An organoid capable of simulating uterine spiral artery remodeling and an in vitro preparation method thereof

Abstract

The present invention relates to an organoid capable of simulating uterine spiral artery remodeling and an in vitro preparation method thereof, and belongs to the field of stem cell technology. The present invention provides an organoid capable of simulating uterine spiral artery remodeling, wherein the organoid capable of simulating uterine spiral artery remodeling is obtained by co-culturing trophoblast stem cells and early vascular cell spheres; the trophoblast stem cells and the early vascular cell spheres are both induced by pluripotent stem cells. The present invention constructs an organoid capable of simulating uterine spiral artery remodeling by co-culturing trophoblast stem cells and early vascular cell spheres. The organoid can simulate the process of uterine spiral artery remodeling in early pregnancy in vitro, and then be used to analyze the causes of adverse pregnancy caused by abnormalities in the process, as well as to induce a research model of pregnancy-related diseases in batches, thereby providing safety testing for the use of drugs for certain clinical pregnant patients.

Description

Technical Field

[0001] The invention relates to an organoid capable of simulating uterine spiral artery remodeling and an in vitro preparation method thereof, and belongs to the technical field of stem cells. Background Art

[0002] During human pregnancy, placental trophoblast cells migrate and invade the maternal uterine spiral arteries, then partially damage vascular-related cells, degrade the extracellular matrix, and dilate the vascular wall, remodeling it from a high-resistance, low-capacity vessel to a low-resistance, high-capacity vessel, ensuring normal fetal growth and development and fetal to maternal waste exchange. This process is called uterine spiral artery remodeling. Abnormal uterine spiral artery remodeling can lead to adverse pregnancy outcomes, including preeclampsia, preeclampsia, intrauterine growth restriction, premature birth, and miscarriage. However, due to technical and ethical restrictions and difficulties in sample acquisition, human samples of uterine spiral artery remodeling are difficult to obtain. Therefore, current research on uterine spiral artery remodeling focuses on animal models (mainly including rodents and non-human primates and other model animals). However, due to species differences between model animals and humans, it is far from enough to use only animal models to study human uterine spiral artery remodeling.

[0003] Organoids refer to tissue analogs with a certain spatial structure formed by three-dimensional (3D) culture of adult stem cells or pluripotent stem cells in vitro. Although organoids are not real human organs, they can simulate real organs in vivo to the greatest extent in terms of structure and function. At present, organoids derived from human stem cells are considered to be a more effective model for exploring the development / disease mechanism of human organs. For example, the establishment of human placental organoids provides an in vitro research model for placental development, and studies have found that human placental organoids are highly similar to human early placentas by performing single-cell sequencing analysis on human placenta, human placental organoid cells, and mammalian placenta. For example, the establishment of human vascular organoids provides an in vitro research model for vascular-related diseases, and studies have constructed a diabetes disease model based on human vascular organoids. If human stem cells can be used to produce organoids that can simulate uterine spiral artery remodeling, it will be very helpful for the study of human uterine spiral artery remodeling. However, at this stage, an organoid model of uterine spiral artery remodeling has not yet been established. Summary of the invention

[0004] To solve the above-mentioned defects, the present invention provides an organoid that can simulate uterine spiral artery remodeling, wherein the organoid that can simulate uterine spiral artery remodeling is obtained by co-culturing trophoblast stem cells (TSCs) and early vascular cell spheres (EVCS); the trophoblast stem cells and the early vascular cell spheres are both induced by pluripotent stem cells (PSCs).

[0005] In one embodiment of the present invention, the pluripotent stem cells include embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPSCs).

[0006] In one embodiment of the present invention, the cell composition of the organoid that can simulate uterine spiral artery remodeling includes cells involved in the process of uterine spiral artery remodeling in vivo and extracellular matrix; the cells involved in the process of uterine spiral artery remodeling in vivo include extravillous trophoblast cells, syncytial trophoblast cells, endothelial cells, smooth muscle cells and mural cells.

[0007] In one embodiment of the present invention, in the organoid that can simulate uterine spiral artery remodeling, cytokeratin 7 (KRT7) and human leukocyte antigen-G (HLA-G)-positive trophoblast cells are alternately arranged with platelet endothelial cell adhesion factor (CD31)-positive endothelial cells, and trophoblast stem cells spontaneously differentiate to produce extravillous trophoblast.

[0008] In one embodiment of the present invention, in the organoid that can simulate uterine spiral artery remodeling, platelet endothelial cell adhesion factor (CD31)-positive endothelial cells are partially lost and replaced by human leukocyte antigen-G (HLA-G)-positive extravillous trophoblast cells, and the extracellular matrix is ​​degraded.

[0009] In one embodiment of the present invention, the method for preparing an organoid that can simulate uterine spiral artery remodeling includes: inducing and culturing pluripotent stem cells to obtain trophoblast stem cells (TSCs); inducing and culturing pluripotent stem cells to obtain early vascular cell spheres (EVCS); on the day when the early vascular cell spheres are formed, co-culturing single cells of the trophoblast stem cells and the early vascular cell spheres using a uterine spiral artery remodeling model culture medium to obtain an organoid that can simulate uterine spiral artery remodeling.

