The cells are in a naturally differentiated state during the culture process, and the primed stem cells can spontaneously differentiate into epiblast and hypoblast cells during the culture process without artificial induction.

[0031] According to an embodiment of the present invention, the mass volume fraction of the N2 supplement in the third culture medium is 0.5-1.5%.

[0032] According to an embodiment of the present invention, the mass volume fraction of the glutaminase in the third culture medium is 0.5-1.5%.

[0033] According to an embodiment of the present invention, the mass volume fraction of the non-essential amino acids in the third culture medium is 0.5-1.5%.

[0034] According to an embodiment of the present invention, the final concentration of heparin in the third culture medium is 0.5-1.5 μg / mL.

[0035] According to an embodiment of the present invention, the mass volume fraction of the penicillin-streptomycin in the third culture medium is 0.5-1.5%.

[0036] According to an embodiment of the present invention, the second culture treatment lasts for 3 to 5 days.

[0037] According to an embodiment of the present invention, before the third culture treatment, the second cell population is mixed with matrigel.

[0038] According to an embodiment of the present invention, the ratio of the volume of the matrix gel to the number of the second cell population is 30 μL matrix gel: 1 second cell population.

[0039] It should be noted that the "second cell population" refers to a formed tissue block, i.e., an embryo-like model, that is, the ratio of the volume of the matrix gel to the number of the embryo-like models is 30 μL matrix gel / 1 embryo-like model (EB / embryoid).

[0040] According to an embodiment of the present invention, the fourth culture medium comprises DMEM-F12 basal medium, neurobasal medium, N2 supplement, glutaminase, non-essential amino acids, insulin, β-mercaptoethanol, penicillin-streptomycin, and B27 supplement without vitamin A. The culture medium contains only nutritional supplements but is serum-free and cytokine-free, so the cells remain in a naturally differentiated state.

[0041] According to an embodiment of the present invention, the fifth culture medium comprises DMEM-F12 basal medium, neurobasal medium, N2 supplement, glutaminase, non-essential amino acids, insulin, β-mercaptoethanol, penicillin-streptomycin, and B27 supplement containing vitamin A. The culture medium contains only nutritional supplements but is serum-free and cytokine-free, so the cells remain in a naturally differentiated state.

[0042] According to an embodiment of the present invention, the volume ratio of the DMEM-F12 basal culture medium to the neural basal culture medium is 1:(0.8-1.2).

[0043] According to an embodiment of the present invention, the mass volume fraction of the N2 supplement in the fourth culture medium or the fifth culture medium is 0.2-0.7%.

[0044] According to an embodiment of the present invention, the mass volume fraction of the glutaminase in the fourth culture medium or the fifth culture medium is 0.5-1.5%.

[0045] According to an embodiment of the present invention, the mass volume fraction of the non-essential amino acids in the fourth culture medium or the fifth culture medium is 0.2-0.7%.

[0046] According to an embodiment of the present invention, the final concentration of the insulin in the fourth culture medium or the fifth culture medium is 1 to 1.5 μg / mL.

[0047] According to an embodiment of the present invention, the final concentration of the β-mercaptoethanol in the fourth culture medium or the fifth culture medium is 0.1-0.25 μM.

[0048] According to an embodiment of the present invention, the mass volume fraction of the penicillin-streptomycin in the fourth culture medium or the fifth culture medium is 0.5-1.5%.

[0049] According to an embodiment of the present invention, the mass volume fraction of the B27 supplement not containing vitamin A in the fourth culture medium is 0.2-0.7%.

[0050] According to an embodiment of the present invention, the mass volume fraction of the B27 supplement containing vitamin A in the fifth culture medium is 0.2-0.7%.

[0051] According to an embodiment of the present invention, the third culturing process includes a third-stage culturing process and a fourth-stage culturing process, the third-stage culturing process is performed in a fourth culture medium, and the fourth-stage culturing process is performed in a fifth culture medium.

[0052] According to an embodiment of the present invention, the duration of the third stage of culturing is 3 to 5 days.

[0053] According to a specific embodiment of the present invention, the duration of the third stage culturing treatment is 4 days.

[0054] According to an embodiment of the present invention, the duration of the fourth stage of culturing treatment is 13 to 18 days.

[0055] According to a specific embodiment of the present invention, the duration of the fourth stage of culturing treatment is 16 days.

[0056] According to an embodiment of the present invention, the fourth stage culturing process is performed on a shaker.

[0057] According to an embodiment of the present invention, the rotation speed of the shaking table is 60-80 RPM.

[0058] According to an embodiment of the present invention, before the naive stem cells are injected into an immunodeficient animal for differentiation, the naive stem cells are mixed with a DMEM-F12 basal culture medium containing matrigel.

[0059] According to an embodiment of the present invention, the volume ratio of the DMEM-F12 basal culture medium to the matrigel is 1:(0.8-1.2).

[0060] According to an embodiment of the present invention, the ratio of the number of the naive stem cells to the volume of the DMEM-F12 containing the matrigel is 5 4 Pieces: (0.8~1.2)μL.

[0061] According to an embodiment of the present invention, the animal is selected from but not limited to immunodeficient mice, immunodeficient rabbits, immunodeficient cows, immunodeficient pigs or immunodeficient monkeys.

[0062] According to an embodiment of the present invention, the naive stem cells are selected from but not limited to at least one of pluripotent stem cells, totipotent stem cells, primitive endoderm cells and trophectoderm cells.

[0063] According to an embodiment of the present invention, the source of the naive stem cells is selected from but not limited to humans, cows, monkeys, pigs or mice.

[0064] According to an embodiment of the present invention, the injection is selected from subcutaneous injection.

[0065] According to an embodiment of the present invention, the in vivo differentiation time is 13 to 70 days.

[0066] According to an embodiment of the present invention, the naive state stem cells are obtained by culturing as follows: the naive state stem cells are subjected to a fourth culture treatment in a sixth culture medium to obtain a fourth cell group; the fourth cell group is subjected to a fifth culture treatment in a seventh culture medium to obtain a fifth cell group; the fifth cell group is subjected to a sixth culture treatment in an eighth culture medium to obtain the naive state stem cells. In some other embodiments, the naive state stem cells can also be obtained by induction using other culture media, such as 5iLAF culture medium, PXGL culture medium, HENSM culture medium, etc. The naive state stem cells prepared by the method do not need to add cytokines and small molecule pathway interference factors in the subsequent preparation of embryo-like models, and do not need gene editing and only use a single starting cell type to obtain an embryo-like model with a relatively complete extra-embryonic lineage.

[0067] According to an embodiment of the present invention, the sixth culture medium includes a complete culture medium for human primed pluripotent stem cells and a Rock pathway inhibitor.

[0068] According to an embodiment of the present invention, the Rock pathway inhibitor includes Y27632.

[0069] According to an embodiment of the present invention, the final concentration of Y27632 in the sixth culture medium is 5 to 15 μM.

[0070] According to an embodiment of the present invention, the fourth culturing treatment lasts for 20 to 27 hours.

[0071] According to an embodiment of the present invention, the seventh culture medium and the eighth culture medium contain neural basal medium, DMEM-F12 basal medium, N2 supplement, B27 supplement, sodium pyruvate, non-essential amino acids, glutaminase, penicillin-streptomycin, DZNep (histone methyltransferase EZH2 inhibitor), TSA (deacetylase inhibitor), MEK inhibitor, WNT signaling pathway inhibitor, recombinant human leukemia inhibitory factor, activin A, L-ascorbic acid and matrigel.

[0072] According to an embodiment of the present invention, the volume ratio of the neural basal culture medium to the DMEM-F12 basal culture medium is 1:1.

[0073] According to an embodiment of the present invention, the mass volume fraction of the N2 supplement in the seventh culture medium or the eighth culture medium is 0.5-1.5%.

[0074] According to an embodiment of the present invention, the mass volume fraction of the B27 additive in the seventh culture medium or the eighth culture medium is 1 to 3%.

[0075] According to an embodiment of the present invention, the mass volume fraction of the sodium pyruvate in the seventh culture medium or the eighth culture medium is 0.5-1.5%.

[0076] According to an embodiment of the present invention, the mass volume fraction of the non-essential amino acids in the seventh culture medium or the eighth culture medium is 0.5-1.5%.

