Culture medium for induced hypoblast stem cells and use thereof

Abstract

The present invention belongs to the technical field of stem cells. Provided are a culture medium for induced hypoblast stem cells and the use thereof. Provided is a culture medium for induced hypoblast stem cells. The components of the culture medium comprise platelet-derived growth factor AA, a leukemia inhibitory factor, fibroblast growth factor 4, GSK-3α / β inhibitor CHIR99021, TGF-β type I receptor inhibitor A83-01, and bone morphogenetic protein 4. The culture medium for induced hypoblast stem cells can accurately regulate the activity of key signaling pathways such as NODAL, BMP, WNT, FGF, and JAK / STAT in the process of somatic cell reprogramming, and ensure that the properties of hypoblast stem cells are maintained in the process of somatic cell reprogramming to support the self-renewal and hypoblast lineage induction of somatic cells in the reprogramming process, thereby realizing the fate transition of somatic cells to hypoblast stem cells.

Description

A culture medium for inducing hypodermal stem cells and its application

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411386158.7, filed on September 30, 2024, entitled “A Culture Medium for Inducing Hypodermic Stem Cells and Its Application”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to a culture medium for inducing hypodermal stem cells and its application, belonging to the field of stem cell technology. Background Technology

[0004] During early human embryonic development, the differentiation of the inner cell mass (ICM) and trophoblastic ectoderm (TE) is a key developmental event. Subsequently, epiblastic stem cells (Epi) and hypoblastic stem cells (Hypo, also known as primitive endoderm stem cells) further develop to form structures such as the fetus and yolk sac. Studying hypoblastic development is crucial for understanding the early processes of human embryonic development.

[0005] Currently, research mainly focuses on inducing human hypodermal-like cells in vitro through chemical induction or transgenic overexpression to study hypodermal development. These methods typically involve... Pluripotent stem cells ( Specific transcription factors or chemicals are introduced into PSCs to induce the generation of hypoblastoid cells. For example, Okubo et al. and Linneberg-Agerholm et al. generated human hypoblastoid cells (hXENs) through chemical induction (see “Okubo, T. et al. Hypoblast from human pluripotent stem cells regulates epiblast development. Nature 626, 357-366 (2024)” and “Linneberg-Agerholm, M. et al. Naive human pluripotent stem cells respond to Wnt, Nodal and LIF signalling to produce expandable naive extra-embryonic endoderm. Development 146 (2019)”).

[0006] However, these methods have multiple defects in the culture and maintenance of hypoblast-like cells. First, the cells produced by these methods usually lack the ability of self-renewal and are difficult to maintain their stem cell state for a long time. Second, the cells produced by these methods are contradictory in the signal requirements of hypoblast. For example, nHyC-7F (see Okubo, T. et al. Hypoblast from human pluripotent stem cells regulates epiblast development. Nature 626, 357-366 (2024).) and PA-FA83X (see Dattani, A. et al. Naive pluripotent stem cell-based models capture FGF-dependent human hypoblast lineage specification. Cell Stem Cell 31, 1058-1071.e5 (2024).) are different from RACL / NACL (see Linneberg-Agerholm, M. et al. human pluripotent stem cells respond to Wnt, Nodal and LIF signalling to produce expandable compared to RCL (see “Oldak, B. et al. Complete human day 14 post-implantation embryo models from naive ES cells. Nature 622, 562-573 (2023).) and FTW (see “Wei, Y. et al. Dissecting embryonic and extraembryonic lineage crosstalk with stem cell co-culture. Cell 186, 5859-5875.e24 (2023).), there are conflicting conclusions about the role of WNT signaling and Activin signaling in hypoblast cell fate determination. Furthermore, the cells generated by these methods are more similar to extraembryonic mesoderm (ExEM) rather than true hypoblast stem cell lineage, limiting their utility in modeling human embryonic development. In addition, the hypoblast-like cells obtained by these methods are not comprehensive in terms of molecular and functional validation, lacking further validation such as human-mouse chimera experiments and single-cell RNA sequencing. That is, the hypoblast-like cells constructed by these methods often lack the self-renewing stem cell properties, and there are contradictions in the signal requirements, or the hypoblast-like cells generated by them are more similar to ectodermal mesoderm (ExEM) rather than true hypoblast lineage, and the identification of hypoblast cell lineage characteristics is insufficient. Therefore, the current in vitro model of hypoblast-like cells still has significant deficiencies in accurately simulating human embryonic development, especially the differentiation and function of hypoblast lineage cells. SUMMARY

[0007] To solve the above-mentioned defects, the present application provides a hypoblast stem cell induction medium, the components of the hypoblast stem cell induction medium comprise platelet-derived growth factor AA (PDGFAA), leukemia inhibitory factor (LIF), fibroblast growth factor 4 (FGF4), GSK-3a / b inhibitor CHIR99021, TGF-beta type I receptor inhibitor A83-01 and bone morphogenetic protein 4 (BMP4).

[0008] In an embodiment of the present application, the concentration of the platelet-derived growth factor AA in the induced hypoblast stem cell culture medium is 10-50 ng / mL; the concentration of the leukemia inhibitory factor in the induced hypoblast stem cell culture medium is 10-50 ng / mL; the concentration of the fibroblast growth factor 4 in the induced hypoblast stem cell culture medium is 25-125 ng / mL; the concentration of the GSK-3a / b inhibitor CHIR99021 in the induced hypoblast stem cell culture medium is 3-15 μM; the concentration of the TGF-β type I receptor inhibitor A83-01 in the induced hypoblast stem cell culture medium is 1-5 μM; and the concentration of the bone morphogenetic protein 4 in the induced hypoblast stem cell culture medium is 10-50 ng / mL.

[0009] In an embodiment of the present application, the components of the induced hypoblast stem cell culture medium further comprise heparin, N2 supplement, B27 supplement, GlutaMAX, non-essential amino acids, 2-mercaptoethanol, and / or penicillin-streptomycin.

[0010] In an embodiment of the present application, the concentration of the N2 supplement in the induced hypoblast stem cell culture medium is 0.5-1% by volume; the concentration of the B27 supplement in the induced hypoblast stem cell culture medium is 1-2% by volume; the concentration of the GlutaMAX in the induced hypoblast stem cell culture medium is 0.5-1% by volume; the concentration of the non-essential amino acids in the induced hypoblast stem cell culture medium is 0.5-1% by volume; the concentration of the penicillin-streptomycin in the induced hypoblast stem cell culture medium is 0.5-1% by volume; the concentration of the heparin in the induced hypoblast stem cell culture medium is 1-5 μg / mL; and the concentration of the 2-mercaptoethanol in the induced hypoblast stem cell culture medium is 0.1-0.5 mM.

[0011] In an embodiment of the present application, the components of the induced hypoblast stem cell culture medium further comprise a matrix; the matrix comprises Neurobasal medium and DMEM / F12 medium; and the volume ratio of Neurobasal medium to DMEM / F12 medium in the matrix is 0.5-1:0.5-1.

[0012] In an embodiment of the present application, the matrix consists of Neurobasal medium and DMEM / F12 medium; and the volume ratio of Neurobasal medium to DMEM / F12 medium in the matrix is 1:1.

[0013] The application also provides a method for constructing induced hypoblast stem cells by somatic cell reprogramming, which comprises: firstly transfecting somatic cells with Yamanaka factors (OSKM), and then inducing and culturing the transfected somatic cells with the above-mentioned induced hypoblast stem cell medium to obtain induced hypoblast stem cells (iHypoblast SCs).

[0014] In an embodiment of the application, the method comprises: after the somatic cells are inoculated into the somatic cell medium, the somatic cells are transfected with Yamanaka factors (OSKM) under the mediation of a transfection vector; after the transfection is completed, the transfected somatic cells are dispersed and inoculated into a cell culture plate with feeder cells, and the cells in the cell culture plate are cultured with the somatic cell medium and the induced hypoblast stem cell medium without additional ROCK-I / II inhibitor Y27632 in sequence; after the culture is completed, the PDGFRA-positive hypoblast-like cells are selected and re-inoculated into a cell culture plate with feeder cells, and the cells in the cell culture plate are cultured with the induced hypoblast stem cell medium with additional ROCK-I / II inhibitor Y27632 and the induced hypoblast stem cell medium without additional ROCK-I / II inhibitor Y27632 in sequence; after the culture is completed, the hypoblast-like colonies in the cell culture plate are transferred to a cell culture plate with feeder cells, and the cells in the cell culture plate are cultured with the induced hypoblast stem cell medium with additional ROCK-I / II inhibitor Y27632 and the induced hypoblast stem cell medium without additional ROCK-I / II inhibitor Y27632 in sequence until the hypoblast-like cells in the cell culture plate reach 80-90% confluence; after the culture is completed, the hypoblast-like cells are dispersed and inoculated into a cell culture plate with feeder cells, and the cells in the cell culture plate are cultured with the induced hypoblast stem cell medium with additional ROCK-I / II inhibitor Y27632 and the induced hypoblast stem cell medium without additional ROCK-I / II inhibitor Y27632 in sequence to obtain induced hypoblast stem cells (iHypoblast SCs).

[0015] In an embodiment of the application, the concentration of the ROCK-I / II inhibitor Y27632 in the induced hypoblast stem cell medium is 5-10 μM.

[0016] In an embodiment of the application, the somatic cells include human fibroblasts (for example, adult dermal fibroblasts and neonatal dermal fibroblasts), amniotic mesenchymal stem cells, cardiac fibroblasts, CD34+ blood cells, mammary epithelial cells, nasal epithelial cells, peripheral blood mononuclear cells, skeletal muscle myoblasts, T cells, umbilical vein epithelial cells and / or urethral epithelial cells.

[0017] In an embodiment of the application, the somatic cell medium comprises fibroblast medium and / or 106 medium.

[0018] In an embodiment of the application, the feeder layer cells comprise inactivated mouse embryonic fibroblasts and / or inactivated human fibroblasts.

[0019] In an embodiment of the application, the Yamanaka factors (OSKM) for reprogramming somatic cells (e.g., human fibroblasts) into a dedifferentiated or pluripotent state comprise OCT3 / 4, c-MYC, KLF4, SOX2, L-MYC, NANOG, LIN28, and / or chemical reprogramming.

[0020] In an embodiment of the application, the transfection vector comprises a virus, a liposome, an mRNA, and / or an exosome.

[0021] In an embodiment of the application, the virus comprises a Sendai virus, an adenovirus, and / or a lentivirus.

[0022] The present application also provides an induced hypoblast stem cell (iHypoblast SCs) constructed by the above method.