[0010] In one embodiment of the present invention, the method for preparing an organoid capable of simulating uterine spiral artery remodeling comprises: inducing and culturing pluripotent stem cells to obtain trophoblast stem cells (Trophoblast stem cells, TSCs); inducing and culturing pluripotent stem cells to obtain early vascular cell spheres (EVCS); on the day of early vascular cell sphere formation, dispersing trophoblast stem cells into single cells and inoculating them together with early vascular cell spheres into a culture container to obtain a mixture of single cells and cell spheres; culturing the mixture of single cells and cell spheres using a uterine spiral artery remodeling model culture medium until the trophoblast cells and early vascular cell spheres form aggregates; mixing matrix gel with collagen to obtain a dual system mixture; mixing the dual system mixture and aggregates to obtain a dual system mixture containing aggregates; after the dual system mixture containing aggregates is condensed and formed, culturing the dual system mixture containing aggregates using a uterine spiral artery remodeling model culture medium until trophoblast cells invade the early vascular cell spheres to obtain organoids; after releasing the organoids from the dual system mixture, culturing the organoids using a uterine spiral artery remodeling model culture medium until vascular remodeling occurs in the vascular cell spheres to obtain organoids that can simulate uterine spiral artery remodeling.

[0011] In one embodiment of the present invention, the time for the trophoblast cells to form aggregates with early vascular cell spheres is 5 to 6 days; the time for the trophoblast cells to invade early vascular cell spheres is 5 to 6 days; and the time for vascular remodeling to occur in the organoids is 3 to 4 days.

[0012] In one embodiment of the present invention, in the mixture, the ratio of the number of trophoblast stem cell single cells to the number of early vascular cell spheres is 500-3000:1.

[0013] In one embodiment of the present invention, the mixing volume ratio of the matrix gel to the collagen is 0.5-2:1.

[0014] In one embodiment of the present invention, the collagen comprises type I collagen.

[0015] In one embodiment of the present invention, the components of the uterine spiral artery remodeling model culture medium include trophoblast organoid culture medium and SFM complete culture medium additionally supplemented with vascular endothelial growth factor (VEGF); the concentration of the vascular endothelial growth factor in the trophoblast organoid culture medium is 20ng / mL~100ng / mL; the volume ratio of the trophoblast organoid culture medium and the SFM complete culture medium is 0.5~1.5:1.

[0016] In one embodiment of the present invention, the components of the trophoblast organoid culture medium include a TGF-β type I receptor inhibitor, a WNT signaling pathway activator and hepatocyte growth factor (HGF).

[0017] In one embodiment of the present invention, the concentration of the TGF-β type I receptor inhibitor in the trophoblast organoid culture medium is 0.1 μM~10 μM; the concentration of the WNT signaling pathway activator in the trophoblast organoid culture medium is 1 μM~6 μM; the concentration of the hepatocyte growth factor in the trophoblast organoid culture medium is 30 ng / mL~100 ng / mL.

[0018] In one embodiment of the present invention, the components of the trophoblast organoid culture medium further include a matrix; the matrix includes N2B27 basal culture medium.

[0019] In one embodiment of the present invention, the method for preparing early vascular cell spheres comprises: inoculating embryonic stem cells into a culture container, and then culturing them in sequence using an enrichment medium, a mesoderm induction medium and a vascular induction medium to obtain early vascular cell spheres.

[0020] In one embodiment of the present invention, the culture time using enriched medium is 20 hours to 26 hours; the culture time using mesoderm induction medium is 3 days to 4 days; and the culture time using vascular induction medium is 2 days to 3 days.

[0021] In one embodiment of the present invention, the components of the enriched medium include serum replacement, L-glutamine substitute Glutamax, non-essential amino acids (NEAA), penicillin-streptomycin and ROCK-I / II inhibitor.

[0022] In one embodiment of the present invention, the concentration of the serum substitute in the enriched medium is 10% to 30% by volume; the concentration of the L-glutamine substitute Glutamax in the enriched medium is 0.5% to 2% by volume; the concentration of the non-essential amino acids in the enriched medium is 0.5% to 2% by volume; the concentration of penicillin-streptomycin in the enriched medium is 0.5% to 2% by volume; the concentration of the ROCK-I / II inhibitor in the enriched medium is 10 μM to 100 μM.

[0023] In one embodiment of the present invention, the components of the enriched medium further include a matrix; the matrix includes DMEM / F12 medium.

[0024] In one embodiment of the present invention, the components of the mesoderm induction medium include a WNT signaling pathway activator and bone morphogenetic protein 4 (BMP4).

[0025] In one embodiment of the present invention, the concentration of the WNT signaling pathway activator in the mesoderm induction medium is 6 μM to 20 μM; the concentration of the bone morphogenetic protein 4 in the mesoderm induction medium is 20 ng / mL to 50 ng / mL.