[0077] According to an embodiment of the present invention, the mass volume fraction of the glutaminase in the seventh culture medium or the eighth culture medium is 0.5-1.5%.

[0078] According to an embodiment of the present invention, the mass volume fraction of the penicillin-streptomycin in the seventh culture medium or the eighth culture medium is 0.5-1.5%.

[0079] According to an embodiment of the present invention, the final concentration of DZNep in the seventh culture medium is 8-12 nM.

[0080] According to an embodiment of the present invention, the final concentration of DZNep in the eighth culture medium is 45-55 nM.

[0081] According to an embodiment of the present invention, the final concentration of TSA in the seventh culture medium is 4-6 nM.

[0082] According to an embodiment of the present invention, the final concentration of TSA in the eighth culture medium is 15-25 nM.

[0083] According to an embodiment of the present invention, the MEK inhibitor includes PD0325901.

[0084] According to an embodiment of the present invention, the final concentration of PD0325901 in the seventh culture medium or the eighth culture medium is 0.5-1.5 μM.

[0085] According to an embodiment of the present invention, the WNT signaling pathway inhibitor includes IWR-1.

[0086] According to an embodiment of the present invention, the final concentration of IWR-1 in the seventh culture medium or the eighth culture medium is 3-6 μM.

[0087] According to an embodiment of the present invention, the final concentration of the recombinant human leukemia inhibitory factor in the seventh culture medium or the eighth culture medium is 15-25 ng / mL.

[0088] According to an embodiment of the present invention, the final concentration of activin A in the seventh culture medium or the eighth culture medium is 15-25 ng / mL.

[0089] According to an embodiment of the present invention, the final concentration of the L-ascorbic acid in the seventh culture medium or the eighth culture medium is 45-55 μg / mL.

[0090] According to an embodiment of the present invention, the volume ratio of the matrix gel in the seventh culture medium or the eighth culture medium is 0.1-0.3%.

[0091] According to an embodiment of the present invention, the fifth culturing treatment lasts for 12 to 24 hours.

[0092] According to an embodiment of the present invention, the sixth culturing treatment lasts for 12 days.

[0093] In a second aspect of the present invention, an embryo-like model is proposed. According to an embodiment of the present invention, the embryo-like model is cultured by the method described in the first aspect. The prepared embryo-like model has a complete pedigree and can simulate embryonic development (especially the human gastrulation period), disease modeling, drug screening, and scientific research.

[0094] In a third aspect of the present invention, a kind of embryo-like model is proposed. According to an embodiment of the present invention, the embryo-like model includes extraembryonic tissue and intraembryonic tissue. Therefore, with all extraembryonic cell lineages and all intraembryonic cell lineages except the trophectoderm, the whole developmental process from the post-implantation embryo of Carnegie 3 to the neural embryo of Carnegie 9 can be simulated, thereby can be used for preparing multiple progenitor cell lineages, disease modeling, drug screening and scientific research and other purposes.

[0095] According to an embodiment of the present invention, the embryo-like model may further include at least one of the following additional technical features:

[0096] According to an embodiment of the present invention, the extraembryonic tissue includes: visceral endoderm, pre-visceral endoderm, yolk sac endoderm, extraembryonic mesoderm, mesothelial cells, allantoic cells, extraembryonic mesoderm-derived hematopoietic cell precursors, mesodermal progenitor cells, extraembryonic mesoderm-derived hemangioblasts, extraembryonic mesoderm-derived endothelial cells, extraembryonic mesoderm-derived arterial endothelial cells, extraembryonic mesoderm-derived hematopoietic cell precursors, extraembryonic mesoderm-derived megakaryocytes, extraembryonic mesoderm-derived red blood cells, extraembryonic mesoderm-derived myeloid cell precursors, amniotic ectoderm cells and amniotic / non-neural ectoderm progenitor cells.

[0097] According to an embodiment of the present invention, the intraembryonic tissues include epiblast, definitive epiblast, primordial germ-like cells, definitive endoderm, primitive streak, emergent mesoderm, late mesoderm, neural epithelium and neural crest-derived cells.

[0098] According to an embodiment of the present invention, the embryo-like model includes epiblast, definitive epiblast, primordial germ-like cells, endoderm lineage, mesoderm lineage, and ectoderm lineage.

[0099] According to an embodiment of the present invention, the endoderm lineage includes intraembryonic tissue and extraembryonic tissue. The intraembryonic tissue includes definitive endoderm; and the extraembryonic tissue includes visceral endoderm, previsceral endoderm and yolk sac endoderm.

[0100] According to an embodiment of the present invention, the mesodermal lineage includes intraembryonic tissues and extraembryonic tissues. The intraembryonic tissues include primitive streak, emergent mesoderm, and late mesoderm; the extraembryonic tissues include extraembryonic mesoderm, mesothelial cells, and allantoic cells, as well as hematopoietic cell precursors derived from the extraembryonic mesoderm, including mesodermal progenitor cells, angioblasts, endothelial cells, arterial endothelial cells, hematopoietic cell precursors, megakaryocytes, erythrocytes, and myeloid cell precursors.

[0101] According to an embodiment of the present invention, the ectodermal lineage includes intraembryonic tissues and extraembryonic tissues. The intraembryonic tissues include neural epithelium and neural crest-derived cells; the extraembryonic tissues include amniotic ectoderm cells and amniotic / non-neural ectoderm progenitor cells.

[0102] According to an embodiment of the present invention, the genes expressed in the visceral endoderm include HNF4A and SOX17; the genes expressed in the anterior visceral endoderm include OTX2 and CXCR4; the genes expressed in the yolk sac endoderm include HNF4A, and low expression or no expression of SOX17; the genes expressed in the extraembryonic mesoderm cells include ANXA1, LUM, NID2 and FRZB; the genes expressed in the mesothelial cells include AQP1; the genes expressed in the allantoic cells include TBX4 and HOXA13; the extraembryonic mesoderm origin The genes expressed in the hematopoietic cell precursor mesodermal progenitor cells include BMP4 and WNT5A; the genes expressed in the extraembryonic mesoderm-derived angioblasts include ETV2, TAL1, BMP4 and WNT5A; the genes expressed in the extraembryonic mesoderm-derived endothelial cells include CDH5 and CD34; the genes expressed in the extraembryonic mesoderm-derived arterial endothelial cells include GJA4 and GJA5; the genes expressed in the extraembryonic mesoderm-derived hematopoietic cell precursors include RUNX1 and SPN; the genes expressed in the extraembryonic mesoderm-derived megakaryocytes include RUNX1 and SPN. The genes expressed in the extraembryonic mesoderm-derived erythrocytes include PPBP; the genes expressed in the extraembryonic mesoderm-derived erythrocytes include GATA1 and HBE1; the genes expressed in the extraembryonic mesoderm-derived myeloid cell precursors include CSF1R; the genes expressed in the amniotic ectoderm cells include ISL1 and GABRP; the genes expressed in the amniotic / non-neural ectoderm progenitor cells include CLDN10, AMR2, PCAT14 and VGLL1; the genes expressed in the epiblast include POU5F1 and DPPA4; the genes expressed in the definitive epiblast include TN FRSF12A and JUND; genes expressed in the primordial germ-like cells include NANOG, NANOS3, TFAP2C and SOX17; genes expressed in the definitive endoderm include HHEX; genes expressed in the primitive streak include TBXT; genes expressed in the emergent mesoderm include LHX1; genes expressed in the late mesoderm include MYL7, and TBXT is lowly expressed or not expressed; genes expressed in the neuroepithelium include PAX6; genes expressed in the neural crest-derived cells include SOX10 and MPZ.

[0103] According to an embodiment of the present invention, the endoderm lineage expresses FOXA2, that is, the definitive endoderm, the visceral endoderm, the previsceral endoderm and the yolk sac endoderm all express FOXA2.