[0023] Characteristics of the induced hypoblast stem cell (iHypoblast SCs) include:

[0024] First, in an undifferentiated, bipotential state, and having the ability to differentiate into cells exhibiting one or more characteristics of endoderm / yolk sac endoderm (VE / YE) like cells and extraembryonic mesoderm (ExEM) like cells;

[0025] Second, a colony appearance of flat epithelial shape;

[0026] Third, expression of one or more biochemical markers of hypoblast stem cells (e.g., PDGFRA, SOX17, GATA6, GATA4, FOXA2, FN1, COL4A1, and Laminin), which can be determined by immunohistochemistry and / or PCR;

[0027] Fourth, single cell omics similar to human embryonic-derived hypoblast cells;

[0028] Fifth, the ability to integrate in the extraembryonic endoderm lineage in human mouse chimeras.

[0029] The application also provides a method for establishing an in vitro model of a hypoblast development related disease, which comprises: first constructing the induced hypoblast stem cell by the above method, and then establishing an in vitro model of a hypoblast development related disease by using the constructed induced hypoblast stem cell.

[0030] Alternatively, the method comprises: establishing an in vitro model of a hypoblast development related disease by using the induced hypoblast stem cell.

[0031] In an embodiment of the application, the establishment of the in vitro model of a hypoblast development related disease comprises: administering SB431542, A83-01 and Activin A drugs to the induced hypoblast stem cell to obtain an in vitro model of Nodal / Activin signaling pathway and hypoblast cell development; or administering CHIR99021, IWP-2 and XAV939 drugs to the induced hypoblast stem cell to obtain an in vitro model of Wnt / β-Catenin signaling pathway and hypoblast cell development; or administering BMP4, LDN-193189 and Noggin drugs to the induced hypoblast stem cell to obtain an in vitro model of BMP signaling pathway and hypoblast cell development; or administering FGF2 and SU5402 drugs to the induced hypoblast stem cell to obtain an in vitro model of FGF signaling pathway and hypoblast cell development.

[0032] The application also provides a method for screening a drug for preventing and / or treating a hypoblast development related disease, which comprises: first constructing the induced hypoblast stem cell by the above method, and then screening the drug for preventing and / or treating a hypoblast development related disease by using the constructed induced hypoblast stem cell as a cell model.

[0033] Alternatively, the method comprises: screening the drug for preventing and / or treating a hypoblast development related disease by using the induced hypoblast stem cell as a cell model.

[0034] Alternatively, the method comprises: first constructing the induced hypoblast stem cell by the above method, then establishing an in vitro model of a hypoblast development related disease by using the constructed induced hypoblast stem cell, and finally screening the drug for preventing and / or treating a hypoblast development related disease by using the in vitro model.

[0035] Alternatively, the method comprises: first establishing an in vitro model of a hypoblast development related disease by using the induced hypoblast stem cell, and then screening the drug for preventing and / or treating a hypoblast development related disease by using the in vitro model.

[0036] In an embodiment of the present application, the hypoblast development related disease comprises yolk sac related disease, genetic disease and / or developmental disorder.

[0037] In an embodiment of the present application, the yolk sac related disease comprises yolk sac agenesis and / or yolk sac hydrops; the genetic disease comprises GATA6 mutation induced genetic pancreatic agenesis, HNF1B related renal dysplasia and / or situs inversus; the developmental disorder comprises endoderm developmental disorder, anterior-posterior axis developmental disorder and / or early embryonic developmental arrest.

[0038] The present application also provides a method for evaluating drug toxicity, which comprises: firstly constructing induced hypoblast stem cells by the above method, then administering the drug to the constructed induced hypoblast stem cells, and evaluating the toxicity of the drug by observing the changes of the indicators of the induced hypoblast stem cells.

[0039] Alternatively, the method comprises: administering the drug to the above induced hypoblast stem cells, and evaluating the toxicity of the drug by observing the changes of the indicators of the induced hypoblast stem cells.

[0040] In an embodiment of the present application, the toxicity of the drug comprises potential embryonic / fetal toxicity, neurotoxicity, immunotoxicity, genotoxicity, endocrine disruption, embryonic lethality and / or teratogenicity.

[0041] The present application also provides the use of the above induced hypoblast stem cell culture medium or the above method or the above induced hypoblast stem cells (iHypoblast SCs) in establishing an in vitro model of hypoblast development related disease, screening drugs for preventing and / or treating hypoblast development related disease or evaluating drug toxicity.

[0042] In an embodiment of the present application, the hypoblast development related disease comprises yolk sac related disease, genetic disease and / or developmental disorder.

[0043] In an embodiment of the present application, the yolk sac related disease comprises yolk sac agenesis and / or yolk sac hydrops; the genetic disease comprises GATA6 mutation induced genetic pancreatic agenesis, HNF1B related renal dysplasia and / or situs inversus; the developmental disorder comprises endoderm developmental disorder, anterior-posterior axis developmental disorder and / or early embryonic developmental arrest.

[0044] In an embodiment of the present application, the toxicity of the drug comprises potential embryonic / fetal toxicity, neurotoxicity, immunotoxicity, genotoxicity, endocrine disruption, embryonic lethality and / or teratogenicity.

[0045] The technical solution of the present application has the following advantages:

[0046] 1. The present application provides an induced hypoblast stem cell culture medium, the composition of the induced hypoblast stem cell culture medium comprises platelet-derived growth factor AA (PDGFAA), leukemia inhibitory factor (LIF), fibroblast growth factor 4 (FGF4), GSK-3α / β inhibitor CHIR99021, TGF-β type I receptor inhibitor A83-01 and bone morphogenetic protein 4 (BMP4). The induced hypoblast stem cell culture medium can accurately regulate the activity of NODAL, BMP, WNT, FGF and JAK / STAT and other key signaling pathways in the process of somatic cell reprogramming, and then ensure that somatic cells maintain the characteristics of hypoblast stem cells during reprogramming to support self-renewal and hypoblast lineage induction of somatic cells during reprogramming, thereby realizing the fate transformation of somatic cells to hypoblast stem cells. Studies have shown that the induced hypoblast stem cells (iHypoblast SCs) constructed using the induced hypoblast stem cell culture medium have self-renewal and differentiation potential, can be cultured in vitro for a long time and maintain their unique hypoblast characteristics (specifically manifested in differentiation ability under in vitro conditions and developmental potential under in vivo conditions), and the induced hypoblast stem cells constructed using the induced hypoblast stem cell culture medium as an in vitro model can significantly improve the accuracy and practicability of simulating the human embryonic development process. Moreover, studies have shown that the induced hypoblast stem cells (iHypoblast SCs) constructed using the induced hypoblast stem cell culture medium can be successfully integrated into the primitive endoderm and visceral endoderm of mouse embryos, demonstrating their lineage contribution and developmental potential in early chimeric embryo development. Therefore, the induced hypoblast stem cell culture medium constructed using the induced hypoblast stem cell culture medium as an in vitro model has very high reliability and applicability in simulating human hypoblast development research, which can provide a powerful platform for human embryology research, and the induced hypoblast stem cell culture medium has great application prospects in the fields of establishing in vitro models of diseases related to hypoblast development, screening drugs for preventing and / or treating diseases related to hypoblast development, or evaluating drug toxicity, etc.

[0047] 2. The application provides a method for constructing induced hypoblast stem cells by somatic cell reprogramming, the method comprising: first transfecting somatic cells with Yamanaka factors (OSKM), and then inducing and culturing the transfected somatic cells with the above-mentioned induced hypoblast stem cell medium to obtain induced hypoblast stem cells (iHypoblast SCs); the components of the induced hypoblast stem cell medium include platelet-derived growth factor AA (PDGFAA), leukemia inhibitory factor (LIF), fibroblast growth factor 4 (FGF4), GSK-3α / β inhibitor CHIR99021, TGF-β type I receptor inhibitor A83-01 and bone morphogenetic protein 4 (BMP4). The method precisely regulates the activity of NODAL, BMP, WNT, FGF and JAK / STAT and other key signaling pathways in the somatic cell reprogramming process by Yamanaka factors (OSKM) and the induced hypoblast stem cell medium, and then ensures that the somatic cells maintain the characteristics of hypoblast stem cells during the reprogramming process to support the self-renewal and hypoblast lineage induction of somatic cells during the reprogramming process, thereby realizing the fate transformation of somatic cells to hypoblast stem cells. Studies have shown that the induced hypoblast stem cells (iHypoblast SCs) constructed using the method have self-renewal and differentiation potential, can be cultured in vitro for a long time and maintain their unique hypoblast characteristics (specifically manifested in differentiation ability under in vitro conditions and developmental potential under in vivo conditions), and the induced hypoblast stem cells constructed using the method as an in vitro model can significantly improve the accuracy and practicability of simulating the human embryonic development process. Moreover, studies have shown that the induced hypoblast stem cells (iHypoblast SCs) constructed using the method can be successfully integrated into the primitive endoderm and visceral endoderm of mouse embryos, demonstrating their lineage contribution and developmental potential in early chimeric embryo development. Therefore, the induced hypoblast stem cell medium constructed using the method as an in vitro model has very high reliability and applicability in simulating human hypoblast development research, can provide a powerful platform for human embryology research, and the method has great application prospects in the fields of establishing in vitro models of diseases related to hypoblast development, screening drugs for preventing and / or treating diseases related to hypoblast development or evaluating drug toxicity, etc.

[0048] 3. The present application provides a kind of induced hypoblast stem cells (iHypoblast SCs), the construction method of the induced hypoblast stem cells (iHypoblast SCs) includes: first using Yamanaka factor (OSKM) to transfect somatic cell, then using the above-mentioned induced hypoblast stem cell culture medium to the somatic cell after transfection is induced culture, obtains induced hypoblast stem cell (iHypoblast SCs);The composition of the induced hypoblast stem cell culture medium includes platelet-derived growth factor AA (PDGFAA), leukemia inhibitory factor (LIF), fibroblast growth factor 4 (FGF4), GSK-3 alpha / beta inhibitor CHIR99021, TGF-beta type I receptor inhibitor A83-01 and bone morphogenetic protein 4 (BMP4).Research shows that the induced hypoblast stem cell (iHypoblast SCs) has self-renewal and differentiation potential, can be cultured in vitro for a long time and keep its unique hypoblast characteristics (specifically embodied in the differentiation ability under in vitro condition and the development potential under in vivo condition), with the induced hypoblast stem cell as in vitro model, the accuracy and practicability of simulating human embryonic development process can be significantly improved.And research shows that the induced hypoblast stem cell (iHypoblast SCs) can be successfully integrated into the original endoderm and visceral endoderm of mouse embryo, and shows its lineage contribution and development potential in early chimeric embryo development.Therefore, the induced hypoblast stem cell as in vitro model has very high reliability and applicability in simulating human hypoblast development research, can provide a strong platform for human embryology research, and the induced hypoblast stem cell has great application prospect in the fields of establishing in vitro model related to hypoblast development, screening drugs for preventing and / or treating diseases related to hypoblast development or evaluating drug toxicity. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1: Inducing human iHypoblast SCs from somatic cells. In Figure 1, (a) schematic diagram of inducing human iHypoblast SCs from somatic cells; (b) FACS screening of the proportion of PDGFRA-positive cells in the 20th day reprogramming intermediate product; (c) morphological changes of 2D cultured reprogramming cells at different passage numbers under phase contrast microscope.

