Culture medium for inducing hypoderm stem cells and application thereof

Abstract

The invention relates to a culture medium for inducing hypoderm stem cells and application thereof, and belongs to the technical field of stem cells. The invention provides a culture medium for inducing hypoderm stem cells. The culture medium for inducing the hypoderm stem cells comprises the following components: a platelet-derived growth factor AA (PDGFAA), a leukemia inhibition factor (LIF), a fibroblast growth factor 4 (FGF4), a GSK-3alpha / beta inhibitor CHIR99021, a TGF-beta I type receptor inhibitor A83-01 and a bone forming protein 4 (BMP4). The culture medium for inducing the hypoderm stem cells can accurately regulate and control the activity of key signal channels such as NODAL, BMP, WNT, FGF and JAK / STAT in the somatic cell reprogramming process, and ensures that the somatic cells maintain the characteristics of the hypoderm stem cells in the reprogramming process so as to support self-renewal and hypoderm lineage induction of the somatic cells in the reprogramming process; therefore, the fate transformation from the somatic cells to the hypoderm stem cells is realized.

Description

Technical Field

[0001] The invention relates to a culture medium for inducing hypoblast stem cells and application thereof, belonging to the technical field of stem cells. Background Art

[0002] During early human embryonic development, the differentiation of the inner cell mass (ICM) and the trophectoderm (TE) is a key developmental event, followed by the further development of epiblast stem cells (Epi) and hypoblast stem cells (Hypo, also known as primitive endoderm stem cells) to form structures such as the fetus and yolk sac. Studying the development of the hypoblast is crucial to understanding the early process of human embryonic development.

[0003] At present, the focus is on inducing human hypoblast-like cells in vitro through chemical induction or transgenic overexpression to study the development of the hypoblast. Pluripotent stem cells ( PSCs) introduce specific transcription factors or chemicals to induce the generation of hypoblast-like cells. For example, Okubo et al. and Linneberg-Agerholm et al. produced human hypoblast-like cells (hXENs) by chemical induction (see document "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). ").

[0004] 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 to self-renew and it is difficult to maintain their stem cell state for a long time. Secondly, the cells produced by these methods have contradictions in the signal requirements of the hypoblast. For example, nHyC-7F (see the literature "Okubo, T. et al. Hypoblast from human pluripotent stem cells regulates epiblast development. Nature 626, 357-366 (2024)") and PA-FA83X (see the literature "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)") and RACL / NACL (see the literature "Linneberg-Agerholm, M. et al. human pluripotent stemcells respond to Wnt,Nodal and LIF signaling to produce expandable extra-embryonic endoderm. Development 146, (2019).” and “Mackinlay, K. et al. An invitro stem cell model of human epiblast and yolk sac interaction. Elife 10, (2021).”), RCL (see the reference “Oldak, B. et al. Complete human day 14 post-implantation embryo models from naive ES cells. Nature 622, 562–573 (2023).”) and FTW (see the reference “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 the fate determination of hypoblast cells. Furthermore, the cells generated by these methods are closer to the extraembryonic mesoderm (ExEM) rather than the true hypoblast stem cell lineage, which limits their utility in simulating human embryonic development. In addition, the hypoblast-like cells obtained by these methods are not comprehensive in molecular and functional validation, and lack further validation such as human-mouse chimera experiments and single-cell RNA sequencing. That is, the hypoblast-like cell in vitro models constructed by these methods usually lack the self-renewal stem cell characteristics and have contradictions in signal requirements, or the hypoblast-like cells induced by them are closer to the ectodermal mesoderm (ExEM) rather than the true hypoblast lineage, and the lineage characteristics of hypoblast cells are insufficiently identified. Therefore, the current hypoblast-like cell in vitro models still have significant deficiencies in accurately simulating human embryonic development, especially in the differentiation and function of hypoblast lineage cells. Summary of the invention

[0005] To solve the above-mentioned defects, the present invention provides an induced hypoblast stem cell culture medium, the components of which 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).

[0006] In one embodiment of the present invention, the concentration of the platelet-derived growth factor AA in the induced hypodermal stem cell culture medium is 10-50 ng / mL; the concentration of the leukemia inhibitory factor in the induced hypodermal stem cell culture medium is 10-50 ng / mL; the concentration of the fibroblast growth factor 4 in the induced hypodermal stem cell culture medium is 25-125 ng / mL; the concentration of the GSK-3α / β inhibitor CHIR99021 in the induced hypodermal stem cell culture medium is 3-15 μM; the concentration of the TGF-β type I receptor inhibitor A83-01 in the induced hypodermal stem cell culture medium is 1-5 μM; the concentration of the bone morphogenetic protein 4 in the induced hypodermal stem cell culture medium is 10-50 ng / mL.

[0007] In one embodiment of the present invention, the components of the culture medium for inducing hypodermal stem cells further include heparin, N2 supplement, B27 supplement, GlutaMAX, non-essential amino acids, 2-mercaptoethanol and / or penicillin-streptomycin.

[0008] In one embodiment of the present invention, 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 penicillin-streptomycin in the induced hypoblast stem cell culture medium is 0.5-1% by volume; the concentration of heparin in the induced hypoblast stem cell culture medium is 1-5 μg / mL; the concentration of 2-mercaptoethanol in the induced hypoblast stem cell culture medium is 0.1-0.5 mM.

[0009] In one embodiment of the present invention, the components of the induced hypodermal stem cell culture medium further include a matrix; the matrix includes Neurobasal culture medium and DMEM / F12 culture medium; in the matrix, the volume ratio of Neurobasal culture medium to DMEM / F12 culture medium is 0.5-1:0.5-1.

[0010] In one embodiment of the present invention, the matrix consists of Neurobasal culture medium and DMEM / F12 culture medium; in the matrix, the volume ratio of Neurobasal culture medium to DMEM / F12 culture medium is 1:1.

[0011] The present invention also provides a method for constructing induced hypoblast stem cells by somatic cell reprogramming, the method comprising: firstly transfecting somatic cells with Yamanaka factor (OSKM), and then inducing and culturing the transfected somatic cells with the above-mentioned induced hypoblast stem cell culture medium to obtain induced hypoblast stem cells (iHypoblast SCs).