[0104] According to an embodiment of the present invention, the genes expressed in the epiblast lineage include POU5F1 and DPPA4; the genes expressed in the definitive epiblast include TNFRSF12A and JUND; the genes expressed in the primordial germ-like cells include NANOG, NANOS3, TFAP2C and SOX17; the genes expressed in the endoderm lineage include FOXA2; the genes expressed in the late mesoderm include TBXT and MYL7; the genes expressed in the allantoic cells include TBX4 and H OXA13; the genes expressed in the amnion / non-neural ectoderm progenitor cells include CLDN10, AMR2, PCAT14 and VGLL1; the genes expressed in the extraembryonic mesoderm include ANXA1, LUM, NID2 and FRZB; the genes expressed in the definitive endoderm include HHEX; the genes expressed in the visceral endoderm include HNF4A and SOX17; the genes expressed in the anterior visceral endoderm include OTX2 and CXCR4; the genes expressed in the yolk sac endoderm include HNF4A and low expression of SOX17; the genes expressed in the primitive streak include TBXT; the genes expressed in the emergent mesoderm include LHX1; the genes expressed in the neuroepithelium include PAX6; the genes expressed in the neural crest-derived cells include SOX10 and MPZ; the genes expressed in the amniotic ectoderm cells include ISL1 and GABRP; the genes expressed in the mesothelial cells include AQP1; the genes expressed in the endothelial cells include CDH5 and CD34; the genes expressed in the arterial endothelial cells include GJA4 and GJA5; the genes expressed in the hematopoietic cell precursor mesodermal progenitor cells include BMP4 and WNT5A; the genes expressed in the angioblasts include ETV2, TAL1, BMP4 and WNT5A; the genes expressed in the hematopoietic cell precursors include RUNX1 and SPN; the genes expressed in the megakaryocytes include PPBP; the genes expressed in the erythrocytes include GATA1 and HBE1; the genes expressed in the myeloid cell precursors include CSF1R.

[0105] According to an embodiment of the present invention, the proportions of each cell type in the extraembryonic tissue are: 0.46%-11.57% of the visceral endoderm, 0.14%-33.17% of the anterior visceral endoderm, 1.19%-4.24% of the yolk sac endoderm, 0.33%-32.35% of the extraembryonic mesoderm, 4.22%-4.33% of the mesothelial cells, 0.14%-4.26% of the allantoic cells, 0.1%-0. 5%, the hemangioblasts 0.1%-0.5%, the endothelial cells 1.40%-4.79%, the arterial endothelial cells 0.04%-0.1%, the hematopoietic cell precursors 0.01%-0.1%, the megakaryocytes 0.01%-0.1%, the erythrocytes 0.1%-0.5% and the myeloid cell precursors 0.01%-0.1%, the amniotic ectoderm cells 0.21%-10.23% and the amniotic / non-neural ectoderm progenitor cells 0.09%-3.00%.

[0106] According to an embodiment of the present invention, the proportions of the main cell types in the embryonic tissue are: 2.18%-33.61% of the epiblast, 2.57%-2.86% of the definitive epiblast, 0.02%-0.19% of the primordial germ-like cells, 0.48%-1.63% of the definitive endoderm, 0.31%-0.90% of the primitive streak, 0.15%-1.83% of the emergent mesoderm, 1.61%-12.81% of the late mesoderm, 0.18%-3.13% of the neural epithelium, and 0.18%-2.84% of the neural crest-derived cells.

[0107] According to an embodiment of the present invention, the proportions of each cell type in the extra-embryonic tissue and intra-embryonic tissue are shown in FIG5 .

[0108] According to an embodiment of the present invention, the embryo-like model can simulate the cell types of the gastrula stage.

[0109] In a fourth aspect of the present invention, use of the embryo-like model described in the second or third aspect in constructing a Carnegie developmental stage model is proposed.

[0110] According to an embodiment of the present invention, the Carnegie developmental stage is selected from the developmental stages from the 3rd stage to the 9th stage.

[0111] In a fifth aspect of the present invention, use of the embryo-like model described in the second or third aspect in drug screening is proposed, wherein the drug is used to intervene in embryonic development during the gastrula stage.

[0112] In a sixth aspect of the present invention, use of the embryo-like model described in the second aspect or the third aspect in constructing a disease model is provided, wherein the disease model is selected from a developmental disease model.

[0113] According to an embodiment of the present invention, the developmental disease model is selected from an embryo implantation defect model or a Down syndrome model. Developmental abnormalities at this stage may lead to miscarriage, organ and brain development defects.

[0114] In the seventh aspect of the present invention, use of the embryo-like model described in the second aspect or the third aspect in obtaining progenitor cells of a specific lineage is proposed.

[0115] According to an embodiment of the present invention, the use includes expanding and culturing a specific lineage in the embryo-like model to obtain progenitor cells of the specific lineage.

[0116] According to an embodiment of the present invention, the method further comprises performing gene editing or cell differentiation on the specific lineage after the expanded culture treatment, or performing cell therapy using the specific lineage after the expanded culture treatment.

[0117] The eighth aspect of the present invention proposes the use of the embryo-like model described in the second aspect or the third aspect in constructing a specific organoid model or a 3D cell aggregate.

[0118] According to an embodiment of the present invention, the use includes inducing the embryo-like model into a specific organoid model or the cell 3D aggregate by induction means.

[0119] According to an embodiment of the present invention, the induction means includes adding small molecule compounds, cell co-culture or adding cytokines.

[0120] In a ninth aspect of the present invention, a method for screening a drug for intervening in gastrula-stage embryonic development is provided. According to an embodiment of the present invention, the drug to be screened is added to the culture process of the embryo-like model constructed according to the first aspect, or the drug to be screened is contacted with the embryo-like model described in the second or third aspect; and the target drug is determined based on whether the embryo-like model obtained by the construction method of the first aspect or the embryo-like model described in the second or third aspect undergoes the expected change.

[0121] According to an embodiment of the present invention, the expected change in the embryo-like model obtained by the construction method described in the first aspect after the addition of the drug compared to before the addition of the drug is an indication that the drug to be screened is the target drug.

[0122] According to an embodiment of the present invention, an expected change in the embryo-like model described in the second aspect or the third aspect after the contact treatment compared to before the contact treatment is an indication that the drug to be screened is a target drug. Beneficial effects:

[0123] The present invention establishes in vivo and in vitro generation of three-dimensional aggregates (embryonic model) methods including intra-embryonic and extra-embryonic tissues. Both the in vivo and in vitro methods do not require cytokines, small molecule pathway interference factors, non-gene editing, and only a single starting cell type. The method is simple and highly reproducible, making it more conducive to large-scale application. The embryonic model in the present invention includes embryonic and extra-embryonic tissues. Compared to the currently available embryonic development model, the pedigree diversification is closer to the state in vivo, which provides a better model for embryonic development disease modeling.

[0124] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0126] FIG1 is a schematic diagram of a method for generating an embryo-like model in vivo or in vitro from pluripotent stem cells / totipotent stem cells according to an embodiment of the present invention;

[0127] FIG2 is a bubble chart showing the expression levels of specific marker genes in different cell types, illustrating single-cell data from in vivo and in vitro experiments according to an embodiment of the present invention. The bubble size represents the expression proportion of the gene in different cell types, and the bubble color represents the relative high and low values ​​of gene expression.

[0128] FIG3 is a single-cell UMAP graph of human embryo (6-19 days) single-cell data according to an embodiment of the present invention, single-cell data of an embryo-like model generated by in vitro 3D culture, and single-cell data of an embryo-like model formed by in vivo teratoma formation, after integration;

[0129] Figure 4 compares the developmental potential of different stem cells differentiated using the in vitro and in vivo methods of the present invention according to an embodiment of the present invention. The upper half is an integrated single-cell UMAP image of primed pluripotent stem cells and naive pluripotent stem cells after differentiation using the in vitro method of the present invention. A uses colors to mark different time points of sampling, and B uses colors to mark different cell types. The lower half is an integrated single-cell UMAP image of primed pluripotent stem cells and naive pluripotent stem cells after differentiation using the teratoma method of the present invention. C uses colors to mark different time points of sampling, and D uses colors to mark different cell types.