[0050] Figure 2: Transcriptome analysis. In Figure 2, (a) Display of defined Epi, Hypo and TE signature scores on the unified manifold approximation and projection (UMAP) integration plot of reprogrammed intermediate cells at day 0, day 4, day 8 and day 21, cells were cultured under fibroblast medium (Fm, fibroblast medium); (b) Display of defined Epi, Hypo and TE signature scores on the bulk RNA-seq data of reprogrammed intermediate cells at day 0, day 3 and day 7, cells were cultured under fibroblast medium; (c) Browser view displaying ATAC peaks of GATA6, SOX17, FOXA2, OTX2, SALL1 and GATA4 in reprogrammed intermediate cells at day 0, day 3 and day 7, cells were cultured under fibroblast medium (highlighted areas demonstrate changes in chromatin accessibility, showing binding of transcription factors associated with hypoblast).

[0051] Figure 3: ATAC-seq analysis. In Figure 3, preview view displaying ATAC peaks of GATA6, SOX17, FOXA2, OTX2, SALL1 and GATA4 in reprogrammed intermediate cells at day 0, day 3 and day 7, cells were cultured under fibroblast medium (highlighted areas demonstrate changes in chromatin accessibility, showing binding of transcription factors associated with hypoblast).

[0052] Figure 4: 33 screening media. In Figure 4, (a) 33 screening media (comprising cytokines and small molecule inhibitors / activators) screened for their ability to maintain human hypoblast stem cells in culture in vitro; (b) Heatmap and score bar chart of relative protein expression of PDGFRA, SOX17, FOXA2, GATA4, NANOG and BST2 corresponding to 33 screening conditions.

[0053] Figure 5: 33 screening media immunofluorescence staining. In Figure 5, representative immunofluorescence staining images of FOXA2, PDGFRA, GATA4, NANOG, SOX17 and BST2 at day 20 of reprogramming, 1# (a), 2# (b), 3# (c), 4# (d), 5# (e), 6# (f) screening conditions.

[0054] Figure 6: 33 screening media immunofluorescence staining. In Figure 6, representative immunofluorescence staining images of FOXA2, PDGFRA, GATA4, NANOG, SOX17 and BST2 at day 20 of reprogramming, 7# (a), 8# (b), 9# (c), 10# (d), 11# (e), 12# (f) screening conditions.

[0055] Figure 7: Immunofluorescence staining of 33 screening media. In Figure 7, representative immunofluorescence staining images of FOXA2, PDGFRA, GATA4, NANOG, SOX17 and BST2 at day 20 of reprogramming, 13# (a), 14# (b), 15# (c), 16# (d), 17# (e), 18# (f) screening conditions.

[0056] Figure 8: Immunofluorescence staining of 33 screening media. In Figure 8, representative immunofluorescence staining images of FOXA2, PDGFRA, GATA4, NANOG, SOX17 and BST2 at day 20 of reprogramming, 19# (a), 20# (b), 21# (c), 22# (d), 23# (e), 24# (f) screening conditions.

[0057] Figure 9: Immunofluorescence staining of 33 screening media. In Figure 9, representative immunofluorescence staining images of FOXA2, PDGFRA, GATA4, NANOG, SOX17 and BST2 at day 20 of reprogramming, 25# (a), 26# (b), 27# (c), 28# (d), 28# (e), 30# (f) screening conditions.

[0058] Figure 10: Immunofluorescence staining of 33 screening media. In Figure 10, representative immunofluorescence staining images of FOXA2, PDGFRA, GATA4, NANOG, SOX17 and BST2 at day 20 of reprogramming, 31# (a), 32# (b), 33#

[0059] (c) screening conditions. FOXA2, PDGFRA, GATA4, NANOG, SOX17 and BST2.

[0060] Figure 11: Brightfield and immunofluorescence staining of 5# and 26# media for donor 1. In Figure 11, (a) Brightfield image of donor 1, 5# hypoblast stem cell selection media passage 15 cells; (b) Representative immunofluorescence staining images of hypoblast markers (PDGFRA, SOX17, FOXA2, GATA4, and E-CAD), ExEM marker (VIM) for donor 1, 5# condition at passage 15; (c) Representative immunofluorescence staining images of hypoblast marker (GATA6), pluripotency marker (NANOG), ExEM (BST2), and proliferation marker (Ki67) for donor 1, 5# condition at passage 15; (d) Brightfield image of donor 1, 26# hypoblast stem cell selection media passage 15 cells; (e) Representative immunofluorescence staining images of hypoblast markers (PDGFRA, SOX17, FOXA2, GATA4, and E-CAD), ExEM marker (VIM) for donor 1, 26# condition at passage 15; (f) Representative immunofluorescence staining images of hypoblast marker (GATA6), pluripotency marker (NANOG), ExEM (BST2), and proliferation marker (Ki67) for donor 1, 26# condition at passage 15.

[0061] Figure 12: Brightfield and immunofluorescence staining of 5# and 26# media for donor 2. In Figure 12, (a) Brightfield image of donor 2, 5# hypoblast stem cell selection media passage 15 cells; (b) Representative immunofluorescence staining images of hypoblast markers (PDGFRA, SOX17, FOXA2, GATA4, and E-CAD), ExEM marker (VIM) for donor 2, 5# condition at passage 15; (c) Representative immunofluorescence staining images of hypoblast marker (GATA6), pluripotency marker (NANOG), ExEM (BST2), and proliferation marker (Ki67) for donor 2, 5# condition at passage 15; (d) Brightfield image of donor 2, 26# hypoblast stem cell selection media passage 15 cells; (e) Representative immunofluorescence staining images of hypoblast markers (PDGFRA, SOX17, FOXA2, GATA4, and E-CAD), ExEM marker (VIM) for donor 2, 26# condition at passage 15; (f) Representative immunofluorescence staining images of hypoblast marker (GATA6), pluripotency marker (NANOG), ExEM (BST2), and proliferation marker (Ki67) for donor 2, 26# condition at passage 15.

[0062] Figure 13: Single-factor removal experiment characterization of 26# culture medium. In Figure 13, (a) representative phase-contrast microscopy images showing the case of removal of each medium component from the 26# condition, respectively (n = 3 independent sets of experiments, scale bar: 100 pm); (b) representative immunofluorescence staining images for the 26# condition, staining for hypoblast markers (PDGFRA, FOXA2, GATA4 and SOX17), pluripotency marker (NANOG), ExEM marker (BST2) (n = 3 independent sets of experiments, scale bar: 100 pm); (c) representative immunofluorescence staining images for the 26# condition after removal of recombinant human PDGFAA, staining for hypoblast markers (PDGFRA, FOXA2, GATA4 and SOX17), pluripotency marker (NANOG), ExEM marker (BST2) (n = 3 independent sets of experiments, scale bar: 100 pm); (d) representative immunofluorescence staining images for the 26# condition after removal of recombinant human LIF, staining for hypoblast markers (PDGFRA, FOXA2, GATA4 and SOX17), pluripotency marker (NANOG), ExEM marker (BST2) (n = 3 independent sets of experiments, scale bar: 100 pm).

[0063] Figure 14: Single factor removal experiment characterization of 26# culture medium. In Figure 14, (a) Representative immunofluorescence staining images of 26# conditions after removal of recombinant human FGF4, staining for hypoblast markers (PDGFRA, FOXA2, GATA4 and SOX17), pluripotency marker (NANOG), ExEM marker (BST2) (n=3 independent experiments in groups, scale bar: 100 pm); (b) Representative immunofluorescence staining images of 26# conditions after removal of CHIR, staining for hypoblast markers (PDGFRA, FOXA2, GATA4 and SOX17), pluripotency marker (NANOG), ExEM marker (BST2) (n=3 independent experiments in groups, scale bar: 100 pm); (c) Representative immunofluorescence staining images of 26# conditions after removal of A83-01, staining for hypoblast markers (PDGFRA, FOXA2, GATA4 and SOX17), pluripotency marker (NANOG), ExEM marker (BST2) (n=3 independent experiments in groups, scale bar: 100 pm); (d) Representative immunofluorescence staining images of 26# conditions after removal of recombinant human BMP4, staining for hypoblast markers (PDGFRA, FOXA2, GATA4 and SOX17), pluripotency marker (NANOG), ExEM marker (BST2) (n=3 independent experiments in groups, scale bar: 100 pm); (e) Hypo score of 26# conditions and after removal of each medium component, based on the relative protein expression levels of FOXA2, PDGFRA, GATA4, SOX17, NANOG and BST2 (mean ± standard error, n=3 independent experiments in groups).

[0064] Figure 15: Karyotype and Sendai virus detection of iHypoblast SCs. In Figure 15, (a) Karyotype detection of iHypoblast SCs (somatic cells derived from two donors, Donor 1 and Donor 2); (b) Relative expression of Sendai virus transgenes and pluripotency markers in iHypoblast SCs and Sendai virus control by RT-PCR detection.

[0065] Figure 16: Single cell sequencing of iHypoblast SCs. In Figure 16, (a) Cartoon representation of the screening of hypoblast-like stem cells in vitro culture and single cell sequencing during reprogramming process; (b) UMAP analysis of iHypoblast SCs with integrated datasets of human pre-implantation blastocysts and in vitro cultured embryos (colors correspond to cell types annotated according to each dataset); (c) UMAP analysis of iHypoblast SCs with integrated datasets of human pre-implantation blastocysts and in vitro cultured embryos (colors correspond to developmental time annotation of embryos in each dataset).

[0066] Figure 17: iHypoblast SCs single cell sequencing. In Figure 17, (a) integrated UMAPs showing reprogramming intermediate cells at day 0, day 4 and day 8, as well as hypoblast-like cells at day 20 (condition 26) and iHypoblast SCs (Fm, fibroblast medium); (b) cell trajectory reconstruction during reprogramming using CytoTRACE and Monocle3; (c) SCENIC analysis showing top 50 differentially active regulators activities in DO, Top 50 differentially active regulators activities in iPS cells, primed iPS cells.