[0012] In one embodiment of the present invention, the method comprises: after inoculating somatic cells into a somatic cell culture medium, transfecting the somatic cells using a Yamanaka factor (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 paved with feeder layer cells, and cultured in sequence using a somatic cell culture medium and an induced hypoblast stem cell culture medium not additionally supplemented with the ROCK-I / II inhibitor Y27632; after the culture is completed, PDGFRA-positive hypoblast-like cells are selected and re-inoculated and re-inoculated into a cell culture plate paved with feeder layer cells, and the induced hypoblast stem cell culture medium additionally supplemented with the ROCK-I / II inhibitor Y27632 and the induced hypoblast stem cell culture medium not additionally supplemented with the ROCK-I / II inhibitor Y27632 are used in sequence to culture the cells in the cell culture plate. The cells are cultured; after the culture is completed, the hypoblast-like colonies in the cell culture plate are selected and transferred to a cell culture plate on which feeder layer cells are laid, and cultured in sequence using an induced hypoblast stem cell culture medium additionally supplemented with ROCK-I / II inhibitor Y27632 and an induced hypoblast stem cell culture medium not additionally supplemented with ROCK-I / II inhibitor Y27632 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 on which feeder layer cells are laid, and cultured in sequence using an induced hypoblast stem cell culture medium additionally supplemented with ROCK-I / II inhibitor Y27632 and an induced hypoblast stem cell culture medium not additionally supplemented with ROCK-I / II inhibitor Y27632 to obtain induced hypoblast stem cells (iHypoblast SCs).

[0013] In one embodiment of the present invention, the concentration of the ROCK-I / II inhibitor Y27632 in the culture medium for inducing hypodermal stem cells is 5-10 μM.

[0014] In one embodiment of the present invention, the somatic cells include human fibroblasts (e.g., adult dermal fibroblasts and neonatal dermal fibroblasts), amniotic fluid 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 urothelial cells.

[0015] In one embodiment of the present invention, the somatic cell culture medium comprises fibroblast culture medium and / or 106 culture medium.

[0016] In one embodiment of the present invention, the feeder layer cells include inactivated mouse embryonic fibroblasts and / or inactivated human fibroblasts.

[0017] In one embodiment of the present invention, the Yamanaka factors (OSKM) used to reprogram somatic cells (e.g., human fibroblasts) to a dedifferentiated or pluripotent state include OCT3 / 4, c-MYC, KLF4, SOX2, L-MYC, NANOG, LIN28 and / or chemical reprogramming.

[0018] In one embodiment of the present invention, the transfection vector comprises virus, liposome, mRNA and / or exosome.

[0019] In one embodiment of the present invention, the virus comprises Sendai virus, adenovirus and / or lentivirus.

[0020] The present invention also provides an induced hypoblast stem cell (iHypoblast SCs), and the induced hypoblast stem cell (iHypoblast SCs) is constructed by the above method.

[0021] The characteristics of the induced hypoblast stem cells (iHypoblast SCs) include:

[0022] First, they are in an undifferentiated, bipotent state and have the ability to differentiate into cells that exhibit one or more characteristics of endoderm / yolk sac endoderm (VE / YE)-like cells and extraembryonic mesoderm (ExEM)-like cells;

[0023] second, the flat, epithelial-shaped colony appearance;

[0024] Third, the expression of one or more biochemical markers of hypoblast stem cells (e.g., PDGFRA, SOX17, GATA6, GATA4, FOXA2, FN1, COL4A1, and Laminin) can be determined by immunohistochemistry and / or PCR assays;

[0025] Fourth, single-cell omics similar to that of hypoblast cells derived from human embryos;

[0026] Fifth, it has the ability to integrate the extraembryonic endoderm lineage in human-mouse chimeras.

[0027] The present invention also provides a method for establishing an in vitro model of a disease related to hypoblast development, the method comprising: first constructing induced hypoblast stem cells using the above method, and then establishing an in vitro model of a disease related to hypoblast development using the constructed induced hypoblast stem cells;

[0028] Alternatively, the method comprises: using the above-mentioned induced hypodermal stem cells to establish an in vitro model of a disease associated with hypodermal development.

[0029] In one embodiment of the present invention, the establishment of an in vitro model of a disease related to hypoblast development comprises: administering SB431542, A83-01 and ActivinA drugs to induced hypoblast stem cells to obtain an in vitro model of Nodal / Activin signaling pathway and hypoblast cell development; or, administering CHIR99021, IWP-2 and XAV939 drugs to induced hypoblast stem cells 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 induced hypoblast stem cells to obtain an in vitro model of BMP signaling pathway and hypoblast cell development; or, administering FGF2 and SU5402 drugs to induced hypoblast stem cells to obtain an in vitro model of FGF signaling pathway and hypoblast cell development.

[0030] The present invention also provides a method for screening drugs for preventing and / or treating diseases related to hypoblast development, the method comprising: firstly constructing induced hypoblast stem cells using the above method, and then using the constructed induced hypoblast stem cells as a cell model to screen drugs for preventing and / or treating diseases related to hypoblast development;

[0031] Alternatively, the method comprises: using the above-mentioned induced hypoblast stem cells as a cell model to screen drugs for preventing and / or treating diseases related to hypoblast development;

[0032] Alternatively, the method comprises: first constructing induced hypoblast stem cells using the above method, then using the constructed induced hypoblast stem cells to establish an in vitro model of a disease associated with hypoblast development, and finally using the in vitro model to screen drugs for preventing and / or treating the disease associated with hypoblast development;

[0033] Alternatively, the method comprises: first using the above-mentioned induced hypoblast stem cells to establish an in vitro model of a disease associated with hypoblast development, and then using the in vitro model to screen drugs for preventing and / or treating the disease associated with hypoblast development.

[0034] In one embodiment of the present invention, the diseases associated with hypoblast development include yolk sac-related diseases, genetic diseases and / or developmental disorders.

[0035] In one embodiment of the present invention, the yolk sac-related diseases include yolk sac dysplasia and / or yolk sac effusion; the genetic diseases include hereditary pancreatic dysplasia caused by GATA6 mutation, HNF1B-related renal dysplasia and / or situs inversus; the developmental disorders include endoderm development disorder, anterior-posterior axis development disorder and / or early embryonic development arrest.

[0036] The present invention also provides a method for evaluating drug toxicity, the method comprising: first constructing induced hypoblast stem cells using the above method, then administering drugs to the constructed induced hypoblast stem cells, and evaluating the toxicity of the drugs by observing changes in indicators of the induced hypoblast stem cells;

[0037] Alternatively, the method comprises: administering a drug to the induced hypodermal stem cells, and evaluating the toxicity of the drug by observing changes in indicators of the induced hypodermal stem cells.

[0038] In one embodiment of the present invention, the toxicity of the drug includes potential embryo / fetal toxicity, neurotoxicity, immunotoxicity, genotoxicity, endocrine disruption, embryolethality and / or teratogenicity.

[0039] The present invention also provides the use of the above-mentioned induced hypoblast stem cell culture medium or the above-mentioned method or the above-mentioned induced hypoblast stem cells (iHypoblast SCs) in establishing an in vitro model of diseases related to hypoblast development, screening drugs for preventing and / or treating diseases related to hypoblast development, or evaluating drug toxicity.