[0130] FIG5 is a bubble chart showing the cell ratios of different stem cells after differentiation using the in vitro and in vivo methods of the present invention according to an embodiment of the present invention;

[0131] FIG6 is a UMAP diagram of subgroups of the epiblast lineage and its derived lineages (left) and a UMAP diagram of subgroups of the hematopoietic-related lineages (right) according to an embodiment of the present invention;

[0132] FIG7 shows the identification of naive stem cells based on real-time fluorescence quantitative PCR and immunofluorescence staining. DETAILED DESCRIPTION

[0133] To facilitate understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

[0134] The term "stem cell" describes a cell that can form cells of multiple tissue types. There are different types of stem cells. When a sperm fertilizes an egg, a single totipotent cell is formed, which has the ability to form an entire organism. In the first hour after fertilization, this cell divides into identical totipotent cells. Approximately four days after fertilization and after several cycles of cell division, these totipotent stem cells begin to specialize. As totipotent cells become more specialized, they are subsequently referred to as "pluripotent." Pluripotent cells can differentiate into every cell type in the body, but cannot form the placenta or support tissues necessary for fetal development. Because the differentiation potential of pluripotent stem cells is not "universal," these cells are not called "totipotent" and are not embryonic. Pluripotent stem cells further specialize into multipotent stem cells, which are dedicated to differentiating into cells of a specific lineage specialized for a specific function. Pluripotent cells can differentiate into the cell types contained in the tissue from which they originate; for example, blood stem cells can only differentiate into red blood cells, white blood cells, and platelets.

[0135] The term "embryonic structure" reflects any structure that occurs during development from fertilization to the fetus, including the morula, blastocyst, and gastrula stages of development. Therefore, the term embryonic structure includes structures that have not yet cavitated into a blastocyst.

[0136] During the second week after fertilization, the inner cell mass proliferates and differentiates, gradually forming a disc-shaped, two-layered blastoderm. This disc is composed of two germ layers and is called a two-layered blastoderm. A layer of columnar cells adjacent to the trophoblast is the epiblast, while a layer of cuboidal cells near the blastocyst cavity is the hypoblast. The close contact between the inner and outer endoblastic cells forms the two-layered blastoderm, which serves as the primordium for embryonic development. In mammalian embryology, when the cuboidal cells of the hypoblast gradually approach the blastocyst cells, and the columnar cells of the epiblast connect with the trophoblast cells, the epiblast is also called the first ectoderm because it can develop into the three subsequent germ layers: the ectoderm, mesoderm, and endoderm. These three germ layers are later associated with gastrulation and development.

[0137] Under natural conditions, cell division in a fertilized egg produces a morula, a spherical embryonic cell structure consisting of 8 to 20 cells. Morula cells are totipotent, meaning they can differentiate into any embryonic or extraembryonic cell type. As the morula matures, a fluid-filled internal cavity forms. This event is associated with the differentiation of the morula cells into an outer layer (trophectoderm) and an inner cell mass (ICM or blastoderm). This hollow cell structure is called a blastocyst. Therefore, the blastocyst is a stage of structural development in the mammalian embryo. The trophoblast cells of the blastocyst surround the inner cell mass (ICM) and the fluid-filled blastocoel. The trophoblast cells of the blastocyst further develop into the exoplacental cone, which ultimately fuses with the uterine lining to form the placenta. The placenta is an organ that connects the developing fetus to the uterine wall, facilitating the absorption of nutrients, elimination, and gas exchange through the mother's blood supply.

[0138] The inner cell mass (ICM) is composed of cells with the ability to differentiate into any tissue of the body, thus possessing pluripotency. Furthermore, cells of the inner cell mass contribute to certain extraembryonic tissues, such as the yolk sac. The blastocyst develops further naturally through the proliferation and differentiation of cells from the trophectoderm and ICM into cells other than embryonic stem cells. The first steps in the development of ICM cells lead to the formation of the epiblast (tissues that ultimately form the animal) and hypoblast (tissues that ultimately form the yolk sac, among others).

[0139] Within the epiblast, cells continue to differentiate by forming three tissue layers (embryonic germ layers). These will collectively form all the different tissue types found in adult animals. The three embryonic germ layers are the endoderm, ectoderm, and mesoderm; the endoderm will develop into, among other things, the gastrointestinal and respiratory tracts; the ectoderm will develop into, among other things, the nervous system, hair, and nails; and the mesoderm will develop into, among other things, muscle and connective tissue. Once the three embryonic germ layers are formed, the embryonic structure becomes the gastrula. Because cells in any embryonic germ layer cannot naturally differentiate to form tissues derived from one of the other embryonic germ layers, cells in the embryonic germ layers are pluripotent, rather than totipotent or multipotent.

[0140] For the scope of the present invention, "embryonic" is defined as any one of a series of developmental structures starting from the blastocyst and including all subsequent developmental stages (including, for example, gastrula) until the formation of the fetus, including, for example, the embryonic development stage, gastrula, until the formation of the fetus. Therefore, the term refers only to those structures that occur in the (natural) development from the blastocyst stage, and includes subsequent structures until the fetal stage. When all organs are substantially formed, the embryo becomes a fetus.

[0141] A fetus is a mammal in the post-embryonic, prenatal developmental stage, in which its organs are fully differentiated but not yet fully developed. Therefore, the terms "embryo" or "embryonic structure" do not encompass a fetus.

[0142] The term "Carnegie stages" refers to a system proposed based on observations and classifications of human embryos, used to describe and compare the characteristics and changes of human embryos at different stages of development. The Carnegie stages divide the human embryonic development process into 23 stages, named after the Carnegie numbers, numbered from 1 to 23. Each stage represents a specific stage and characteristic of the embryonic development, including embryonic size, morphological characteristics, organ formation, and cell differentiation. The 23 stages are specifically divided into the following stages: Stages 1-2: Formation of the zygote and cell division; Stage 3: Blastocyst expansion and implantation; Stages 4-5: Formation of the endoderm and ectoderm; Stages 6-7: Gastrulation; Stages 8-9: Neuralization; Stages 10-11: Formation of organ primordia; Stages 12-13: Formation of organ systems; Stages 14-15: Emergence of fetal characteristics; Stages 16-17: Development of the nervous and muscular systems; Stages 18-19: Refinement of organs and enhanced function; and Stages 20-23: The final stage of embryonic development.

[0143] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0144] Example 1 Obtaining naive stem cells and early embryonic-like totipotent stem cells

[0145] To obtain naive stem cells ( Human primed PSCs (including commercial human pluripotent stem cell lines such as H9 and H1, or induced pluripotent stem cells) were washed once with DPBS and then stained with TrypLE TM Express: Human primed PSCs were dissociated into single cells using 0.5 mM EDTA (1:1 volume ratio) and plated at 1000–1500 cells / cm 2The cells were plated at a density of 10 μM in a culture dish containing complete culture medium for human primed pluripotent stem cells with a concentration of 10 μM Y-27632. After 24 hours, the culture medium was replaced with 4CL medium, which consisted of neurobasal medium (Gibco, 21103049) and DMEM / F12 basal medium (Gibco, 12634028), 0.5-1.5% N2 supplement (Gibco, 17502048), 1-3% B27 supplement (Gibco, 17502048), 0.5-1.5% sodium pyruvate (Corning, 25000CL), 0.5-1.5% non-essential amino acids (Corning, 25025CL), 0.5-1.5% glutaminase (Gibco, 35050061), 0.5-1.5% penicillin-streptomycin (HyClone, SV30010), 10 nM DZNep (Selleck, S7120), 5nM TSA (Vetec, V900931), 1μM PD0325901 (Axon, 1408), 5μM IWR-1 (Sigma, I0161), 20ng / mL recombinant human leukemia inhibitory factor (Peprotech, 300-05), 20ng / mL activin A (Peprotech, 120-14E), 50μg / ml L-ascorbic acid (Sigma, A8960) and 0.2% (v / v) Geltrex or Matrigel. 4CL medium was changed every day, and cells were passaged every 3-4 days (1:5 to 1:8). After about 12 days of culture (passage 3), 4CL naive stem cells ( PSCs).