[0067] Figure 18: iHypoblast SCs single cell sequencing UMAP marker genes expression. In Figure 18, (a) expression of basal lamina markers (FN1, COL4A1, LAMA1, LAMB) in iHypoblast SCs UMAP; (b) expression of ectoderm markers (DCN, FOXF1, HAND2, BST2) in iHypoblast SCs UMAP; (c) expression of hypoblast markers (PDGFRA, BMP2, GATA6, FOXA2, HNF4A, GATA4) in iHypoblast SCs UMAP; (d) expression of pluripotency markers (NANOG, OCT3 / 4) in iHypoblast SCs UMAP.

[0068] Figure 19: iHypoblast SCs characterization analysis. In Figure 19, (a) UMAP analysis of iHypoblast SCs integrated data with in vitro ExEM-like cells; (b) principal component analysis (PCA) of RNA sequencing (bulk RNA-seq) and single cell RNA sequencing (scRNA-seq) data in this study and published studies; (c) correlation analysis of RNA sequencing hypoblast and single cell RNA sequencing data in this study and published studies and human embryo data.

[0069] Figure 20: BMP4-induced differentiation. In Figure 20, (a) representative immunofluorescence staining images of COL6A1, FOXA1 and GATA6 in iHypoblast SCs at day 5 of BMP4-induced differentiation; (b) RT-qPCR analysis of visceral endoderm markers (AFP and HNF4A), hypoblast markers (GATA6 and PDGFRA) and parietal endoderm markers (PLAT and SPARC) in iHypoblast SCs at day 5 of BMP4-induced differentiation; (c) determination of AFP and APOA1 concentrations in iHypoblast SCs culture supernatants at day 5 of BMP4-induced differentiation by ELISA.

[0070] Figure 21: FBS-induced differentiation. In Figure 21, (a) Representative immunofluorescence staining images of COL6A1, FOXA1 and GATA6 in FBS-induced differentiated iHypoblast SCs at day 5; (b) RT-qPCR analysis of visceral endoderm markers (AFP and HNF4A), hypoblast markers (GATA6 and PDGFRA) and parietal endoderm markers (PLAT and SPARC) in FBS-induced differentiated iHypoblast SCs at day 5; (c) Determination of AFP and APOA1 concentrations in FBS-induced differentiated iHypoblast SCs culture supernatants at day 5 by ELISA.

[0071] Figure 22: Basement membrane markers in iHypoblast SCs. In Figure 22, representative immunofluorescence staining images of basement membrane markers (FN1, COL4A1 and Laminin), hypoblast markers (GATA6, GATA4 and SOX17), pluripotency marker (SOX2) and ecto-endoderm markers (FOXF1 and VIM) in iHypoblast SCs (n = 3 independent groups of experiments, scale bar: 100 pm).

[0072] Figure 23: Chimeric experiments. In Figure 23, (a) Cartoon chimeric experiment schematic: td-Tomato labeled iHypoblasts microinjected into mouse E3.5 blastocysts (scale bar, 25 pm); (b) Td-Tomato labeled iHypoblasts microinjected into mouse E3.5 blastocysts, cultured in vitro for about 24 hours, and chimeric efficiency statistics collected at E4.5 (arrows indicate successful integration of human td-Tomato+ iHypoblast SCs into mouse primitive endoderm, n = 48 mouse embryos, scale bar: 100 pm); (c) Td-Tomato labeled iHypoblasts microinjected into mouse E3.5 blastocysts, implanted into pseudopregnant foster mothers and developed in vivo to E6.5, E6.5 chimeras collected (arrows indicate successful integration of human td-Tomato+ iHypoblast SCs into mouse visceral endoderm, n = 25 mouse embryos, scale bar: 100 pm).

[0073] Figure 24: Chimeric experiment immunofluorescence staining. In Figure 24, (a) Immunofluorescence staining images of E4.5 human-mouse chimera embryos for detection of human antigen, td-Tomato and SOX17 (arrows indicate human td-Tomato+ iHypoblast SCs successfully integrated into mouse primitive endoderm, n = 3 independent experiments, scale bar: 25 pm); (b) Immunofluorescence staining images of E6.5 human-mouse chimera embryos for detection of human antigen, td-Tomato and SOX17 (arrows indicate human td-Tomato+ iHypoblast SCs successfully integrated into mouse visceral endoderm, n = 3 independent experiments, scale bar: 65 pm).

[0074] Figure 25: 33 screening media. In Figure 25, GO enrichment analysis was performed on single-cell transcriptome sequencing (scRNA-seq) data obtained from day 20 of 5# condition (7F) and 26# condition (inducible hypoblast-like stem cells, iHypoblast SCs).

[0075] Figure 26: Cellular plasticity of iHypoblast SCs. In Figure 26, (a) Heatmap of WNT signaling pathway related genes was plotted based on human embryonic hypoblast single-cell transcriptome sequencing (scRNA-seq) data in Radley literature dataset, reprogrammed day 20 of 5# and 26# conditions (corresponding to 7F and iHypoblast SCs, respectively), and reprogrammed passage 15 cells of 5# and 26# conditions (corresponding to 7F and iHypoblast SCs, respectively); (b) Representative images of immunofluorescence staining of hypoblast marker (GATA6), pluripotency marker (OCT3 / 4) and proliferation marker (Ki67) of cells obtained from day 0 H9 PXGL culture system under 5# and 26# conditions, respectively. n = 3 independent experiments, scale bar: 100 pm; (c) Representative images of immunofluorescence staining of pluripotency markers (OCT3 / 4, NANOG) and related markers (KLF17) of iHypoblast-like stem cells (iHypoblast SCs) under 5# and 26# conditions after 5 days of induction in primitive endoderm (PE) state. n = 3 independent experiments, scale bar: 100 pm; (d) Representative images of immunofluorescence staining of trophoblast stem cell (TSC) related markers (GATA3, CK7 and NR2F2) of iHypoblast-like stem cells (iHypoblast SCs) under 5# and 26# conditions after 5 days of induction in trophoblast endoderm (TE) state. n = 3 independent experiments, scale bar: 100 pm.

[0076] ​​​Figure 27: iHypoblast SCs characterization analysis. In Figure 27, (a) Heatmap of OCT3 / 4 downstream target genes; (b) H3K27ac, H3K4mel and H3K4me3 modification profiles of OCT3 / 4 upstream regions in iHypoblast stem cells, and compared with primed state human pluripotent stem cells (hPSCs); (c) Chromatin open peak distribution of representative hypoblast genes regions in iHypoblast stem cells, hPSCs, primed hPSCs and human embryos using IGV; (d) Chromatin open peak distribution of representative hypoblast genes in iHypoblast stem cells, hPSCs, primed hPSCs and human embryos using IGV; (e) Gene overlap of chromatin open peak of different enhancer regions with nearest transcription start site (TSS) annotation analysis of differentially expressed genes (DEGs) in human pre-implantation embryos in vivo; (f) Clustered heatmap of DNA methylation levels of hypoblast lineage specific genes.

[0077] Figure 28: Immunofluorescence staining for chimerism experiments. In Figure 28, (a) immunofluorescence staining images of E4.5 stage human-mouse chimeric embryos (empty injection group) to detect the expression of human antigen, td-Tomato, and SOX17. Experimental replicates n=3, scale bar: 25μm; (b) immunofluorescence staining images of E4.5 stage human-mouse chimeric embryos (iPSCs group) to detect the expression of human antigen, td-Tomato, and SOX17. Experimental replicates n=3, scale bar: 2μm; (c) Immunofluorescence staining images of E4.5 human-mouse chimeric embryos (iHypoblast SCs group), used to detect human antigen, td-Tomato, and SOX17 (arrows indicate successful integration of human td-Tomato + iHypoblast SCs into the mouse primitive endoderm, n=3 independent experiments, scale bar: 25μm); (d) Immunofluorescence staining images of E6.5 human-mouse chimeric embryos (empty injection group), used to detect the expression of human antigen, td-Tomato, and SOX17. Experimental replicates n=3, scale bar: 50μm; (e) Immunofluorescence staining images of E6.5 human-mouse chimeric embryos (iPSCs group), used to detect the expression of human antigen, td-Tomato, and SOX17. Experimental replicates n=3, scale bar: 25μm; (f) Immunofluorescence staining images of E6.5 human-mouse chimeric embryos (iHypoblast SCs group) used to detect human antigen, td-Tomato and SOX17 (arrows indicate that human td-Tomato+iHypoblast SCs were successfully integrated into the mouse organ endoderm, n=3 independent experiments, scale bar: 65μm).

[0078] Figure 29: Immunofluorescence staining for blastocyst experiments. In Figure 29, (a) schematically illustrates the process... Cell aggregates constructed from human embryonic stem cells (hESCs) and iHypoblast SCs. Arrows in the figure indicate successful integration of human td-Tomato-positive iHypoblast SCs into blastoid structures. Scale bar: 50 μm; (b) Quantitative results of blastoid cavity formation efficiency on day 6. Data are expressed as mean ± standard error (sem), n = 6 independent experiments; (c) From Immunofluorescence staining image of day 6 cell aggregates constructed from human embryonic stem cells, targeting OCT3 / 4, td-Tomato, and GATA6; (d) From Figure 6: Immunofluorescence staining images of day 6-like cystic embryos co-constructed with human embryonic stem cells and iHypoblast SCs, targeting OCT3 / 4, td-Tomato and GATA6. n = 3 independent experiments. Scale bar: 50 μm; (e) Representative images of day 10 cell aggregates co-constructed with human embryonic stem cells and iHypoblast SCs, targeting OCT3 / 4, td-Tomato, T and GATA6. n = 3 independent experiments. Scale bar: 50 μm; (f) Immunofluorescence staining images of day 10-like cystic embryos co-constructed with human embryonic stem cells and iHypoblast stem cells, targeting OCT3 / 4, td-Tomato, T and GATA6. n = 3 independent experiments. Scale bar: 50 μm. Figure 6: Immunofluorescence staining images of day 6-like cystic embryos co-constructed with human embryonic stem cells and iHypoblast SCs, targeting OCT3 / 4, td-Tomato and GATA6. n = 3 independent experiments. Scale bar: 50 μm; (e) Representative images of day 10 cell aggregates co-constructed with human embryonic stem cells and iHypoblast SCs, targeting OCT3 / 4, td-Tomato, T and GATA6. n = 3 independent experiments. Scale bar: 50 μm; (f) Immunofluorescence staining images of day 10-like cystic embryos co-constructed with human embryonic stem cells and iHypoblast stem cells, targeting OCT3 / 4, td-Tomato, T and GATA6. n = 3 independent experiments. Scale bar: 50 μm. Figure 6: Immunofluorescence staining images of day 6-like cystic embryos co-constructed with human embryonic stem cells and iHypoblast SCs, targeting OCT3 / 4, td-Tomato and GATA6. n = 3 independent experiments. Scale bar: 50 μm; (e) Representative images of day 10 cell aggregates co-constructed with human embryonic stem cells and iHypoblast SCs, targeting OCT3 / 4, td-Tomato, T and GATA6. n = 3 independent experiments. Scale bar: 50 μm; (f) Immunofluorescence staining images of day 10-like cystic embryos co-constructed with human embryonic stem cells and iHypoblast stem cells, targeting OCT3 / 4, td-Tomato, T and GATA6. n = 3 independent experiments. Scale bar: 50 μm.