[0040] In one embodiment of the present invention, the diseases associated with hypoblast development include yolk sac-related diseases, genetic diseases and / or developmental disorders.

[0041] In one embodiment of the present invention, the yolk sac-related diseases include yolk sac dysplasia and / or yolk sac effusion; the genetic diseases include hereditary pancreatic dysplasia caused by GATA6 mutation, HNF1B-related renal dysplasia and / or situs inversus; the developmental disorders include endoderm development disorder, anterior-posterior axis development disorder and / or early embryonic development arrest.

[0042] In one embodiment of the present invention, the toxicity of the drug includes potential embryo / fetal toxicity, neurotoxicity, immunotoxicity, genotoxicity, endocrine disruption, embryolethality and / or teratogenicity.

[0043] The technical solution of the present invention has the following advantages:

[0044] 1. The present invention provides an induced hypoblast stem cell culture medium, the components of which 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 induced hypoblast stem cell culture medium can accurately regulate the activity of key signaling pathways such as NODAL, BMP, WNT, FGF and JAK / STAT during somatic cell reprogramming, thereby ensuring that somatic cells maintain the characteristics of hypoblast stem cells during reprogramming, so as to support the self-renewal of somatic cells during reprogramming and hypoblast lineage induction, 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 reflected in the differentiation ability under in vitro conditions and the developmental potential under in vivo conditions). 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 practicality of simulating the human embryonic development process. In addition, 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 embryonic development. Therefore, the induced hypoblast stem cell culture medium constructed using the induced hypoblast stem cell culture medium has extremely high reliability and applicability as an in vitro model in simulating human hypoblast development research, and can provide a powerful platform for human embryology research. In addition, 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.

[0045] 2. The present invention provides a method for constructing induced hypodermal stem cells by somatic cell reprogramming, the method comprising: firstly transfecting somatic cells with Yamanaka factor (OSKM), and then inducing and culturing the transfected somatic cells with the above-mentioned induced hypodermal stem cell culture medium to obtain induced hypodermal stem cells (iHypoblast SCs); the components of the induced hypodermal stem cell culture 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 key signaling pathways such as NODAL, BMP, WNT, FGF and JAK / STAT during somatic cell reprogramming through Yamanaka factors (OSKM) and induced hypoblast stem cell culture medium, thereby ensuring that 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 achieving 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 reflected in the differentiation ability under in vitro conditions and the developmental potential under in vivo conditions). The induced hypoblast stem cells constructed using the method as an in vitro model can significantly improve the accuracy and practicality of simulating the human embryonic development process. Furthermore, 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 embryonic development. Therefore, the induced hypoblast stem cell culture medium constructed using the method has extremely high reliability and applicability as an in vitro model in simulating human hypoblast development research, and can provide a powerful platform for human embryology research. In addition, 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.

[0046] 3. The present invention provides an induced hypoblast stem cell (iHypoblast SCs). The construction method of the induced hypoblast stem cell (iHypoblast SCs) comprises: firstly transfecting somatic cells with Yamanaka factor (OSKM), and then inducing and culturing the transfected somatic cells with the above-mentioned induced hypoblast stem cell culture medium to obtain induced hypoblast stem cells (iHypoblast SCs); the components of the induced hypoblast stem cell culture 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). Studies have shown that the induced hypoblast stem cells (iHypoblast SCs) have self-renewal and differentiation potential, can be cultured in vitro for a long time and maintain its unique hypoblast characteristics (specifically embodied in the differentiation ability under in vitro conditions and the developmental potential under in vivo conditions), using the induced hypoblast stem cells as an in vitro model, can significantly improve the accuracy and practicality of simulating the human embryonic development process. In addition, studies have shown that the induced hypoblast stem cells (iHypoblast SCs) can be successfully integrated into the primitive endoderm and visceral endoderm of mouse embryos, showing its lineage contribution and developmental potential in early chimeric embryonic development. Therefore, the induced hypoblast stem cells have extremely high reliability and applicability in simulating human hypoblast development research as an in vitro model, can provide a powerful platform for human embryology research, and the induced hypoblast stem cells have great application prospects in the fields of establishing in vitro models of diseases related to hypoblast development, screening for the prevention and / or treatment of diseases related to hypoblast development or evaluating drug toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 : Induction of human iHypoblast SCs from somatic cell reprogramming. Figure 1 In the figure, (a) Schematic diagram of inducing human iHypoblast SCs from somatic cell reprogramming; (b) FACS screening of the proportion of cells positive for PDGFRA, an intermediate of reprogramming, on day 20; (c) Observation of morphological changes of reprogrammed cells in 2D culture at different passage numbers under phase contrast microscopy.

[0048] Figure 2 : Transcriptome analysis. Figure 2Figure 2. (a) Display of defined Epi, Hypo, and TE signature scores on the unified manifold approximation and projection (UMAP) integrative map of reprogramming intermediate cells at day 0, day 4, day 8, and day 21, cultured in fibroblast medium (Fm); (b) Display of defined Epi, Hypo, and TE signature scores from the Radley dataset on bulk RNA-seq data of reprogramming intermediate cells at day 0, day 3, and day 7, cultured in fibroblast medium; (c) Browser view showing ATAC peaks of GATA6, SOX17, FOXA2, OTX2, SALL1, and GATA4 in reprogramming intermediate cells at day 0, day 3, and day 7, cultured in fibroblast medium (highlighted regions demonstrate changes in chromatin accessibility, indicating binding of hypoblast-associated transcription factors).

[0049] Figure 3 :ATAC sequencing analysis. Figure 3 In the figure, the preview shows ATAC peaks for GATA6, SOX17, FOXA2, OTX2, SALL1, and GATA4 in reprogramming intermediate cells at day 0, day 3, and day 7, when cells were cultured in fibroblast medium (highlighted areas demonstrate changes in chromatin accessibility, indicating binding of hypoblast-associated transcription factors).

[0050] Figure 4 : 33 screening media. Figure 4 (a) Screening of 33 screening culture media (including cytokines and small molecule inhibitors / activators) that can maintain human hypoblast stem cells in vitro; (b) Heat map and scoring bar chart of relative protein expression of PDGFRA, SOX17, FOXA2, GATA4, NANOG and BST2 corresponding to the 33 screening conditions.

[0051] Figure 5 : Immunofluorescence staining of 33 screening media. Figure 5 In the figure, on the 20th day of reprogramming, representative immunofluorescence staining images of FOXA2, PDGFRA, GATA4, NANOG, SOX17 and BST2 were obtained for screening conditions 1#(a), 2#(b), 3#(c), 4#(d), 5#(e), and 6#(f).