[0146] There are two methods to obtain early embryonic-like totipotent stem cells. The first method is to dissociate the primitive stem cells into single cells and culture them at a density of 2000–3000 cells / cm. 2 The cells were plated at a density of 100 μM in a culture dish containing 4CL medium with a concentration of 5 μM Y-27632. After culturing for 24 hours, the culture dish was replaced with e4CL medium. The e4CL medium has the same composition as the 4CL medium, but the concentration of DZNep is 50 nM and the concentration of TSA is 20 nM. The culture was carried out for 5 days, and the e4CL medium was replaced every day. The second method is to dissociate human primed embryonic stem cells into single cells and plate them at a density of 2000–3000 cells / cm2 in order to make the primitive stem cells directly generate early embryonic-like totipotent stem cells in e4CL medium. 2Cells were plated at a density of 10 μM in culture dishes containing complete human primed pluripotent stem cell culture medium or TeSR-E8 medium supplemented with 10 μM Y-27632. After 24 hours of culture, the medium was changed to e4CL medium, with daily medium changes. On day 4, cells were digested into single cells and passaged. Culture was continued in e4CL conditions for another 4 days to obtain early embryonic-like totipotent stem cells.

[0147] Example 2 Identification of 4CL Naive Stem Cells

[0148] The naive stem cells obtained by culture in Example 1 were tested using RT-qPCR to detect the expression levels of naive marker genes such as DPPA3, DPPA5, KLF17, DNMT3L, and MAEL. As shown in Figure 7, compared with primed cells, the expression levels of DPPA3, DPPA5, KLF17, DNMT3L, and MAEL in naive stem cells were significantly increased, while the expression levels of pluripotency genes OCT4 and NANOG were basically stable. At the same time, immunofluorescence experiments detected that naive stem cells highly expressed the related protein KLF17, proving that the cells were in a naive state.

[0149] Example 3: Generation of intra- and extra-embryonic tissues in an in vitro 3D environment

[0150] According to the method shown in Figure 1, in vitro culture is performed, and the primitive stem cells or early embryonic-like totipotent stem cells obtained by culture in Example 1 are cultured in an in vitro three-dimensional environment. An embryo-like model is produced by a 3D culture organoid method. This method does not require cytokines, small molecule pathway interference factors, or gene editing. Specifically, the method includes cell preparation, using embryoid body culture medium in a low-adsorption round-bottom 96-well plate to make the cells spherical, then using induction culture medium to allow the cells to differentiate, and then long-term culture in a long-term differentiation culture medium. The specific steps are:

[0151] Day 0: When the cells (naive stem cells or early embryonic-like totipotent stem cells obtained by culture in Example 1) have grown to 70-80% of the culture plate area, the clones are digested into single cells using Accutase (Gibco). 9,000 cells are resuspended in 150 μL of embryoid body medium (specific ingredients are shown in Table 1) and seeded into a 96-well low-adhesion culture plate.

[0152] Table 1. Embryoid body culture medium

[0153] Day 1: After 24 hours, small embryoid bodies with clear boundaries can be observed under a tissue culture microscope. The embryoid bodies are then cultured in a cell culture incubator at 37°C and 5% CO2.

[0154] Day 2: Remove the old embryoid body medium without touching the embryoid bodies on the bottom of the culture plate, and add 150 μL of fresh embryoid body medium to the culture plate.

[0155] Day 4: Fresh embryoid body medium (ie, consisting of mTesR and 1% penicillin-streptomycin) without Y27632 was added to the culture plates.

[0156] Day 6: Use a 200 μL pipette tip to transfer the embryoid bodies to a 24-well low-adhesion plate and add 500 μL of induction medium (specific ingredients are shown in Table 2).

[0157] Table 2. Induction medium

[0158] Day 8: Remove the old induction medium and add 500 μL of fresh induction medium to the culture plate.

[0159] Day 10: The embryoid bodies have formed into balls and become tissue blocks. The tissue blocks are transferred to Matrigel matrix gel.

[0160] 1) Dissolve Matrigel at 4°C for 30 minutes.

[0161] 2) Place the parafilm over the 200 μL pipette tip, creating a (4 x 4) 16-well area. Press the parafilm with your finger to create a small depression. Sterilize under UV for 30 minutes.

[0162] 3) Use a 200 μL pipette tip to transfer the tissue chunks into each small groove prepared in advance.

[0163] 4) Carefully remove the excess culture medium from each well using a 200 μL pipette tip.

[0164] 5) Immediately add about 30 μL of growth factor-reduced Matrigel (BD Biosciences, 356230) onto the tissue block in each groove to fill the groove.

[0165] 6) Use a 10 μL pipette tip to move the tissue block to the center of the droplet as much as possible.

[0166] 7) Place the groove device at 60mm 2 Incubate at 37°C in a culture dish for 20-30 minutes to allow the Matrigel droplets to wrap tightly around the tissue pieces.

[0167] 8) To 60mm 2 Add 5 mL of long-term differentiation medium containing B27 supplement without vitamin A to the culture plate (see Table 3 for specific ingredients).

[0168] 9) Use sterile forceps to rotate the Parafilm and shake the culture dish to remove the Matrigel droplets. If any droplets remain on the Parafilm, hold one end of the Parafilm with forceps and shake it in the culture medium to remove the remaining droplets. Place the removed Matrigel droplets in the cell culture incubator and continue culturing.

[0169] Day 12: Add 5 mL of long-term differentiation medium containing B27 supplement without vitamin A to the culture dish.

[0170] Day 14: Add 5 mL of long-term differentiation medium containing B27 supplement with vitamin A (see Table 3 for specific ingredients) to the culture plate and transfer the culture plate to an incubator with a shaker at 70 rpm. Change the medium every 3-4 days.

[0171] Table 3. Long-term differentiation culture medium

[0172] Example 4 In vivo generation of intra- and extra-embryonic tissues

[0173] In vivo culture was performed according to the method shown in Figure 1, and teratomas were formed by cell transplantation into immunodeficient mice to produce cell aggregates. This method includes mixing the naive stem cells or early embryonic-like totipotent stem cells obtained in Example 1 with Matrigel, and then injecting the mixture into the mice to form cell aggregates with a 3D structure. The specific steps are as follows:

[0174] 1) Animal Preparation

[0175] Male NOD-scid-IL2Rg_ / _ mice (WuXi AppTec) were cultured and maintained in an SPF facility until they were 4-6 weeks old.

[0176] 2) Cell preparation

[0177] One million naive stem cells or early embryonic-like totipotent stem cells obtained by culture in Example 1 were separated from the culture medium and collected and then incubated with 200 μL of pre-cooled DMEM / F12 basal culture medium (Invitrogen cat. no. 11330-032 or 31330-038) with a volume ratio of 1:1. The mixture of Matrigel and cells is resuspended to obtain a resuspension. The resuspension is placed on ice before transplantation. Transplantation should be performed immediately.

[0178] 3) Transplantation experiments

[0179] The resuspended solution in step 2) was collected into a 1 ml syringe and injected subcutaneously into 4-6 week old male NOD-scid-IL2Rg_ / _ mice.

[0180] To avoid stress, mice should be prepared at least one week in advance. For teratoma formation, count 1 million cells in 200 μl of a 1:1 pre-chilled mixture of DMEM / F12 basal medium and Matrigel in a 1 mL sterile syringe. Expose the mouse's abdomen for injection and insert the needle of a sterile syringe obliquely into the mouse's abdominal skin. Inject the cell suspension subcutaneously into 4-6 week-old male NOD-scid IL2Rg_ / _ mice. Slowly withdraw the needle of the sterile syringe to prevent the cell suspension from escaping the injection site.

[0181] 4) Animal monitoring during teratoma formation

[0182] Mice were maintained in an SPF facility for 2-3 weeks after injection. Teratomas growing at the injection site were visually inspected for size. Typically, teratomas were isolated 2-3 weeks after injection.

[0183] 5) Collecting human cells from teratomas

[0184] Mice are sacrificed 2-3 weeks after injection, and teratomas are isolated for cryosectioning, cell isolation, single-cell transcriptome analysis, or other purposes.