[0079] Figure 30: Brightfield and immunofluorescence staining of mesenchymal stem cells (MSCs) and peripheral blood mononuclear cells (PBMCs) reprogrammed for 20 days in 26# medium. In Figure 30, (a) Representative phase-contrast microscopic images of MSCs and PBMCs reprogrammed for 20 days in 26# medium. n = 3 independent experiments. Scale bar: 100 μm (b) Immunofluorescence images of MSCs and PBMCs reprogrammed for 20 days in 26# medium, stained for hypoblast markers (PDGFRA, FOXA2, GATA4 and SOX17), pluripotency marker (NANOG), and ExEM marker (BST2) (n = 3 independent experiments, scale bar: 100 μm).

[0080] Figure 31 : 26# medium induces differentiation in embryonic stem cells (H9). In Figure 31, (a) Representative images of immunofluorescence staining of human embryonic stem cells (H9) in PXGL state at passage 0 (P0) induction to condition #26, detecting markers including primitive endoderm-related markers (PDGFRA, SOX17, FOXA2, GATA4 and E-CAD), pluripotency marker (NANOG), and extraembryonic mesoderm (ExEM) markers (VIM and BST2). n = 3 independent experiments. Scale bar: 100 μm; (b) Representative images of immunofluorescence staining of human embryonic stem cells (H9) in PXGL state at passage 3 (P3) induction to condition #26, detecting markers including primitive endoderm-related markers (PDGFRA, GATA6, SOX17, FOXA2, GATA4 and E-CAD), pluripotency markers (NANOG and OCT3 / 4), extraembryonic mesoderm (ExEM) markers (VIM and BST2), and proliferation-related marker (Ki67). n = 3 independent experiments. Scale bar: 100 μm. DETAILED DESCRIPTION

[0081] The following examples are provided to better enable those skilled in the art to further understand the application, and are not intended to limit the content and scope of the application. Any person skilled in the art who learns the content of the present application or combines the present application with other prior art features will be able to obtain any product that is the same or similar to the present application, and such product falls within the scope of the present application.

[0082] The following examples do not specify the specific experimental procedures or conditions, which can be performed according to the conventional experimental procedures described in the literature in the art. The reagents or instruments used are not specified by the manufacturer, and are conventional reagents that can be obtained commercially.

[0083] The materials and reagents involved in the following examples are shown in Table 1.

[0084] Table 1 Experimental materials and reagents

[0085] Example 1: A hypoblast stem cell culture medium

[0086] The present embodiment provides a hypoblast stem cell culture medium, which is an N2B27 basic medium containing 10 ng / mL human PDGFAA, 10 ng / mL human LIF, 25 ng / mL human FGF4, 1 μg / mL heparin (promoting FGF4 binding to its receptor, optimizing FGF4 biological function), 3 μM CHIR99021, 1 μM A83-01 and 10 ng / mL human BMP4; the N2B27 basic medium is a mixed medium containing 1% (v / v) N2, 2% (v / v) B27, 1% (v / v) GlutaMAX, 1% (v / v) non-essential amino acids, 0.1 mM 2-mercaptoethanol and 1% (v / v) penicillin-streptomycin; the mixed medium is composed of Neurobasal medium and DMEM / F12 at a volume ratio of 1:1.

[0087] Example 2: A method for constructing hypoblast stem cells by somatic cell reprogramming

[0088] The present embodiment provides a method for constructing hypoblast stem cells by somatic cell reprogramming, which is as follows (see Fig. 1a for specific operation process):

[0089] Step one: reprogram primary human adult skin fibroblasts (including donor 1 and donor 2) using CytoTune-iPS 2.0 Sendai Reprogramming Kit according to the instructions. The reprogramming process is as follows: human fibroblasts are seeded in mouse embryonic fibroblast medium at a cell density of 5 x 105cells / mL and transfected using Sendai virus at a multiplicity of infection (MOI) of 5 (KOS), 5 (c-Myc), and 6 (KLF4). 4

[0090] Step two: on day 7 of transfection, dissociate the transfected human fibroblasts in fibroblast medium using TryPLE Select for 5 minutes at room temperature (25) (TryPLE Select dosage 0.5 mL / well) and reseed at a seeding amount of 6 x 105cells / well in a cell culture plate with feeder layer cells prepared 1 day in advance (mouse embryonic fibroblasts are used as feeder layer cells, and the plating method of the feeder layer cells is described in the literature “Liu, X. et al. Reprogramming roadmap reveals route to human induced trophoblast stem cells. Nature 586, 101-107 (2020).”) (in addition to the feeder layer cells, 1 mL / well of mouse embryonic fibroblast medium is added to the cell culture plate), and culture in a 37°C cell culture incubator with 5% (v / v) CO2, 5% (v / v) O2. 4

[0091] Step three: after 24 hours of culture, replace the medium in the cell culture plate with the induced hypoblast stem cell medium of Example 1, and continue to culture in a 37°C cell culture incubator with 5% (v / v) CO2, 5% (v / v) O2.

[0092] Step four: after 20 days of culture, enrich and screen for PDGFRA-high-expressing hypoblast-like stem cells (the proportion of cells positive for PDGFRA in the reprogramming intermediate product is shown in Figure 1b) according to the expression of PDGFRA by FACS (flow cytometry sorting technology).

[0093] Step five: select PDGFRA-positive hypoblast-like cells and reseed at a seeding amount of 6 x 105cells / well in a cell culture plate with feeder layer cells prepared 1 day in advance (in addition to the feeder layer cells, 1 mL / well of the induced hypoblast stem cell medium of Example 1 supplemented with 10 μM ROCK-I / II inhibitor Y27632 is added to the cell culture plate), and culture in a 37°C cell culture incubator with 5% (v / v) CO2, 5% (v / v) O2. 4 ​​​

[0094] Step six: After 24 hours of culture, replace the medium in the cell culture plate with the induced hypoblast stem cell medium of Example 1 without additional supplement of ROCK-I / II inhibitor Y27632, and continue to culture in a 37°C cell culture incubator with 5% (v / v) CO2, 5% (v / v) O2.

[0095] Step seven: After 5 days of culture, pick the hypoblast-like colonies in the cell culture plate and transfer them to a cell culture plate with feeder cells prepared 1 day in advance (in addition to the feeder cells, 1 mL / well of the induced hypoblast stem cell medium of Example 1 supplemented with 10 mM ROCK-I / II inhibitor Y27632 is added) at a seeding amount of 6 x 105cells / well, and culture in a 37°C cell culture incubator with 5% (v / v) CO2, 20% (v / v) O2. Replace the medium every day during the culture. 4

[0096] Step eight: When the hypoblast-like cells in the cell culture plate reach 80-90% confluence, dissociate the hypoblast-like cells in the cell culture plate with Accutase at room temperature (25°C) for 5 minutes (the amount of Accutase used is 0.5 mL / well) and transfer them to a cell culture plate with feeder cells prepared 1 day in advance (in addition to the feeder cells, 1 mL / well of the induced hypoblast stem cell medium of Example 1 supplemented with 10 mM ROCK-I / II inhibitor Y27632 is added) at a seeding amount of 6 x 105cells / well, and culture in a 37°C cell culture incubator with 5% (v / v) CO2, 20% (v / v) O2. After 24 hours of culture, the construction of induced hypoblast stem cells (iHypoblast SCs) is successful. 4

[0097] Step nine: After the construction of induced hypoblast stem cells (iHypoblast SCs) is successful, subculture the induced hypoblast stem cells (iHypoblast SCs) at a passage ratio of 1:5 every 3 days, and the subculture process is the same as step eight. During the subculture process, observe the morphological changes of the reprogrammed cells at different passages under a phase contrast microscope, and the observation results are shown in Fig. 1c.

[0098] wherein the mouse embryonic fibroblast medium (MEF medium) is a DMEM medium containing 10% (v / v) fetal bovine serum (FBS), 1% (v / v) non-essential amino acids, 1% (v / v) GlutaMAX, 1% (v / v) penicillin-streptomycin, 0.1 mM 2-mercaptoethanol, and 1% (v / v) sodium pyruvate.

[0099] ​​Experimental Example 1: Exploration of the construction conditions of induced hypoblast stem cells (iHypoblast SCs) and verification of the performance of induced hypoblast stem cells (iHypoblast SCs)

[0100] The present experimental example provides an experiment of exploring the construction conditions of induced hypoblast stem cells (iHypoblast SCs) and verifying the performance of induced hypoblast stem cells (iHypoblast SCs), and the experimental process is as follows:

[0101] 1. Reprogramming fibroblasts into induced hypoblast stem cells (iHypoblast SCs)

[0102] Based on the previous work, in the process of reprogramming human fibroblasts into induced pluripotent stem cells (iPS cells) (see the literature “Liu, X. et al. Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming. Nat. Methods 14, 1055-1062 (2017).” and “Liu, X. et al. Reprogramming roadmap reveals route to human induced trophoblast stem cells. Nature 586, 101-107 (2020).”), the present study noticed that the expression of related hypoblast genes was significantly up-regulated on the 8th day of reprogramming (Figure 2a). This result was further verified by RNA-seq and ATAC-seq data (Figure 2b, Figure 3). These results showed that the 8th day of reprogramming intermediate state cells provided a reasonable window for capturing human hypoblast cell fate in vitro.

[0103] In order to develop culture conditions for capturing and maintaining these hypoblast-like stem cells, the present study investigated the signal requirements of hypoblast cells in human embryos. The present study found that NODAL, BMP, WNT, FGF and JAK / STAT signal receptors were highly expressed in hypoblast cells, indicating that they played a key role in the determination and maintenance of hypoblast cell fate (Figure 2c). Based on these findings, the present study designed a screening experiment to test 33 combinations of activators or inhibitors targeting NODAL, BMP and WNT signaling pathways (chemical compounds or cytokines) (Figure 4a).