[0052] Figure 6 : Immunofluorescence staining of 33 screening media. Figure 6In the figure, on day 20 of reprogramming, 7#(a), 8#(b), 9#(c), 10#(d), 11#(e), and 12#(f) screening conditions, representative immunofluorescence staining images of FOXA2, PDGFRA, GATA4, NANOG, SOX17, and BST2.

[0053] Figure 7 : Immunofluorescence staining of 33 screening media. Figure 7 In the figure, on the 20th day of reprogramming, 13#(a), 14#(b), 15#(c), 16#(d), 17#(e), and 18#(f) screening conditions, representative immunofluorescence staining images of FOXA2, PDGFRA, GATA4, NANOG, SOX17, and BST2.

[0054] Figure 8 : Immunofluorescence staining of 33 screening media. Figure 8 In the figure, on the 20th day of reprogramming, 19#(a), 20#(b), 21#(c), 22#(d), 23#(e), and 24#(f) screening conditions, representative immunofluorescence staining images of FOXA2, PDGFRA, GATA4, NANOG, SOX17, and BST2.

[0055] Fig. 9 : Immunofluorescence staining of 33 screening media. Fig. 9 In the figure, on the 20th day of reprogramming, 25#(a), 26#(b), 27#(c), 28#(d), 28#(e), and 30#(f) screening conditions, representative immunofluorescence staining images of FOXA2, PDGFRA, GATA4, NANOG, SOX17, and BST2.

[0056] Fig.10 : Immunofluorescence staining of 33 screening media. Fig.10 In the figure, on day 20 of reprogramming, 31#(a), 32#(b), and 33#(c) screening conditions, representative immunofluorescence staining images of FOXA2, PDGFRA, GATA4, NANOG, SOX17, and BST2.

[0057] Fig.11 : Bright field and immunofluorescence staining of 5# and 26# culture medium of donor 1. Fig.11In the figure, (a) bright field image of cells in donor 1, 5# hypoblast stem cell screening culture medium at passage 15; (b) representative immunofluorescence staining images of hypoblast markers (PDGFRA, SOX17, FOXA2, GATA4 and E-CAD) and ExEM marker (VIM) at passage 15 of donor 1, 5#; (c) representative immunofluorescence staining images of hypoblast markers (GATA6), pluripotency markers (NANOG), ExEM (BST2) and proliferation markers (Ki67) at passage 15 of donor 1, 5#; (d) Bright field image of cells at passage 15 of donor 1, 26# hypoblast stem cell screening culture medium; (e) Representative immunofluorescence staining images of hypoblast markers (PDGFRA, SOX17, FOXA2, GATA4 and E-CAD) and ExEM marker (VIM) at passage 15 of donor 1, 26# conditions; (f) Representative immunofluorescence staining images of hypoblast markers (GATA6), pluripotency markers (NANOG), ExEM (BST2) and proliferation markers (Ki67) at passage 15 of donor 1, 26# conditions.

[0058] Fig.12 : Bright field and immunofluorescence staining of 5# and 26# culture medium of donor 2. Fig.12 In the figure, (a) bright field image of cells in donor 2, 5# hypoblast stem cell screening medium at passage 15; (b) representative immunofluorescence staining images of hypoblast markers (PDGFRA, SOX17, FOXA2, GATA4 and E-CAD) and ExEM marker (VIM) at passage 15 of donor 2, 5#; (c) representative immunofluorescence staining images of hypoblast markers (GATA6), pluripotency markers (NANOG), ExEM (BST2) and proliferation markers (Ki67) at passage 15 of donor 2, 5#; (d) Bright field image of cells at passage 15 of donor 2, 26# hypoblast stem cell screening culture medium; (e) Representative immunofluorescence staining images of hypoblast markers (PDGFRA, SOX17, FOXA2, GATA4 and E-CAD) and ExEM marker (VIM) at passage 15 of donor 2, 26# conditions; (f) Representative immunofluorescence staining images of hypoblast markers (GATA6), pluripotency markers (NANOG), ExEM (BST2) and proliferation markers (Ki67) at passage 15 of donor 2, 26# conditions.

[0059] Fig.13 : Single factor elimination experimental characterization of 26# culture medium. Fig.13(a) Representative phase contrast microscopy images showing the removal of each medium component from the 26# condition (n=3 independent experiments, scale bar: 100 μm); (b) Representative immunofluorescence staining images under the 26# condition, staining for hypoblast markers (PDGFRA, FOXA2, GATA4 and SOX17), pluripotency marker (NANOG), and ExEM marker (BST2) (n=3 independent experiments, scale bar: 100 μm); (c) Representative immunofluorescence staining images under the 26# condition after the removal of recombinant human PDGFAA, staining for (d) Representative immunofluorescence staining images under 26# condition after removal of recombinant human LIF, staining for hypodermal markers (PDGFRA, FOXA2, GATA4 and SOX17), pluripotency marker (NANOG), and ExEM marker (BST2) (n=3 independent experiments, scale bar: 100 μm).

[0060] Fig.14 : Single factor elimination experimental characterization of 26# culture medium. Fig.14(a) Representative immunofluorescence staining images of 26# condition 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, scale bar: 100μm); (b) Representative immunofluorescence staining images of 26# condition after removal of CHIR, staining for hypoblast markers (PDGFRA, FOXA2, GATA4 and SOX17), pluripotency marker (NANOG), ExEM marker (BST2) (n=3 independent experiments, scale bar: 100μm); (c) Representative immunofluorescence staining images of 26# condition after removal of A83-01, staining for hypoblast markers (PDGFR A, staining for FOXA2, GATA4 and SOX17), pluripotency markers (NANOG), and ExEM markers (BST2) (n=3 independent experiments, scale bar: 100 μm); (d) Representative immunofluorescence staining images under 26# condition after removal of recombinant human BMP4, staining for hypoblast markers (PDGFRA, FOXA2, GATA4 and SOX17), pluripotency markers (NANOG), and ExEM markers (BST2) (n=3 independent experiments, scale bar: 100 μm); (e) Hypo scores under 26# condition and after removal of various culture medium components, based on the relative protein expression levels of FOXA2, PDGFRA, GATA4, SOX17, NANOG and BST2 (mean ± standard error, n=3 independent experiments).

[0061] Fig.15 : Karyotype and Sendai virus detection of iHypoblast SCs. Fig.15 (a) Karyotype detection of iHypoblast SCs (somatic cells were 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.