[0185] Example 5 Single-cell sequencing identifies intra- and extra-embryonic cell types generated by in vivo and in vitro differentiation methods

[0186] To systematically analyze the entire cell lineage in the embryo-like and teratoma models derived from naive stem cells, single-cell RNA sequencing (scRNA-seq) analysis was performed on embryo-like (days 10, 20, and 30) and teratoma (days 14 and 21) using the DNBelab C4 droplet platform. After quality control, 75,326 single cells were obtained from embryo-like and 397,684 single cells were obtained from teratoma for further analysis. As shown in Figure 2, unified manifold approximation and projection (UMAP) analysis identified embryo-like cell types, including epiblast (Epi, POU5F1+ and DPPA4+), committed epiblast (Committed Epiblast, TNFRSF12A+ / JUND+), specifically including primordial germ cell-like cells (PGCLC, NANOG+ / NANOS3+ / TFAP2C+ / SOX17+); endoderm lineage (FOXA2+) including definitive endoderm (DE, HHEX+), intraembryonic endoderm (VE, HNF4A+ / SOX17+), anterior intraembryonic endoderm (AVE, OTX2+ / CXCR4+), and yolk sac endoderm (YE, HNF4A+ / SOX17-); mesoderm lineage including primitive streak (PS, TBXT+), emergent mesoderm (EM, LHX1+); late mesoderm cells (AM, TBXT- / MYL7+), allantoic cells (AI, TBX4+ / HOXA13+), and amnion / non-neural ectoderm progenitors (CLDN10, AMR2+ / PCAT14+ / VGLL1+). , and extraembryonic mesoderm cells (EXMCs) express mesodermal characteristics (ANXA1, LUM, NID2 / FRZB) but do not express markers of any embryonic counterparts; mesodermal-derived mesothelial cells (AQP1+), endothelial cells (CDH5+ / CD34+), arterial endothelial cells (GJA4+ / GJA5+), hematopoietic cell precursors mesodermal progenitors (BMP4+ / WNT5A+), angioblasts (ETV2+ / TAL1+ / BMP4+ / WNT5A+), hematopoietic cell precursors (RUNX1+ / SPN+), megakaryocytes (PPBP), erythroid cells (GATA1+ / HBE1+) and myeloid cell precursors (CSF1R+); ectodermal lineages include neuroepithelium (NE, PAX6+), neural crest-derived cells (NCC, SOX10+ / MPZ+) and amniotic ectoderm cells (ISL1+ / GABRP+).Further subgrouping, as shown in Figure 6, also identified cell types such as committed epiblast (TNFRSF12A+ / JUND+), arterial endothelial cells (GJA4+ / GJA5+), hematopoietic cell precursor mesoderm progenitors (BMP4+ / WNT5A+), angioblasts (ETV2+ / TAL1+ / BMP4+ / WNT5A+), hematopoietic cell precursors (RUNX1+ / SPN+), megakaryocytes (PPBP), erythrocytes (GATA1+ / HBE1+) and myeloid cell precursors (CSF1R+).

[0187] To evaluate the developmental progression of embryoids and teratomas derived from naive stem cells, scRNAseq data were integrated with a human embryo reference dataset, including human blastocysts at the pre-gastrulation stage (E6-E14), and Carnegie stage 7 (CS7; E16-E19) embryos of human gastrulation. As shown in Figure 3, the UMAP projection not only shows an excellent match of cell type characteristics of embryonic and extraembryonic lineages, but also shows that cell type differentiation follows the same process as development. It is worth noting that the model of the present invention includes neuroectodermal cells, as well as all other cell types found during CS7 gastrulation, indicating that the model of the present invention has surpassed the gastrulation stage and entered the early organogenesis stage. The identity of the neuroectodermal lineage was further verified by integrating monkey embryo datasets (CS7 to CS11). Neither teratomas nor embryoids contain the trophectoderm lineage (which later develops into the placenta). This limitation means that the model of the present invention cannot complete a complete embryo.

[0188] Example 6 Comparison of Differentiation Potential of Embryonic Stem Cells in In Vitro 3D Culture Methods

[0189] Cell preparation: Human primary PSCs were cultured using mTeSR medium (STEM CELL). When the primary human PSCs reached 70-80% of their growth rate and were ready for passage or in vitro 3D culture experiments, the medium was removed and 1 mL of PBS was added along the wall of the culture dish. The cells were gently washed to remove the residual medium and the PBS was removed. 1 mL of TrypLE was added. TMExpress: 0.5mM EDTA (volume ratio of 1:1) digestion solution, place in a 37°C incubator for digestion for about 5 minutes, and then observe under a microscope; when the cell digestion shows clear edges, use a pipette to draw 1mL of fresh culture medium to blow the cells, and transfer the cell suspension to a 1.5mL EP tube; centrifuge at 300g for 3min. Remove the supernatant, resuspend the cells with an appropriate amount of fresh culture medium, and count them with a counting plate or cell counter. Resuspend 9000 cells in 150μL embryoid body culture medium (specific components are shown in Table 1) and inoculate them into a 96-well low-adsorption culture plate. The specific steps of subsequent in vitro 3D culture are shown in Example 3.

[0190] In order to compare the differentiation potential of primed stem cells and naive stem cells in vitro, 3D products derived from primed and naive stem cells cultured to specific time points were stained with TrypLE TMExpress: 0.5mM EDTA (1:1 volume ratio) digestion into single cells, and then single-cell RNA sequencing (scRNA-seq) library analysis was performed on 3D differentiation products (days 10, 20, and 30) using the DNBelab C4 droplet platform (ref). As shown in Figures 4 and 5, the embryonic cell type epiblast cell type is predominant in the differentiation products derived from primed stem cells and naive stem cells, while extraembryonic cell types, such as the hypoblast-derived anterior visceral endoderm and visceral endoderm, are only present in the differentiation products derived from naive stem cells and are completely absent in the differentiation products derived from primed stem cells. In addition, Figure 5 shows that the proportions of each cell type in extraembryonic tissues in primitive stem cells are as follows: visceral endoderm 0.46%-11.57%, anterior visceral endoderm 0.14%-33.17%, yolk sac endoderm 1.19%-4.24%, extraembryonic mesoderm 0.33%-32.35%, mesothelial cells 4.22%-4.33%, allantoic cells 0.14%-4.26%, extraembryonic mesoderm-derived hematopoietic cell precursors mesodermal progenitor cells 0.1%-0.5%, angioblasts 0.1%-0.5%, endothelial cells 1.40%-4.79%, arterial endothelial cells 0.04%-0.1%, hematopoietic cell precursors 0.01%-0.1%, and megakaryocytes 0.01%-0. 1%, erythrocytes 0.1%-0.5% and, myeloid cell precursors 0.01%-0.1%, amniotic ectoderm cells 0.21%-10.23%, amniotic / non-neural ectoderm progenitor cells 0.09%-3.00%; the proportions of the major cell types in the embryonic tissues are: epiblast 2.18%-33.61%, definitive epiblast 2.57%-2.86%, primordial germ-like cells 0.02%-0.19%, definitive endoderm 0.48%-1.63%, primitive streak 0.31%-0.90%, emergent mesoderm 0.15%-1.83%, late mesoderm 1.61%-12.81%, neuroepithelium 0.18%-3.13%, neural crest-derived cells 0.18%-2.84%.

[0191] This unique differentiation ability of naive stem cells mimics the characteristics of the inner cell mass of the blastocyst, which can simultaneously generate epiblast and hypoblast lineages. In addition, on days 20 and 30, naive stem cell-derived differentiation products contain more lineages, such as extraembryonic lineages and primordial germ-like cell types, while primed stem cell-derived differentiation products are mainly composed of neuroectodermal cell types. This shows that naive stem cells have superior differentiation potential to primed stem cells under in vitro 3D culture methods.

[0192] Example 7 Comparison of the differentiation potential of different embryonic stem cells in teratoma formation

[0193] When the primary PSCs have grown to 70-80%, add 1 mL of PBS, gently wash the cells, wash away the residual culture medium, remove the PBS, and add 1 mL of TrypLE TM Express: 0.5 mM EDTA (volume ratio 1:1).

[0194] Digest them into single cells, take 1 million human PSCs and use 100 μL pre-cooled DMEM / F12 basal medium (Invitrogen cat. no. 11330-032 or 31330-038 and The mixture of matrigel is resuspended to obtain a resuspension. The resuspension is placed on ice before transplantation. Transplantation should be performed immediately. The subsequent transplantation experiment and specific experimental procedures are shown in Example 4.