[0104] To evaluate the indicators of successful construction of hypoblast-like stem cells, the present study referred to a recent pseudo-time analysis of blastocyst development, which detailed the sequential expression of hypoblast markers—PDGFRA, SOX17, FOXA2, and GATA4 (see Corujo-Simon, E., Radley, A. H. & Nichols, J. Evidence implicating sequential commitment of the founder lineages in the human blastocyst by order of hypoblast gene activation. Development 150, (2023).). Notably, once these markers were expressed, the expression of NANOG in hypoblast cells was undetectable. Furthermore, while there was partial overlap in gene expression between extraembryonic mesoderm (ExEM) cells and hypoblast cells, BST2 was identified as a specific surface marker for ExEM cells (see Pham, T. X. A. et al. Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells. Cell Stem Cell 29, 1346-1365.e10 (2022).). Based on the above findings, the present study selected PDGFRA, SOX17, FOXA2, and GATA4 as positive markers, and NANOG and BST2 as negative markers. This combination enabled the present study to calculate a “Hypo Score,” which evaluated the proportion of hypoblast-like cells through the relative protein expression levels of the six markers (Fig. 4b, Figs. 5-10). Among the conditions tested, the present study found that conditions 5# and 26# performed best in generating hypoblast-like cells (Fig. 4b).

[0105] To determine whether conditions 5# and 26# can support long-term stabilization of hypoblast-like stem cells, the present study performed serial passaging of cells maintained in these media. Under condition 5#, cells gradually lost epithelial morphology at passage 15, accompanied by a significant decrease in proliferative potential (Figures 11a-11c, 12a-12c). Immunofluorescence staining showed loss of hypoblast markers including PDGFRA, GATA6, SOX17, FOXA2, and GATA4 expression (Figures 11a-11c, 12a-12c). In contrast, condition 26# was able to stably maintain hypoblast stem cell-like state up to passage 15, manifested as Ki67-positive, flat epithelial colonies, and expression of all hypoblast markers (Figures 11b-11d, 12b-12d). Notably, cells cultured long-term under condition 5# eventually developed into putative ExEM-like cells, which can be evidenced by upregulated expression of BST2 and VIM (Figures 11a-11c, 12a-12c). To determine the key components in condition 26#, the present study performed single component removal experiments, and found that A83-01 and CHIR99021 are the key components to maintain hypoblast stem cell properties (Figures 13, 14). These findings indicate that condition 26# is the optimal culture formula to maintain hypoblast cells in self-renewal, stem cell-like state. As these stem cells are generated through somatic cell reprogramming, the present study named them as induced hypoblast stem cells (iHypoblast SCs), and the 26# medium was named as induced hypoblast stem cell medium. Importantly, iHypoblast SCs can be reproducibly generated from fibroblasts of multiple biologically independent donors, maintaining karyotype stability and free of reprogramming transgenes (Figures 15a-15b). In summary, the present study developed a novel culture condition that can generate and long-term maintain iHypoblast SCs through fibroblast reprogramming. This marks the first successful establishment of scalable human hypoblast stem cells.

[0106] Table 2. Candidate conditions for hypoblast culture

[0107] 2. Single-cell transcriptomic study of induced hypoblast stem cells (iHypoblast SCs)

[0108] To gain insight into the generation mechanism of iHypoblast SCs, Chromium 10X single-cell RNA sequencing (scRNA-seq) analysis was performed at different time points during their generation (Figure 16a).To assess whether iHypoblast SCs recapitulate key features of the human embryonic hypoblast lineage, the scRNA-seq data of this study were integrated with published human embryonic datasets covering stages from pre-implantation (see references ‘Petropoulos, S. et al. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos. Cell 165, 1012-1026 (2016).’, ‘Guo, G. et al. Human naive epiblast cells possess unrestricted lineage potential. Cell Stem Cell 28, 1040-1056.e6 (2021).’, ‘Meistermann, D. et al. Integrated pseudotime analysis of human pre-implantation embryo single-cell transcriptomes reveals the dynamics of lineage specification. Cell Stem Cell 28, 1625-1640.e6 (2021).’ and ‘Yu, L. et al. Large-scale production of human blastoids amenable to modeling blastocyst development and maternal-fetal cross talk. Cell Stem Cell 30, 1246-1261.e9 (2023).’) to post-implantation (E8-E14) (see references ‘Xiang, L. et al. A developmental landscape of 3D-cultured human pre-gastrulation embryos. Nature 577, 537-542 (2020).’ and ‘Ai, Z. et al. Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids. Cell Res. 33, 661-678 (2023).’) (Fig. 16b).By unsupervised uniform manifold approximation and projection (UMAP) clustering analysis based on previously defined lineage-specific markers, the present study identified multiple cell types including 8-cell stage, morula, pre-lineage, inner cell mass (ICM), trophoblast ectoderm (TE), ectoderm (Epi), hypoblast (Hypo), primitive streak (PS), ectoderm mesoderm (ExEM), and trophoblast (TrB) (Fig. 2a). Remarkably, iHypoblast SCs tightly clustered with in vivo human hypoblast cells, indicating that their transcriptional signatures were highly consistent with in vivo hypoblast cells (Fig. 16b). Further analysis showed that iHypoblast SCs highly expressed hypoblast-specific genes PDGFRA, BMP2, GATA6, FOXA2, HNF4A, and GATA4, but not the pluripotency marker NANOG, and ExEM markers DCN, FOXF1, HAND2, and BST2 (Fig. 18b-d). Likewise, integrated analysis with in vitro ExEM-like cells further confirmed that iHypoblast SCs were different from ExEM clusters of human embryos (Fig. 19a).

[0109] To determine the developmental stage of iHypoblast SCs, the present study performed subpopulation analysis and dimensionality reduction analysis according to previous annotations of each dataset, thereby mapping different developmental stages onto the integrated UMAP atlas of the present study. Compared with in vivo hypoblast lineage cells, iHypoblast SCs formed a cluster close to E5-E7 human embryo hypoblast cells, indicating that they were similar to pre-implantation hypoblast (Fig. 16c). Likewise, analysis of hypoblast-like cells in previous studies (see Okubo, T. et al. Hypoblast from human pluripotent stem cells regulates epiblast development. Nature 626, 357-366 (2024); Dattani, A. et al. Naive pluripotent stem cell-based models capture FGF-dependent human hypoblast lineage specification. Cell Stem Cell 31, 1058-1071.e5 (2024); Linneberg-Agerholm, M. et al. human pluripotent stem cells respond to Wnt, Nodal and LIF signalling to produce expandableextra-embryonic endoderm. Development 146, (2019).”, “Mackinlay, K.M. et al. An in vitro stem cell model of human epiblast and yolk sac interaction. Elife 10, (2021).”, “Oldak, B. et al. Complete human day 14 post-implantation embryo models from naive ES cells. Nature 622, 562-573 (2023).”, “Wei, Y. et al. Dissecting embryonic and extraembryonic lineage crosstalk with stem cell co-culture. Cell 186, 5859-5875.e24 (2023).”, “Pham, T.X.A. et al. Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells. Cell Stem Cell 29, 1346-1365.e10 (2022).”, and “Chu, L.-F. et al. Single-cell RNA-seq reveals novel regulators of human embryonic stem cell differentiation to definitive endoderm. Genome Biol. 17, 173 (2016).”) and scRNA-seq data of human embryos (see “Petropoulos, S. et al. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos. Cell 165, 1012-1026 (2016).”, “Guo, G. et al. Human naive epiblast cells possess unrestricted lineage potential. Cell Stem Cell 28, 1040-1056.e6 (2021).”, “Meistermann, D.et al. Integrated pseudotime analysis of human pre-implantation embryo single-cell transcriptomes reveals the dynamics of lineage specification. Cell Stem Cell 28, 1625-1640.e6 (2021). “Yu, L. et al. Large-scale production of human blastoids amenable to modeling blastocyst development and maternal-fetal cross talk. Cell Stem Cell 30, 1246-1261.e9 (2023).” “Xiang, L. et al. A developmental landscape of 3D-cultured human pre-gastrulation embryos. Nature 577, 537-542 (2020).” and “Ai, Z. et al. Dissecting peri-implantation development using cultured human embryos and embryo-like assembloids. Cell Res. 33, 661-678 (2023).”) principal component analysis (PCA) showed that iHypoblast SCs, The gene expression profiles of hPSC-derived hypoblast-like cells (nHyCs) and PXGL-RACLs were similar to E5 human embryonic hypoblast cells (Figure 19b). Correlation analysis further confirmed the high consistency between iHypoblast SCs and human embryonic hypoblast cells (Figure 19c). Moreover, pseudo-time trajectory analysis using Monocle3 and CytoTrace algorithms further supported the concept that hypoblast cell fate could be directly captured from the 8th day reprogrammed intermediate cells (Figures 17a-b). Given that transcription factors (TFs) control cell fate by binding to cis-regulatory regions, thereby forming gene regulatory networks (GRNs), the study reconstructed the GRNs in iHypoblast SCs using single-cell regulatory network inference and clustering (SCENIC) analysis. The expected regulators GATA6, HNF4A, HNF1B, FOXA2, and GATA4 were found to be active in iHypoblast SCs, indicating their regulatory activity for hypoblast identity (Figure 17c). In summary, the single-cell transcriptome analysis of the present study showed that iHypoblast SCs faithfully recapitulated the molecular features of pre-implantation human embryonic hypoblast cells.

[0110] 3. Functional characterization of induced hypoblast stem cells (iHypoblast SCs)

[0111] Next, the present study elucidated the cellular characteristics and developmental potential of iHypoblast SCs through various in vitro and in vivo functional experiments. One of the features of hypoblast is its ability to generate basement membrane components between the ectoderm (Epi) and hypoblast (Hypo) to facilitate early embryonic development. Similar to human embryonic hypoblast cells, scRNA-seq analysis showed that iHypoblast SCs strongly expressed basement membrane factors such as FN1, COL4A1, LAMA1, and LAMB1 (Figure 18a). Further immunostaining analysis also confirmed that iHypoblast SCs generated basement membrane components, including FN1, COL4A1, and Laminin (Figure 22).

[0112] Previous studies have shown that human Naive ectoderm endoderm (nEnd), blastoid nEnd, and ectoderm endoderm (XENs) added with BMP4 or FBS can be transformed into visceral endoderm / yolk sac endoderm (VE / YE)-like cells and ExEM-like cells (see literature “Linneberg-Agerholm, M. et al. human pluripotent stem cells respond to Wnt, Nodal and LIF signalling to produce expandable extra-embryonic endoderm. Development 146, (2019).” and “Yu, L. et al. Blastocyst-like structures generated from human pluripotent stem cells. Nature 591, 620-626 (2021).”) Therefore, we performed BMP4 or FBS-induced differentiation experiments on iHypoblast SCs (FOXA1+GATA6+COL6A1-), and found that both differentiation cultures generated VE / YE-like cells (FOXA1+GATA6-COL6A1-) and ExEM-like cells (FOXA1-GATA6+COL6A1+) in vitro (Fig. 20a, Fig. 21a). Further RT-qPCR analysis confirmed the upregulation of VE markers AFP and HNF4A, downregulation of hypoblast markers GATA6 and PDGFRA, and downregulation of parietal layer markers PLAT and SPARC (Fig. 20b, Fig. 21b). In addition, AFP and APOA1 expression was elevated under both hypoblast differentiation methods, which are proteins generated and secreted by hypoblast and yolk sac tissue (Fig. 20c, Fig. 21c).