[0062] Fig.16 :iHypoblast SCs single-cell sequencing. Fig.16 (a) Cartoon illustration of in vitro culture and single-cell sequencing for screening hypoblast-like stem cells during reprogramming; (b) UMAP analysis of integrated datasets of iHypoblast SCs, human preimplantation blastocysts, and in vitro cultured embryos (colors correspond to cell types annotated according to the respective datasets); (c) UMAP analysis of integrated datasets of iHypoblast SCs, human preimplantation blastocysts, and in vitro cultured embryos (colors correspond to developmental time annotations of embryos in the respective datasets).

[0063] Fig.17 :iHypoblast SCs single-cell sequencing. Fig.17 In the figure, (a) the integrated UMAP shows the reprogramming intermediate cells at 0 days, 4 days and 8 days, as well as the hypoblast-like cells (condition 26) and iHypoblast SCs (Fm, fibroblast culture medium) at 20 days; (b) CytoTRACE and Monocle3 were used to reconstruct the cell trajectory during the reprogramming process; (c) SCENIC analysis showed that D0, Activities of the top 50 differentially active regulators in iPS cells and primed iPS cells.

[0064] Fig.18 :iHypoblast SCs single cell sequencing UMAP marker gene expression. Fig.18 (a) Expression of basement membrane markers (FN1, COL4A1, LAMA1, LAMB) in iHypoblastSCs UMAP; (b) Expression of ectoderm markers (DCN, FOXF1, HAND2, BST2) in iHypoblast SCs UMAP; (c) Expression of hypodermal markers (PDGFRA, BMP2, GATA6, FOXA2, HNF4A, GATA4) in iHypoblast SCs UMAP; (d) Expression of pluripotency markers (NANOG, OCT3 / 4) in iHypoblast SCs UMAP.

[0065] Fig.19 :Characterization and analysis of iHypoblast SCs. Fig.19 (a) UMAP analysis of integrated data of iHypoblast SCs and 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 those in published studies; (c) correlation analysis of RNA sequencing hypoblast and single-cell RNA sequencing data in this study with those in published studies and human embryo data.

[0066] Fig. 20 : BMP4 induced differentiation. Fig. 20(a) Representative immunofluorescence staining images of COL6A1, FOXA1 and GATA6 in iHypoblast SCs differentiated by BMP4 to day 5; (b) RT-qPCR analysis of visceral endoderm markers (AFP and HNF4A), hypodermal markers (GATA6 and PDGFRA) and parietal endoderm markers (PLAT and SPARC) in iHypoblast SCs differentiated by BMP4 to day 5; (c) ELISA was used to measure the concentrations of AFP and APOA1 in the culture supernatant of iHypoblast SCs differentiated by BMP4 to day 5.

[0067] Fig.21 : FBS induced differentiation. Fig.21 (a) Representative immunofluorescence staining images of COL6A1, FOXA1 and GATA6 in iHypoblast SCs differentiated by FBS to day 5; (b) RT-qPCR analysis of visceral endoderm markers (AFP and HNF4A), hypodermal markers (GATA6 and PDGFRA) and parietal endoderm markers (PLAT and SPARC) in iHypoblast SCs differentiated by FBS to day 5; (c) ELISA was used to measure the concentrations of AFP and APOA1 in the culture supernatant of iHypoblast SCs differentiated by FBS to day 5.

[0068] Fig. 22 :Basement membrane markers in iHypoblast SCs. Fig. 22 Representative immunofluorescence staining images of basement membrane markers (FN1, COL4A1, and Laminin), hypodermal markers (GATA6, GATA4, and SOX17), pluripotency marker (SOX2), and ectodermal and endodermal markers (FOXF1 and VIM) in iHypoblast SCs (n = 3 independent experiments, scale bar: 100 μm).

[0069] Fig.23 : Chimera experiment. Fig.23In the figure, (a) Cartoon schematic diagram of chimera experiment: td-Tomato-labeled iHypoblasts were microinjected into mouse E3.5 blastocysts (scale bar, 25 μm); (b) Td-Tomato-labeled iHypoblasts were microinjected into mouse E3.5 blastocysts, cultured in vitro for about 24 hours, and chimera efficiency statistics were collected at E4.5 (arrows indicate that human td-Tomato+iHypoblast SCs were successfully integrated into the mouse primitive endoderm, n=48 mouse embryos, scale bar: 100 μm); (c) Td-Tomato-labeled iHypoblasts were microinjected into mouse E3.5 blastocysts, transplanted into pseudopregnant mothers and developed until E6.5, and E6.5 chimeras were collected (arrows indicate that human td-Tomato+iHypoblast SCs were successfully integrated into the mouse visceral endoderm, n=25 mouse embryos, scale bar: 100 μm).

[0070] Fig.24 : Immunofluorescence staining of chimera experiment. Fig.24 (a) Immunofluorescence staining images of E4.5 human-mouse chimeric embryos, used to detect human antigens, td-Tomato, and SOX17 (arrows indicate that human td-Tomato+iHypoblast SCs have been successfully integrated into the mouse primitive endoderm, n=3 independent experiments, scale bar: 25 μm); (b) Immunofluorescence staining images of E6.5 human-mouse chimeric embryos, used to detect human antigens, td-Tomato, and SOX17 (arrows indicate that human td-Tomato+iHypoblast SCs have been successfully integrated into the mouse visceral endoderm, n=3 independent experiments, scale bar: 65 μm). DETAILED DESCRIPTION

[0071] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0072] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0073] The materials and reagents involved in the following examples are shown in Table 1.

[0074] Table 1 Experimental materials and reagents

[0075]

[0076]

[0077] Example 1: A culture medium for inducing hypoblast stem cells

[0078] The present embodiment provides an induced hypoblast stem cell (iHypoblast SCs) culture medium, wherein the induced hypoblast stem cell culture medium is an N2B27 basal culture medium containing 10 ng / mL human PDGFAA, 10 ng / mL human LIF, 25 ng / mL human FGF4, 1 μg / mL heparin (promoting the binding of FGF4 to its receptor and optimizing the biological function of FGF4), 3 μM CHIR99021, 1 μM A83-01 and 10 ng / mL human BMP4; the N2B27 basal culture medium is a mixed culture 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 culture medium is composed of Neurobasal culture medium and DMEM / F12 in a volume ratio of 1:1.

[0079] Example 2: A method for constructing induced hypoblast stem cells by somatic cell reprogramming

[0080] This example provides a method for constructing induced hypoblast stem cells by somatic cell reprogramming. The method is as follows (see the specific operation process for details). Figure 1 a) in:

[0081] Step 1: Use the CytoTune-iPS2.0Sendai reprogramming kit to reprogram primary human adult skin fibroblasts (including donor 1 and donor 2) according to the instructions. The reprogramming process is: human fibroblasts are cultured at 5×10 4 Cells were plated at a density of 100 cells / mL in mouse embryonic fibroblast culture medium and transfected with Sendai virus at a multiplicity of infection (MOI) of 5 (KOS), 5 (c-Myc), and 6 (KLF4).