[0195] In order to compare the differentiation potential of primed stem cells and naive stem cells under the teratoma formation method. The obtained teratomas (days 14 and 21) were subjected to single-cell RNA sequencing (scRNA-seq) library construction analysis. As shown in Figures 4 and 5, the teratoma lineage derived from primed stem cells is comparable to the lineage of in vitro 3D culture differentiation products derived from naive stem cells; compared with the teratoma lineage derived from naive stem cells, the teratoma lineage derived from primed stem cells mainly contains neuroectodermal cell types and a small amount of extraembryonic lineages, while the teratomas derived from naive stem cells have more extraembryonic lineages and primitive germ-like cell types. In addition, Figure 5 also shows that the proportion of each cell type in the extraembryonic tissues in the teratoma derived from primitive stem cells is as follows: visceral endoderm 0.46%-11.57%, anterior visceral endoderm 0.14%-33.17%, yolk sac endoderm 1.19%-4.24%, extraembryonic mesoderm 0.33%-32.35%, mesothelial cells 4.22%-4.33%, allantoic cells 0.14%-4.26%, extraembryonic mesoderm-derived hematopoietic cell precursors mesodermal progenitor cells 0.1%-0.5%, hemangioblasts 0.1%-0.5%, endothelial cells 1.40%-4.79%, arterial endothelial cells 0.04%-0.1%, hematopoietic cell precursors 0.01%-0.1%, megakaryocytes 0.01%. -0.1%, erythrocytes 0.1%-0.5% and, myeloid cell precursors 0.01%-0.1%, amniotic ectoderm cells 0.21%-10.23%, amniotic / non-neural ectoderm progenitor cells 0.09%-3.00%; the proportions of the major cell types in the embryonic tissues are: epiblast 2.18%-33.61%, definitive epiblast 2.57%-2.86%, primordial germ-like cells 0.02%-0.19%, definitive endoderm 0.48%-1.63%, primitive streak 0.31%-0.90%, emergent mesoderm 0.15%-1.83%, late mesoderm 1.61%-12.81%, neuroepithelium 0.18%-3.13%, neural crest-derived cells 0.18%-2.84%.

[0196] This shows that the differentiation potential of naive stem cells is superior to that of primed stem cells under the teratoma formation method.

[0197] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0198] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for constructing an embryo-like model, characterized in that: include: The embryo-like model is obtained by culturing the naive stem cells in vitro or injecting the naive stem cells into immunodeficient animals for differentiation.

2. The method according to claim 1, characterized in that The in vitro culture comprises: Subjecting the naive stem cells to a first culture treatment in a first culture medium and a second culture medium to obtain a first cell population; subjecting the first cell population to a second culture treatment in a third culture medium to obtain a second cell population; The second cell population is subjected to a third culture treatment in a fourth culture medium and a fifth culture medium to obtain the embryo-like model.

3. The method according to claim 2, characterized in that Pre-culture the naive stem cells, wherein the coverage of the naive stem cells in the culture dish after the pre-culture treatment is 70-80%; Optionally, the naive stem cells after the pre-culture treatment are subjected to a digestion treatment; Optionally, the digestion process is carried out in TrypLE TM Express enzyme in the presence of the 4. The method according to claim 2, characterized in that: The first culture medium comprises complete culture medium of human primed pluripotent stem cells, penicillin-streptomycin and Rock pathway inhibitor; Optionally, the second culture medium comprises complete culture medium for human primed pluripotent stem cells and penicillin-streptomycin; Optionally, the mass volume fraction of penicillin-streptomycin in the first culture medium or the second culture medium is 0.5-1.5%; Optionally, the Rock pathway inhibitor includes Y27632; Optionally, the final concentration of Y27632 in the first culture medium is 45-55 μM; Optionally, the first culture treatment is performed in a low-adsorption round-bottom 96-well plate, a 3D scaffold, or a microfluidic device; Optionally, the first culture treatment period is 5 to 7 days, preferably 5 days; Optionally, the first culturing treatment includes a first-stage culturing treatment and a second-stage culturing treatment, the first-stage culturing treatment is performed in the first culture medium, and the second-stage culturing treatment is performed in the second culture medium; Optionally, the first stage of culturing treatment lasts for 3 days. Optionally, after the naive stem cells are subjected to the first stage of culturing treatment in the first culture medium for 1 day, the first culture medium is replaced, and the naive stem cells are subjected to the first stage of culturing treatment in the first culture medium for another 2 days. Optionally, the second stage of culture treatment lasts for 2 to 4 days, preferably 2 days.

5. The method according to claim 2, characterized in that: The third culture medium comprises DMEM-F12 basal medium, N2 supplement, glutaminase, non-essential amino acids, heparin and penicillin-streptomycin; Optionally, the mass volume fraction of the N2 supplement in the third culture medium is 0.5-1.5%; Optionally, the mass volume fraction of the glutaminase in the third culture medium is 0.5-1.5%; Optionally, the mass volume fraction of the non-essential amino acids in the third culture medium is 0.5-1.5%; Optionally, the final concentration of the heparin in the third culture medium is 0.5 to 1.5 μg / mL; Optionally, the mass volume fraction of penicillin-streptomycin in the third culture medium is 0.5-1.5%; Optionally, the second culture treatment period is 3 to 5 days.

6. The method according to claim 2, characterized in that Prior to the third culture treatment, the second cell population is mixed with Matrigel.

7. The method according to any one of claims 2 to 6, characterized in that: The fourth culture medium comprises DMEM-F12 basal medium, neural basal medium, N2 supplement, glutaminase, non-essential amino acids, insulin, β-mercaptoethanol, penicillin-streptomycin and B27 supplement without vitamin A; Optionally, the fifth culture medium includes DMEM-F12 basal medium, neural basal medium, N2 supplement, glutaminase, non-essential amino acids, insulin, β-mercaptoethanol, penicillin-streptomycin and B27 supplement containing vitamin A; Optionally, the volume ratio of the DMEM-F12 basal medium to the neural basal medium is 1:(0.8-1.2); Optionally, the mass volume fraction of the N2 supplement in the fourth culture medium or the fifth culture medium is 0.2-0.7%; Optionally, the mass volume fraction of the glutaminase in the fourth culture medium or the fifth culture medium is 0.5 to 1.5%; Optionally, the mass volume fraction of the non-essential amino acid in the fourth culture medium or the fifth culture medium is 0.2-0.7%; Optionally, the final concentration of the insulin in the fourth culture medium or the fifth culture medium is 1 to 1.5 μg / mL; Optionally, the final concentration of the β-mercaptoethanol in the fourth culture medium or the fifth culture medium is 0.1 to 0.25 μM; Optionally, the mass volume fraction of penicillin-streptomycin in the fourth culture medium or the fifth culture medium is 0.5-1.5%; Optionally, the mass volume fraction of the B27 supplement not containing vitamin A in the fourth culture medium is 0.2-0.7%; Optionally, the mass volume fraction of the B27 supplement containing vitamin A in the fifth culture medium is 0.2-0.7%; Optionally, the third culture treatment includes a third-stage culture treatment and a fourth-stage culture treatment, the third-stage culture treatment is carried out in a fourth culture medium, and the fourth-stage culture treatment is carried out in a fifth culture medium; Optionally, the third stage of culturing treatment lasts for 3 to 5 days, preferably 4 days; Optionally, the duration of the fourth stage of culturing treatment is 13 to 18 days, preferably 16 days; Optionally, the fourth stage culturing process is performed on a shaker; Optionally, the shaking table has a rotation speed of 60 to 80 RPM.

8. The method according to claim 1, characterized in that Before injecting the naive stem cells into an immunodeficient animal for differentiation, the naive stem cells are mixed with a DMEM-F12 basal medium containing matrigel; Optionally, the volume ratio of the DMEM-F12 basal medium to the matrix gel is 1:(0.8-1.2); Optionally, the ratio of the number of the naive stem cells to the volume of the DMEM-F12 basal medium containing the matrigel is 5 4 Pieces: (0.8~1.2)μL.

9. The method according to claim 1 or 8, characterized in that: The animal is selected from immunodeficient mice, immunodeficient rabbits, immunodeficient cattle, immunodeficient pigs or immunodeficient monkeys; Optionally, the primitive stem cells are selected from at least one of pluripotent stem cells, totipotent stem cells, primitive endoderm cells and trophectoderm cells; Optionally, the source of the naive stem cells is selected from humans, cows, monkeys, pigs or mice; Optionally, the injection is selected from subcutaneous injection; Optionally, the in vivo differentiation time is 13 to 70 days.