[0113] To further assess the in vivo developmental potential of iHypoblast SCs, human-mouse chimera experiments were performed. By labeling iHypoblast SCs with tdTomato fluorescent protein, the present study was able to track the cells injected into mouse embryos (Fig. 23, Fig. 24). Within 24 hours after injection of 10-15 td-Tomato labeled iHypoblast SCs into mouse blastocysts, the present study observed that iHypoblast SCs effectively integrated into the primitive endoderm (PrE) of the inner cell mass (ICM) and maintained SOX17 expression, indicating their contribution to extraembryonic endoderm lineage development (Fig. 23b, Fig. 24a). As a control group, the present study performed blank injection and confirmed that no td-Tomato cells were observed in the PrE layer of the blastocyst (Fig. 23a, Fig. 24a-24b). Importantly, after transferring the chimeric embryos into pseudopregnant surrogate mice, the present study observed that iHypoblast SCs contributed to the visceral endoderm (VE) layer at day 6.5 of embryonic development without integrating into the ectoderm or extraembryonic ectoderm, indicating that iHypoblast SCs were functional in the chimeras and able to develop into downstream cell types in the human-mouse chimera (Fig. 23c, Fig. 24b). Overall, iHypoblast SCs recapitulated the molecular characteristics of hypoblast cells and exhibited their lineage contribution and developmental potential in vivo, which can be classified as hypoblast stem cells (SCs) of the extraembryonic endoderm lineage in humans.

[0114] Experimental Example 2: WNT signaling pathway-related gene expression analysis of 26# medium

[0115] We performed single-cell transcriptome sequencing (scRNA-seq) on the induced hypoblast-like stem cells (iHypoblast SCs) obtained on day 20 of reprogramming under 5# and 26# screening conditions (corresponding to 7F and iHypoblast SCs, respectively), and carried out GO enrichment analysis on the data. The analysis results showed that iHypoblast SCs under 26# condition exhibited a unique enrichment pattern in the expression of multiple biology process-related genes, which were mainly involved in key biological processes such as cell differentiation, cell proliferation, cell migration, and cell-to-cell signal transduction, indicating that the cells induced under this condition were highly consistent with the biological characteristics of hypoblast stem cells in terms of gene expression, had stronger cell plasticity and developmental potential, and could provide strong support for subsequent cell fate conversion and tissue and organ development, further confirming the advantages and effectiveness of the induced hypoblast stem cell medium of the present application in precisely regulating cell fate conversion (Fig. 25).

[0116] Based on the single-cell transcriptome sequencing (scRNA-seq) data of human embryonic hypoblast from Radley literature dataset, we plotted the heatmap of WNT signaling pathway related genes of 5# and 26# screening conditions at day 20 of reprogramming (corresponding to 7F and iHypoblast SCs, respectively) and 5# and 26# screening conditions at passage 15 of reprogramming. From the heatmap, it can be clearly observed that iHypoblast SCs under 26# screening condition are highly similar to human embryonic hypoblast cells in the expression of key genes of WNT signaling pathway, and compared with 5# screening condition, the expression pattern of these genes is more stable and coordinated. This indicates that the hypoblast stem cell culture medium of the application can effectively activate and maintain the WNT signaling pathway, thereby promoting the conversion of cells to a stable hypoblast stem cell state and providing key signal support for cell self-renewal and differentiation (Figure 26a). To further verify the above results, the cells obtained by induction from H9 PXGL culture system at passage 0 were continuously subjected to immunofluorescence staining. The results show that iHypoblast SCs obtained under 26# screening condition express hypoblast marker (GATA6), pluripotency marker (OCT3 / 4) and proliferation marker (Ki67) at the same time, and the expression level is high and the distribution is uniform. This indicates that the cells induced by the culture medium of the application not only have typical hypoblast characteristics, but also retain a certain degree of pluripotency, which can provide more possibilities for further differentiation and tissue construction of cells (Figure 26b).

[0117] Experimental Example 3: Cell plasticity study

[0118] Previous studies have found that mouse OCT4-positive primitive endoderm cells exhibit lineage plasticity in vivo and in vitro (see the literature “Linneberg-Agerholm et al. The primitive endoderm supports lineage plasticity to enable regulative development. Cell 187, 1-20 (2024).”). To evaluate the plasticity of iHypoblast SCs, we placed them in the culture conditions of pluripotent stem cells and trophoectoderm stem cells. Culture conditions of pluripotent stem cells and trophoectoderm stem cells. In After 5 days of induction in the state, immunofluorescence staining was performed on iHypoblast SCs obtained under 5# and 26# screening conditions to detect pluripotency markers (OCT3 / 4, NANOG) and related markers (KLF17). The results show that cells under 26# condition express pluripotency markers and ​The expression of the related markers under the 5# condition is better than that under the 26# condition, indicating that the cells induced under the 5# condition are closer to the iHypoblast SCs have higher developmental potential and plasticity, and can better simulate the cell state in the early embryonic development process (Fig. 26c).

[0119] After 5 days of trophoblast ectoderm (TE) state induction, iHypoblast SCs obtained under the 5# and 26# screening conditions were subjected to immunofluorescence staining to detect trophoblast stem cell (TSC) related markers (GATA3, CK7 and NR2F2). The results show that the cells under the 26# condition are better than those under the 5# condition in the expression of trophoblast stem cell related markers, indicating that the cells induced under the 26# condition are closer to trophoblast stem cells, indicating that iHypoblast SCs have higher developmental potential and plasticity, and can better simulate the cell state in the early embryonic development process (Fig. 26d).

[0120] Experimental Example 4: Gene regulatory network (GRN) and chromatin openness analysis

[0121] 4.1 OCT3 / 4 downstream target gene expression analysis

[0122] By detecting the expression of OCT3 / 4 downstream target genes, we found that the expression level of OCT3 / 4 downstream target genes in iHypoblast SCs under the 26th condition was similar to that of human pre-implantation hypoblast cells, and the expression of these target genes was similar to that of primed hPSCs. hPSCs are different. This indicates that the hypoblast stem cell culture medium induced in the present application can effectively activate the key transcription factor OCT3 / 4 and its downstream gene network, thereby regulating the pluripotency state and developmental direction of the cells, laying a solid gene regulation foundation for stable induction and lineage differentiation of the cells, making the cells induced in the gene expression regulation level closer to natural hypoblast stem cells, having stronger developmental potential and plasticity (Fig. 27a).

[0123] 4.2 Chromatin modification map and openness analysis

[0124] (1) Comparison of H3K27ac, H3K4me1 and H3K4me3 modification maps

[0125] To understand the epigenetic characteristics of iHypoblast SCs, histone modifications (H3K27ac, H3K4me1, H3K4me3, and H3K27me3) and DNA methylation were analyzed using targeted cut & tag (CUT&T) and whole-genome sulfite sequencing (WGBS). H3K27ac and H3K4me1 represent active and initiating histone markers, respectively. H3K4me3 is a conserved marker for promoters and transcription initiation, while H3K27me3 is a repressive histone marker. Using defined... Enhancer annotation of type I and Primed human pluripotent stem cells (hPSCs) revealed that in iHypoblast SCs, neither the proximal nor distal enhancers of OCT3 / 4 underwent acetylation modification, suggesting the possible existence of an alternative OCT3 / 4 enhancer element in early human hypodermis, a feature similar to that of mouse OCT3 / 4-positive primitive endoderm (Fig. 27b).

[0126] (2) Display of chromatin open peak distribution

[0127] Using IGV to demonstrate iHypoblast SCs, hPSCs, primed hPSCs, and representative hypoblasts in human embryos Peak distribution of pluripotency-related genes. Analysis revealed that H3K27ac was specifically deposited on hypodermal genes (such as GATA6 and PDGFRA) in iHypoblast SCs, while primitive pluripotency-specific genes (such as NANOG and KLF17) exhibited the opposite epigenetic state. This further confirms the advantages of the hypodermal stem cell induction medium proposed in this application in regulating cell chromatin state, effectively inducing chromatin opening and providing a favorable regulatory environment for gene transcription and expression (Figure 27c-d).

[0128] 4.3 Gene overlap analysis

[0129] Subsequently, enhancer states were defined according to different histone modification signatures: active enhancers (coexistence of H3K27ac and H3K4ml), primed enhancers (H3K4ml only), and bivalent enhancers (coexistence of H3K4ml and H3K27me3) (Figure 27e). To link enhancer regulatory states to transcriptional plasticity, enhancer peaks were annotated to their nearest transcription start sites (±20 kb) and compared to differentially expressed genes (DEGs) from human embryonic Epi, Hypo, and TE single-cell RNA sequencing datasets. Genes associated with active enhancers of iHypoblast SCs overlapped with hypoblast-specific genes, while genes associated with active enhancers of primed pluripotent stem cells (hPSCs) were enriched in epiblast-specific genes (Figure 27e), further emphasizing their distinct cell lineage identities. Moreover, genes regulated by primed enhancers of iHypoblast SCs also overlapped with epiblast- and trophoblast-specific genes, supporting their ability to convert to primed pluripotent stem cell-like cells and trophoblast-like cells during development (Figure 27e). This indicates that the induced hypoblast stem cell medium of the present application can effectively activate the gene expression regulatory network closely related to early embryonic development, induce cells to express a series of genes similar to the process of pre-implantation embryonic development, and thus make the induced cells more similar to natural hypoblast stem cells in terms of gene expression regulation, and better mimic the gene expression patterns and cell behaviors during embryonic development (Figure 27e).

[0130] 4.4 Cluster analysis of DNA methylation levels

[0131] We performed a cluster heat map analysis of the DNA methylation levels of hypoblast lineage-specific genes, and the results showed that iHypoblast SCs under condition 26 exhibited a similar hypomethylation state to human embryos in the DNA methylation levels of these genes, which was significantly different from hPSCs, primed hPSCs, and D0-fibroblasts. The hypomethylation state is usually associated with the active expression of genes, which further confirms the advantages of the induced hypoblast stem cell medium of the present application in maintaining the developmental potential and pluripotency of cells, effectively inducing cells to enter a hypomethylation state similar to the early embryonic development stage, providing a more favorable gene regulation environment for the further differentiation and tissue construction of cells, and enhancing the plasticity and developmental potential of cells (Figure 27f).