[0082] Step 2: On the 7th day after transfection, the transfected human fibroblasts in the fibroblast culture medium were dissociated using TryPLE Select at room temperature (25°C) for 5 minutes (TryPLE Select dosage 0.5 mL / well) and then 6×10 4The cells were re-inoculated at a rate of 1 mL / well into a cell culture plate (in addition to the feeder layer cells, 1 mL / well of mouse embryonic fibroblast culture medium was added to the cell culture plate) with feeder layer cells (mouse embryonic fibroblasts were used as feeder layer cells, and the method of laying feeder layer cells can be found in the literature “Liu, X. et al. Reprogramming roadmap reveals route to human induced trophoblast stem cells. Nature 586, 101–107 (2020)”) laid one day in advance, and cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2.

[0083] Step 3: After culturing for 24 hours, the culture medium in the cell culture plate was replaced with the induced hypodermal stem cell culture medium of Example 1, and the cells were continued to be cultured in a 37° C. cell culture incubator with 5% (v / v) CO 2 and 5% (v / v) O 2 .

[0084] Step 4: After 20 days of culture, FACS (flow cytometry) was used to screen hypoblast-like stem cells with high PDGFRA expression based on PDGFRA expression (the proportion of cells positive for reprogramming intermediate PDGFRA is shown in Figure 1 b) in the above.

[0085] Step 5: Select PDGFRA-positive hypoblast-like cells and count them at 6×10 4 The cells were re-seeded at an inoculum size of 1 mL / well in a cell culture plate on which feeder layer cells had been laid one day in advance (in addition to the feeder layer cells, the cell culture plate also contained 1 mL / well of the induced hypodermal stem cell culture medium of Example 1 supplemented with 10 μM ROCK-I / II inhibitor Y27632), and cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2.

[0086] Step 6: After culturing for 24 hours, the culture medium in the cell culture plate was replaced with the induced hypoblast stem cell culture medium of Example 1 without additional supplementation of ROCK-I / II inhibitor Y27632, and the culture was continued in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2.

[0087] Step 7: After 5 days of culture, the hypoblast-like colonies in the cell culture plate were selected and cultured at 6×10 4The inoculated amount of cells / well was transferred to a cell culture plate with feeder layer cells laid one day in advance (in addition to the feeder layer cells, the cell culture plate also contained 1 mL / well of the induced hypoblast stem cell culture medium of Example 1 supplemented with 10 μM ROCK-I / II inhibitor Y27632), and cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 20% (v / v) O2. During the culture period, the culture medium was replaced once a day.

[0088] Step 8: When the hypoblast-like cells in the cell culture plate reached 80-90% confluence, the hypoblast-like cells in the cell culture plate were dissociated with Accutase at room temperature (25°C) for 5 minutes (Accutase dosage 0.5 mL / well) and 6×10 4 The inoculated amount of cells / well was transferred to a cell culture plate with feeder layer cells laid one day in advance (in addition to the feeder layer cells, the cell culture plate also added 1 mL / well of the induced hypoblast stem cell culture medium of Example 1 supplemented with 10 μM ROCK-I / II inhibitor Y27632), and cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 20% (v / v) O2. After 24 hours of culture, induced hypoblast stem cells (iHypoblast SCs) were successfully constructed.

[0089] Step 9: After the iHypoblast SCs were successfully constructed, the iHypoblast SCs were subcultured every 3 days at a subculture ratio of 1:5. The subculture process was the same as step 8. During the subculture process, the morphological changes of the 2D cultured reprogrammed cells at different subculture times were observed under a phase contrast microscope. The results are shown in Figure 1 c in.

[0090] Among them, the mouse embryonic fibroblast culture medium (MEF culture medium) is a DMEM culture 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.

[0091] Experimental Example 1: Exploration of the construction conditions of induced hypodermal stem cells (iHypoblast SCs) and verification of the performance of induced hypodermal stem cells (iHypoblast SCs)

[0092] This experimental example provides an exploration of the construction conditions of induced hypoblast stem cells (iHypoblast SCs) and a verification experiment of the performance of induced hypoblast stem cells (iHypoblast SCs). The experimental process is as follows:

[0093] 1. Reprogramming fibroblasts into induced hypodermal stem cells (iHypoblast SCs)

[0094] Based on 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)"), this study noticed that on the 8th day of reprogramming, the expression of related hypodermal genes was significantly upregulated ( Figure 2 a). This result was further verified by RNA-seq and ATAC-seq data ( Figure 2 b. Figure 3 ). These results suggest that day 8 reprogramming intermediates provide a reasonable window for capturing human hypoblast cell fate in vitro.

[0095] To develop culture conditions to capture and maintain these hypoblast-like stem cells, this study investigated the signaling requirements of hypoblast cells in human embryos. This study found that NODAL, BMP, WNT, FGF, and JAK / STAT signaling receptors are highly expressed in hypoblast cells, indicating that they play a key role in the determination and maintenance of hypoblast cell fate ( Figure 2 c). Based on these findings, this study designed screening experiments to test 33 combinations of activators or inhibitors (chemical compounds or cytokines) targeting NODAL, BMP, and WNT signaling pathways ( Figure 4 a).

[0096] To assess indicators of successful construction of hypoblast-like stem cells, this 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 the reference "Corujo-Simon, E., Radley, AH & Nichols, J. Evidence implicating sequential commitment of the founder lineages in the human blastocyst by order of hypoblast gene activation. Development 150, (2023)."). It is worth noting that once these markers are expressed, the expression of NANOG in hypoblast cells cannot be detected. In addition, although there is some overlap in the gene expression of extraembryonic mesoderm (ExEM) cells and hypoblast cells, BST2 was identified as a specific surface marker for ExEM cells (see the literature “Pham, TXA et al. Modeling human extraembryonic mesoderm cells using naive pluripotent stem cells. Cell Stem Cell 29, 1346–1365.e10 (2022)”). Based on the above research results, this study selected PDGFRA, SOX17, FOXA2 and GATA4 as positive markers, and NANOG and BST2 as negative markers. This combination enabled this study to calculate the “Hypo score” to evaluate the proportion of hypoblast-like cells by the relative protein expression levels of these six markers ( Figure 4 b. Figures 5 to 10 Among the conditions tested, this study found that conditions 5# and 26# performed best in generating hypoblast-like cells ( Figure 4 b).