10. An embryo-like model, characterized in that: The method is obtained by culturing according to any one of claims 1 to 9.

11. An embryo-like model, characterized in that: Includes extra-embryonic tissues and intra-embryonic tissues.

12. The embryo-like model according to claim 11, characterized in that: The extraembryonic tissues include: visceral endoderm, pre-visceral endoderm, yolk sac endoderm, extraembryonic mesoderm, mesothelial cells, allantoic cells, mesodermal progenitor cells of hematopoietic cell precursors derived from extraembryonic mesoderm, hemangioblasts derived from extraembryonic mesoderm, endothelial cells derived from extraembryonic mesoderm, arterial endothelial cells derived from extraembryonic mesoderm, hematopoietic cell precursors derived from extraembryonic mesoderm, megakaryocytes derived from extraembryonic mesoderm, erythrocytes derived from extraembryonic mesoderm, myeloid cell precursors derived from extraembryonic mesoderm, amniotic ectoderm cells, and amniotic / non-neural ectoderm progenitor cells; Optionally, the intraembryonic tissue includes epiblast, definitive epiblast, primordial germ-like cells, definitive endoderm, primitive streak, emergent mesoderm, late mesoderm, neural epithelium and neural crest-derived cells.

13. The embryo-like model according to claim 12, characterized in that: The genes expressed in the visceral endoderm include HNF4A and SOX17; the genes expressed in the anterior visceral endoderm include OTX2 and CXCR4; the genes expressed in the yolk sac endoderm include HNF4A, and SOX17 is lowly expressed or not expressed; the genes expressed in the extraembryonic mesoderm cells include ANXA1, LUM, NID2 and FRZB; the genes expressed in the mesothelial cells include AQP1; the genes expressed in the allantoic cells include TBX4 and HOXA13; the genes in the hematopoietic cell precursors derived from the extraembryonic mesoderm include ANXA1, LUM, NID2 and FRZB; the genes expressed in the mesothelial cells include AQP1; the genes expressed in the allantoic cells include TBX4 and HOXA13; The genes expressed in the germ layer progenitor cells include BMP4 and WNT5A; the genes expressed in the angioblasts derived from the extraembryonic mesoderm include ETV2, TAL1, BMP4 and WNT5A; the genes expressed in the endothelial cells derived from the extraembryonic mesoderm include CDH5 and CD34; the genes expressed in the arterial endothelial cells derived from the extraembryonic mesoderm include GJA4 and GJA5; the genes expressed in the hematopoietic cell precursors derived from the extraembryonic mesoderm include RUNX1 and SPN; the genes expressed in the megakaryocytes derived from the extraembryonic mesoderm include The genes expressed in the extraembryonic mesoderm-derived erythrocytes include GATA1 and HBE1; the genes expressed in the extraembryonic mesoderm-derived myeloid cell precursors include CSF1R; the genes expressed in the amniotic ectoderm cells include ISL1 and GABRP; the genes expressed in the amniotic / non-neural ectoderm progenitor cells include CLDN10, AMR2, PCAT14 and VGLL1; the genes expressed in the epiblast include POU5F1 and DPPA4; the genes expressed in the definitive epiblast include TNFRS F12A and JUND; the genes expressed in the primordial germ-like cells include NANOG, NANOS3, TFAP2C and SOX17; the genes expressed in the definitive endoderm include HHEX; the genes expressed in the primitive streak include TBXT; the genes expressed in the emergent mesoderm include LHX1; the genes expressed in the late mesoderm include MYL7, and TBXT is lowly expressed or not expressed; the genes expressed in the neuroepithelium include PAX6; the genes expressed in the neural crest-derived cells include SOX10 and MPZ; Optionally, the definitive endoderm, the visceral endoderm, the pre-visceral endoderm and the yolk sac endoderm co-express FOXA2.

14. The embryo-like model according to claim 12, characterized in that: The proportions of each cell type in the extraembryonic tissue are as follows: 0.46%-11.57% of the visceral endoderm, 0.14%-33.17% of the anterior visceral endoderm, 1.19%-4.24% of the yolk sac endoderm, 0.33%-32.35% of the extraembryonic mesoderm, 4.22%-4.33% of the mesothelial cells, 0.14%-4.26% of the allantoic cells, 0.1%-0.5% of the hematopoietic cell precursor mesoderm progenitor cells derived from the extraembryonic mesoderm, and 0.2%-0.6% of the progenitor cells. Angioblasts 0.1%-0.5%, endothelial cells 1.40%-4.79%, arterial endothelial cells 0.04%-0.1%, hematopoietic cell precursors 0.01%-0.1%, megakaryocytes 0.01%-0.1%, erythrocytes 0.1%-0.5%, myeloid cell precursors 0.01%-0.1%, amniotic ectoderm cells 0.21%-10.23%, amniotic / non-neural ectoderm progenitor cells 0.09%-3.00%; Optionally, the proportions of the main cell types in the embryonic tissue are: the epiblast 2.18%-33.61%, the definitive epiblast 2.57%-2.86%, the primordial germ-like cells 0.02%-0.19%, the definitive endoderm 0.48%-1.63%, the primitive streak 0.31%-0.90%, the emergent mesoderm 0.15%-1.83%, the late mesoderm 1.61%-12.81%, the neural epithelium 0.18%-3.13%, the neural epithelium 0.19%-1.20%, the neural epithelium 0.16%-1.30%, the neural epithelium 0.16 ... Crest-derived cells: 0.18%-2.84%.

15. The embryo-like model according to any one of claims 11 to 14, characterized in that: The proportions of each cell type in the extra-embryonic tissue and intra-embryonic tissue are shown in FIG5 .

16. The embryo-like model according to any one of claims 10 to 15, characterized in that: The embryo-like model can simulate the cell types in the gastrula stage.

17. Use of the embryo-like model according to any one of claims 10 to 16 in constructing a Carnegie developmental stage model; Optionally, the Carnegie developmental stage is selected from the developmental stages of Stage 3 to Stage 9.

18. Use of the embryo-like model according to any one of claims 10 to 16 in drug screening, wherein the drug is used to intervene in embryonic development during the gastrula stage.

19. Use of the embryo-like model according to any one of claims 10 to 16 in constructing a disease model, wherein the disease model comprises a developmental disease model; Optionally, the developmental disease model comprises an implantation defect model or a Down syndrome model.

20. Use of the embryo-like model according to any one of claims 10 to 16 in obtaining progenitor cells of a specific lineage.

21. The use according to claim 20, characterized in that The method comprises expanding and culturing the specific lineage in the embryo-like model to obtain the progenitor cells of the specific lineage; Optionally, the method further comprises performing gene editing or cell differentiation on the specific lineage after the expansion culture treatment, or performing cell therapy using the specific lineage after the expansion culture treatment.

22. Use of the embryo-like model according to any one of claims 10 to 16 in constructing a specific organoid model or a 3D cell aggregate.

23. The use according to claim 22, characterized in that Including inducing the embryo-like model into a specific organoid model or the cell 3D aggregate by induction means; Optionally, the inducing means comprises adding small molecule compounds, co-culturing cells or adding cytokines.

24. A method for screening a drug for intervening in the development of gastrula embryos, characterized in that: Adding the drug to be screened to the culture process of the method for constructing the embryo-like model according to any one of claims 1 to 9 or contacting the drug to be screened with the embryo-like model according to any one of claims 10 to 16; The target drug is determined based on whether the embryo-like model obtained by the construction method described in any one of claims 1 to 9 or the embryo-like model described in any one of claims 10 to 16 undergoes expected changes.

25. The method according to claim 24, characterized in that The expected change in the embryo-like model obtained by the construction method according to any one of claims 1 to 9 after adding the drug compared to before adding the drug is an indication that the drug to be screened is the target drug.

26. The method according to claim 24, characterized in that The expected change of the embryo-like model according to any one of claims 10 to 16 after the contact treatment compared to before the contact treatment is an indication that the drug to be screened is a target drug.

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