[0132] Experimental Example 5, Chimeric experiments and blastocyst-like experiments

[0133] 5.1 Human-mouse chimeric experiments

[0134] (1) E4.5 chimeric experiments

[0135] By labeling iHypoblast SCs with td-Tomato fluorescent protein and injecting them into mouse blastocysts, we observed that iHypoblast SCs effectively integrated into the primitive endoderm (PrE) of the mouse inner cell mass (ICM) on day 4.5 of embryonic development, while maintaining SOX17 expression. Immunofluorescence staining results showed positive expression of human antigen, td-Tomato, and SOX17, indicating that iHypoblast SCs successfully integrated into the mouse primitive endoderm and made a significant contribution to extraembryonic endoderm lineage development. This result further confirms that the cells induced by the hypodermal stem cell culture medium of this application possess strong in vivo developmental potential and can exert their biological functions as hypodermal stem cells in xenogeneic chimeras, providing a powerful tool for studying human embryonic development mechanisms and related diseases (Figures 28a-c).

[0136] (2) E6.5 Chimera Experiment

[0137] After transferring chimeric embryos into pseudopregnant surrogate mice, we observed that on day 6.5 of embryonic development, iHypoblast SCs further contributed to the visceral endoderm (VE) layer of the mouse embryo, but did not integrate into the ectoderm or ectoectoderm-ectoderm. Immunofluorescence staining results showed positive expression of human antigen, td-Tomato, and SOX17, indicating that iHypoblast SCs are functional in the chimera and can develop into downstream cell types in the human-mouse chimera. This further demonstrates their lineage contribution and developmental potential during in vivo development, providing a more accurate model for studying cell fate transformation and tissue and organ formation during human embryonic development (Figure 28d-f).

[0138] 5.2 Blastocyst Experiment

[0139] (1) Efficiency of blastocyst construction and cavity formation

[0140] We will Human embryonic stem cells (hESCs) and iHypoblast SCs were co-cultured to construct blastoid structures. Experimental results showed that... Cell aggregates co-constructed from human embryonic stem cells and iHypoblast SCs successfully formed blastocyst cavities on day 6, with a significantly higher cavity formation efficiency than those constructed solely from human embryonic stem cells and iHypoblast SCs. Cell aggregates constructed from human embryonic stem cells alone. This indicates that iHypoblast SCs play a key role in blastocyst construction, promoting the formation and development of blastocyst structures, and providing a powerful model for studying cell interactions and tissue construction during early human embryonic development (Fig. 29a-b).

[0141] (2) Immunofluorescence staining analysis of blastocysts

[0142] We are by Immunofluorescence staining analysis was performed on blastocysts co-constructed with human embryonic stem cells and iHypoblast SCs to detect biomarkers including OCT3 / 4, td-Tomato, T, and GATA6. The results showed that these biomarkers were positively expressed in blastocysts at days 6 and 10, indicating that iHypoblast SCs successfully integrated into the blastocyst structure and were able to interact with it. Human embryonic stem cells develop synergistically to form blastocysts with typical embryonic structural features. This result further confirms the strong ability of cells induced by hypoblastocyst culture medium in this application to construct blastocyst structures in vitro, providing a more accurate in vitro model for studying human embryonic development mechanisms and related diseases, and also providing a stronger theoretical basis for the clinical application of cells (Figures 29c-f).

[0143] Experiment Example 6: Reprogramming and Induced Differentiation Experiments of Different Cell Types

[0144] 6.1 Reprogramming of Mesenchymal Stem Cells (MSCs) and Peripheral Blood Mononuclear Cells (PBMCs)

[0145] We performed reprogramming experiments on mesenchymal stem cells (MSCs) and peripheral blood mononuclear cells (PBMCs), culturing them in 26# medium until day 20. Bright-field and immunofluorescence staining results showed that the reprogrammed MSCs and PBMCs successfully expressed hypodermal markers (PDGFRA, FOXA2, GATA4, and SOX17), pluripotency marker (NANOG), and extraembryonic mesoderm (ExEM) marker (BST2), with high expression levels and uniform distribution. This indicates that the hypodermal stem cell induction medium of this application is not only suitable for the reprogramming of human fibroblasts, but can also effectively induce other types of somatic cells (such as MSCs and PBMCs) to transform into induced hypodermal stem cells, demonstrating broad applicability to various cell types. This provides strong support for the induction and research of hypodermal stem cells using somatic cells from different sources, further expanding the application scope and potential of this application (Figure 30).

[0146] 6.2 Induction of differentiation of human embryonic stem cells (H9)

[0147] We performed induction differentiation experiments on human embryonic stem cells (H9) in PXGL state, and induced them to 26# culture medium condition at the 0th passage (P0) and the 3rd passage (P3), respectively. The results of immunofluorescence staining showed that during the induction of the 0th passage and the 3rd passage, H9 cells could successfully express primitive endoderm related markers (PDGFRA, SOX17, FOXA2, GATA4 and E-CAD), proliferation related markers (Ki67), not express pluripotency markers (NANOG) and extraembryonic mesoderm (ExEM) markers (VIM and BST2). This indicates that the induced hypoblast stem cell culture medium of the present application can effectively induce embryonic stem cells to transform into induced hypoblast stem cells, providing strong support for the induction and research of hypoblast stem cells using embryonic stem cells, and further verifying the high efficiency and stability of the culture medium of the present application in cell induction and differentiation (Figure 31).

[0148] The above supplementary experimental data further verify the strong advantages and wide applicability of the induced hypoblast stem cell culture medium of the present application in precisely regulating cell fate transformation, maintaining cell developmental potential and pluripotency, simulating embryonic development process and constructing in vitro model.

[0149] Obviously, the above embodiments are only examples for clarity and do not limit the embodiments. Based on the above description, those skilled in the art can make other different forms of changes or modifications. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or modifications derived therefrom are still within the protection scope of the present application.

Claims

A hypoblast stem cell induction medium characterized in that, The components of the hypoblast stem cell induction medium comprise platelet-derived growth factor AA, leukemia inhibitory factor, fibroblast growth factor 4, GSK-3α / β inhibitor CHIR99021, TGF-β type I receptor inhibitor A83-01 and bone morphogenetic protein 4. The hypoblast stem cell culture medium of claim 1, wherein The concentration of the platelet-derived growth factor AA in the hypoblast stem cell induction medium is 10-50 ng / mL; the concentration of the leukemia inhibitory factor in the hypoblast stem cell induction medium is 10-50 ng / mL; the concentration of the fibroblast growth factor 4 in the hypoblast stem cell induction medium is 25-125 ng / mL; the concentration of the GSK-3α / β inhibitor CHIR99021 in the hypoblast stem cell induction medium is 3-15 μM; the concentration of the TGF-β type I receptor inhibitor A83-01 in the hypoblast stem cell induction medium is 1-5 μM; and the concentration of the bone morphogenetic protein 4 in the hypoblast stem cell induction medium is 10-50 ng / mL. The hypoblast stem cell culture medium according to claim 1 or 2, characterized in that The components of the hypoblast stem cell induction medium further comprise heparin, N2 supplement, B27 supplement, GlutaMAX, non-essential amino acids, 2-mercaptoethanol and / or penicillin-streptomycin. The hypoblast stem cell culture medium of claim 3, wherein The concentration of the N2 supplement in the hypoblast stem cell induction medium is 0.5-1% by volume; the concentration of the B27 supplement in the hypoblast stem cell induction medium is 1-2% by volume; the concentration of the GlutaMAX in the hypoblast stem cell induction medium is 0.5-1% by volume; the concentration of the non-essential amino acids in the hypoblast stem cell induction medium is 0.5-1% by volume; the concentration of the penicillin-streptomycin in the hypoblast stem cell induction medium is 0.5-1% by volume; the concentration of the heparin in the hypoblast stem cell induction medium is 1-5 μg / mL; and the concentration of the 2-mercaptoethanol in the hypoblast stem cell induction medium is 0.1-0.5 mM. The hypoblast stem cell culture medium of claim 3, wherein The components of the hypoblast stem cell induction medium further comprise a matrix; the matrix comprises Neurobasal medium and DMEM / F12 medium; and the volume ratio of Neurobasal medium to DMEM / F12 medium in the matrix is 0.5-1:0.5-1. A method for constructing induced hypoblast stem cells by somatic cell reprogramming, characterized in that, The method comprises: first transfecting somatic cells using Yamanaka factors, and then inducing and culturing the transfected somatic cells using the hypoblast stem cell induction medium according to any one of claims 1-5 to obtain induced hypoblast stem cells. An induced hypoblast stem cell, characterized in that, The induced hypoblast stem cells are constructed by the method according to claim 6. A method of establishing an in vitro model of a disease associated with hypoblast development, characterized in that, The method comprises: first constructing induced hypoblast stem cells using the method according to claim 6, and then establishing an in vitro model of a disease related to hypoblast development using the constructed induced hypoblast stem cells. Alternatively, the method comprises: establishing an in vitro model of a disease related to hypoblast development using the induced hypoblast stem cells according to claim 7. A method of screening for a drug for preventing and / or treating a disease associated with hypoblast development, characterized by, The method comprises: constructing the induced hypoblast stem cell using the method of claim 6, and screening a drug for preventing and / or treating a disease related to hypoblast development using the constructed induced hypoblast stem cell as a cell model; Alternatively, the method comprises: screening a drug for preventing and / or treating a disease related to hypoblast development using the induced hypoblast stem cell of claim 7 as a cell model; Alternatively, the method comprises: constructing the induced hypoblast stem cell using the method of claim 6, establishing an in vitro model of a disease related to hypoblast development using the constructed induced hypoblast stem cell, and screening a drug for preventing and / or treating the disease related to hypoblast development using the in vitro model; Alternatively, the method comprises: establishing an in vitro model of a disease related to hypoblast development using the induced hypoblast stem cell of claim 7, and screening a drug for preventing and / or treating the disease related to hypoblast development using the in vitro model. A method of evaluating toxicity of a drug, characterized by, The method comprises: constructing the induced hypoblast stem cell using the method of claim 6, administering a drug to the constructed induced hypoblast stem cell, and evaluating toxicity of the drug by observing changes in indexes of the induced hypoblast stem cell; Alternatively, the method comprises: administering a drug to the induced hypoblast stem cell of claim 7, and evaluating toxicity of the drug by observing changes in indexes of the induced hypoblast stem cell. The induced hypoblast stem cell medium of any one of claims 1 to 5 or the method of claim 6 or the induced hypoblast stem cell of claim 7 is used for establishing an in vitro model of a disease related to hypoblast development, screening a drug for preventing and / or treating a disease related to hypoblast development, or evaluating toxicity of a drug.

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