[0097] To determine whether conditions 5# and 26# could support long-term stabilization of hypoblast-like stem cells, cells maintained in these media were serially passaged. Under condition 5#, as the passages progressed, the cells gradually lost their epithelial morphology at passage 15, accompanied by a significant decrease in proliferation potential ( Fig.11 a~ Fig.11 c. Fig.12 a~ Fig.12 c). Immunofluorescence staining showed loss of expression of hypoblast markers including PDGFRA, GATA6, SOX17, FOXA2, and GATA4 ( Fig.11 a~ Fig.11 c. Fig.12a~ Fig.12 c). In contrast, condition 26# was able to stably maintain the hypoblast stem cell-like state until passage 15, showing Ki67-positive, flat epithelial colonies and expressing all hypoblast markers ( Fig.11 b~ Fig.11 d. Fig.12 b~ Fig.12 d). Notably, cells cultured for a long time under condition 5# eventually developed into putative ExEM-like cells, as evidenced by the upregulated expression of BST2 and VIM ( Fig.11 a~ Fig.11 c. Fig.12 a~ Fig.12 c). In order to identify the key components in condition 26#, this study conducted a single component removal experiment and found that A83-01 and CHIR99021 are the key components for maintaining the characteristics of hypoblast stem cells ( Fig.13 , Fig.14 ). These findings indicate that condition 26# is the optimal culture formula for maintaining hypoblast cells in a self-renewing, stem cell-like state. Because these stem cells are generated by somatic cell reprogramming, this study named them induced hypoblast stem cells (iHypoblast SCs) and medium 26# was named induced hypoblast stem cell medium. Importantly, iHypoblast SCs can be reproducibly generated from fibroblasts of multiple biologically independent donors, maintain karyotype stability, and do not contain reprogramming transgenes ( Fig.15 a~ Fig.15 b) In conclusion, this study developed a novel culture condition that enables the generation and long-term maintenance of iHypoblast SCs via fibroblast reprogramming. This marks the first successful establishment of expandable human hypoblast stem cells.

[0098] Table 4 Candidate conditions for hypoblast culture

[0099]

[0100]

[0101]

[0102] 2. Single-cell transcriptomics study of induced hypoblast stem cells (iHypoblast SCs)

[0103] To gain a deeper understanding of the generation mechanism of iHypoblast SCs, this study performed Chromium 10X single-cell RNA sequencing (scRNA-seq) analysis at different time points during their generation. Fig.16a). To assess whether iHypoblast SCs recapitulate key features of the human embryonic hypodermal lineage, this study integrated the scRNA-seq data of this study with published human embryonic datasets covering the entire human embryonic period from preimplantation (see the literature "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 the post-implantation (E8–E14) stage (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). Through unsupervised unified manifold approximation and projection (UMAP) cluster analysis based on previously defined lineage-specific markers, this study identified multiple cell types including the 8-cell stage, morula, anterior lineage, inner cell mass (ICM), trophectoderm (TE), epiblast (Epi), hypoblast (Hypo), primitive streak (PS), ectomesoderm (ExEM), and trophoblast (TrB) ( Figure 2 a). Strikingly, iHypoblast SCs clustered closely with in vivo human hypoblast cell clusters, indicating that their transcriptional signatures are highly consistent with in vivo hypoblast cells ( Fig.16 b). Further analysis revealed that iHypoblast SCs highly expressed hypodermal-specific genes—PDGFRA, BMP2, GATA6, FOXA2, HNF4A, and GATA4—but did not express the pluripotency marker NANOG and the ExEM markers DCN, FOXF1, HAND2, and BST2 ( Fig.18 b~ Fig.18 d). Similarly, integration analysis with in vitro ExEM-like cells further confirmed that iHypoblast SCs are distinct from human embryonic ExEM clusters ( Fig.19 a).

[0104] To determine the developmental stage of iHypoblast SCs, we performed subpopulation analysis and dimensionality reduction analysis based on previous annotations of each dataset, thereby mapping different developmental stages onto our integrated UMAP atlas. Compared with the in vivo hypoblast lineage cells, iHypoblast SCs formed a cluster close to E5–E7 human embryonic hypoblast cells, indicating that they are similar to preimplantation hypoblast ( Fig.16 c). Similarly, for hypoblast-like cells in previous studies (see the literature "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 specificity. Cell Stem Cell 31, 1058–1071. e5 (2024).", "Linneberg-Agerholm, M. et al. human pluripotent stem cells respond to Wnt,Nodal and LIF signallingto produce expandable extra-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 the literature “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 StemCell 28,1625–1640.e6(2021).", "Yu, L. et al. Large-scale production of human blastoids amenable to modeling blastocyst development and maternal-fetalcross 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. Principal component analysis (PCA) of Res.33,661–678(2023).") showed that iHypoblast SCs,. The gene expression profiles of hPSC-derived hypoblast-like cells (nHyCs) and PXGL-RACL are similar to those of E5 human embryonic hypoblast cells ( Fig.19 b). Correlation analysis further confirmed the high consistency between iHypoblast SCs and human embryonic hypoblast cells ( Fig.19 c). In addition, pseudo-time trajectory analysis using Monocle3 and CytoTrace algorithms further supported the concept that hypoblast cell fate can be captured directly from day 8 reprogramming intermediate cells ( Fig.17 a~ Fig.17b). Given that transcription factors (TFs) control cell fate by binding to cis-regulatory regions, thus forming gene regulatory networks (GRNs), this study reconstructed 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 that they have regulatory activities for hypoblast identity ( Fig.17 c) In conclusion, our single-cell transcriptome analysis demonstrated that iHypoblast SCs faithfully recapitulate the molecular signature of preimplantation human hypoblast cells.

[0105] 3. Functional characterization of induced hypoblast stem cells (iHypoblast SCs)

[0106] Next, this study used a variety of in vitro and in vivo functional experiments to elucidate the cellular characteristics and developmental potential of iHypoblast SCs. A characteristic of the hypoblast is its ability to generate basement membrane components between the ectoderm (Epi) and hypoblast (Hypo) to promote 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 ( Fig.18 a). Further immunostaining analysis also confirmed that iHypoblast SCs produced basement membrane components, including FN1, COL4A1, and Laminin ( Fig. 22 ).

[0107] Previous studies have shown that human naive ectodermal endoderm (nEnd), embryoid body nEnd and ectodermal endoderm (XENs) can be transformed into visceral endoderm / yolk sac endoderm (VE / YE)-like cells and ExEM-like cells by adding BMP4 or FBS (see the reference "Linneberg-Agerholm, M. et al. human pluripotent stem cells respond to Wnt,Nodal and LIF signaling 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, this study 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+)( Fig. 20 a. Fig.21 a). Further RT-qPCR analysis confirmed the upregulation of VE markers AFP and HNF4A, downregulation of hypoblast markers GATA6 and PDGFRA, and downregulation of parietal markers PLAT and SPARC ( Fig. 20 b. Fig.21 b). In addition, the expression of AFP and APOA1, which are proteins produced and secreted by the hypoblast and yolk sac tissue, was increased in both hypoblast differentiation methods ( Fig. 20 c. Fig.21 c).

[0108] To further evaluate the in vivo developmental potential of iHypoblast SCs, we performed human-mouse chimera experiments. By labeling iHypoblast SCs with tdTomato fluorescent protein, we were able to track the cells injected into mouse embryos. Fig.23 , Fig.24 Within 24 hours after 10-15 td-Tomato-labeled iHypoblast SCs were injected into mouse blastocysts, we observed that iHypoblast SCs were effectively integrated into the primitive endoderm (PrE) of the inner cell mass (ICM) and maintained SOX17 expression, indicating their contribution to the development of the extraembryonic endoderm lineage ( Fig.23 b. Fig.24 a). As a control group, blank injection was performed in this study and it was confirmed that no td-Tomato cells were observed in the PrE layer of blastocysts ( Fig.23 a. Fig.24 a~ Fig.24b). Importantly, after transplanting chimeric embryos into pseudopregnant surrogate mice, this study observed that iHypoblastSCs contributed to the visceral endoderm (VE) layer at embryonic day 6.5 without integrating into the epiblast or ectoderm, indicating that iHypoblastSCs are functional in chimeras and are able to develop into downstream cell types in human-mouse chimeras ( Fig.23 c. Fig.24 b). Overall, iHypoblast SCs recapitulate the molecular features of hypoblast cells and demonstrate their lineage contribution and developmental potential in vivo, which can be classified as hypoblast stem cells (SCs) of the human extraembryonic endoderm lineage.

[0109] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A culture medium for inducing hypoblast stem cells, characterized in that: The components of the culture medium for inducing hypoblast stem cells include 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.

2. The culture medium for inducing hypoblast stem cells according to claim 1, characterized in that The concentration of the platelet-derived growth factor AA in the induced hypodermal stem cell culture medium is 10 to 50 ng / mL; the concentration of the leukemia inhibitory factor in the induced hypodermal stem cell culture medium is 10 to 50 ng / mL; the concentration of the fibroblast growth factor 4 in the induced hypodermal stem cell culture medium is 25 to 125 ng / mL; the concentration of the GSK-3α / β inhibitor CHIR99021 in the induced hypodermal stem cell culture medium is 3 to 15 μM; the concentration of the TGF-β type I receptor inhibitor A83-01 in the induced hypodermal stem cell culture medium is 1 to 5 μM; and the concentration of the bone morphogenetic protein 4 in the induced hypodermal stem cell culture medium is 10 to 50 ng / mL.

3. The culture medium for inducing hypoblast stem cells according to claim 1 or 2, characterized in that The components of the culture medium for inducing hypoblast stem cells further include heparin, N2 supplement, B27 supplement, GlutaMAX, non-essential amino acids, 2-mercaptoethanol and / or penicillin-streptomycin.

4. The culture medium for inducing hypoblast stem cells according to claim 3, characterized in that In terms of volume percentage, the concentration of the N2 supplement in the induced hypodermal stem cell culture medium is 0.5-1%; in terms of volume percentage, the concentration of the B27 supplement in the induced hypodermal stem cell culture medium is 1-2%; in terms of volume percentage, the concentration of the GlutaMAX in the induced hypodermal stem cell culture medium is 0.5-1%; in terms of volume percentage, the concentration of the non-essential amino acids in the induced hypodermal stem cell culture medium is 0.5-1%; in terms of volume percentage, the concentration of penicillin-streptomycin in the induced hypodermal stem cell culture medium is 0.5-1%; in terms of volume percentage, the concentration of heparin in the induced hypodermal stem cell culture medium is 1-5 μg / mL; and the concentration of 2-mercaptoethanol in the induced hypodermal stem cell culture medium is 0.1-0.5 mM.

5. The culture medium for inducing hypoblast stem cells according to claim 3, characterized in that The components of the induced hypodermal stem cell culture medium also include a matrix; the matrix includes Neurobasal culture medium and DMEM / F12 culture medium; in the matrix, the volume ratio of Neurobasal culture medium to DMEM / F12 culture medium is 0.5-1:0.5-1.

6. A method for constructing induced hypoblast stem cells by somatic cell reprogramming, characterized in that: The method comprises: firstly transfecting somatic cells with Yamanaka factors, and then inducing and culturing the transfected somatic cells with the induced hypoblast stem cell culture medium according to any one of claims 1 to 5 to obtain induced hypoblast stem cells.

7. An induced hypoblast stem cell, characterized in that: The induced hypoblast stem cells are constructed by the method according to claim 6.

8. A method for 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 of claim 6, and then using the constructed induced hypoblast stem cells to establish an in vitro model of a disease related to hypoblast development; Alternatively, the method comprises: using the induced hypodermal stem cells according to claim 7 to establish an in vitro model of a disease associated with hypodermal development.

9. A method for screening drugs for preventing and / or treating diseases associated with hypoblast development, characterized in that: The method comprises: first constructing induced hypoblast stem cells using the method of claim 6, and then using the constructed induced hypoblast stem cells as a cell model to screen drugs for preventing and / or treating diseases related to hypoblast development; Alternatively, the method comprises: using the induced hypoblast stem cells according to claim 7 as a cell model to screen drugs for preventing and / or treating diseases related to hypoblast development; Alternatively, the method comprises: first constructing induced hypoblast stem cells using the method of claim 6, then using the constructed induced hypoblast stem cells to establish an in vitro model of a disease associated with hypoblast development, and finally using the in vitro model to screen drugs for preventing and / or treating the disease associated with hypoblast development; Alternatively, the method comprises: first using the induced hypoblast stem cells of claim 7 to establish an in vitro model of a disease associated with hypoblast development, and then using the in vitro model to screen drugs for preventing and / or treating the disease associated with hypoblast development.

10. A method for evaluating drug toxicity, characterized in that: The method comprises: first constructing induced hypoblast stem cells using the method of claim 6, then administering a drug to the constructed induced hypoblast stem cells, and evaluating the toxicity of the drug by observing changes in indicators of the induced hypoblast stem cells; Alternatively, the method comprises: administering a drug to the induced hypodermal stem cells according to claim 7, and evaluating the toxicity of the drug by observing changes in indicators of the induced hypodermal stem cells.

11. Use of the induced hypoblast stem cell culture medium according to any one of claims 1 to 5, the method according to claim 6, or the induced hypoblast stem cells according to claim 7 in establishing an in vitro model of a disease associated with hypoblast development, screening drugs for preventing and / or treating a disease associated with hypoblast development, or evaluating drug toxicity.

Images