[0026] In one embodiment of the present invention, the components of the mesoderm induction medium further include a matrix; the matrix includes N2B27 basal medium.

[0027] In one embodiment of the present invention, the components of the vascular induction medium include an adenylate cyclase activator and vascular endothelial growth factor (VEGF).

[0028] In one embodiment of the present invention, the concentration of the adenylate cyclase activator in the vascular induction medium is 1 μM to 3 μM; the concentration of the vascular endothelial growth factor in the vascular induction medium is 50 ng / mL to 100 ng / mL.

[0029] In one embodiment of the present invention, the components of the vascular induction medium further include a matrix; the matrix includes N2B27 basal medium.

[0030] In one embodiment of the present invention, the preparation method of the trophoblast stem cells comprises: after inoculating induced pluripotent stem cells into a culture container, first using E8 complete medium and trophoblast stem cell medium additionally supplemented with bone morphogenetic protein 4 (BMP4) for induction culture in sequence, and then using trophoblast stem cell medium without bone morphogenetic protein 4 for maintenance culture to obtain trophoblast stem cells.

[0031] In one embodiment of the present invention, the induction culture time is 3 to 5 days; the maintenance culture time is 7 to 12 days.

[0032] In one embodiment of the present invention, the concentration of bone morphogenetic protein 4 in the trophoblast stem cell culture medium is 4 ng / mL to 30 ng / mL.

[0033] In one embodiment of the present invention, the components of the E8 complete medium include E8 basal medium (Essential 8 TM Basal Medium) and E8 supplement (Essential 8 TM Supplement (50X)); the E8 basal medium (Essential 8 TMBasal Medium) and E8 supplement (Essential 8 TM The volume ratio of Supplement (50X) is 30~80:1.

[0034] In one embodiment of the present invention, the components of the trophoblast stem cell culture medium include bovine serum albumin (BSA), insulin-transferrin-selenium-aminoethanol (Insulin-Transferrin-Selenium-X, ITS-X), 2-mercaptoethanol (2-mercaptoethanol), penicillin-streptomycin (penicillin-streptomycin), fetal bovine serum, antioxidants, TGF-β type I receptor inhibitor, valproic acid and ROCK-I / II inhibitor.

[0035] In one embodiment of the present invention, the concentration of bovine serum albumin in the trophoblast stem cell culture medium is 0.1% to 0.8% by volume; the concentration of insulin-transferrin-selenium-aminoethanol in the trophoblast stem cell culture medium is 0.5% to 2% by volume; the concentration of penicillin-streptomycin in the trophoblast stem cell culture medium is 0.3% to 0.8% by volume; the concentration of fetal bovine serum in the trophoblast stem cell culture medium is 0.05% to 0.5% by volume; The concentration of the 2-mercaptoethanol in the trophoblast stem cell culture medium is 0.05mM~0.2mM; the concentration of the antioxidant in the trophoblast stem cell culture medium is 0.5μg / mL~10μg / mL; the concentration of the TGF-βI type receptor inhibitor in the trophoblast stem cell culture medium is 0.5μM~10μM; the concentration of the valproic acid in the trophoblast stem cell culture medium is 0.5mM~1.5mM; the concentration of the ROCK-I / II inhibitor in the trophoblast stem cell culture medium is 2μM~10μM.

[0036] In one embodiment of the present invention, the components of the trophoblast stem cell culture medium further include a matrix; the matrix includes DMEM / F12 culture medium.

[0037] In one embodiment of the present invention, the TGF-β type I receptor inhibitor (TGF-β type I receptor is the type I receptor of the transforming growth factor β superfamily) includes SB431542 (selective activin receptor-like kinase 5 inhibitor), A83-01 (activin receptor-like kinase 4 / 5 / 7 inhibitor), galensertib (LY2157299, also known as Galunisertib) and / or Repsox (E-616452); the WNT signaling pathway activator includes a GSK-3α / β inhibitor (GSK-3 is an ATP-competitive glycogen synthase Kinase 3), WNT3a (secreted protein) and / or R-Spondins (secreted protein); the ROCK-I / II inhibitors (two isoforms of Rho-associated coiled-coil-forming protein kinases) include Y27632, Thiazovivin (N-benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide) and / or GSK429286A; the adenylate cyclase activator includes Forskolin and / or Colforsin; the antioxidant includes vitamin C and / or vitamin C derivatives.

[0038] In one embodiment of the present invention, the GSK-3α / β inhibitor includes CHIR99021, BIO (6-bromoindirubin-3'-oxime, a specific inhibitor of bisindole and glycogen synthase kinase 3), lithium chloride (LiCl), SB216763, SB415286 (2-(1H-reduced)-3-cyano-4-(dimethylamino)cholic acid), AZD1080 (2-hydroxy-3-[5-(morpholine-4-methyl)pyridin-2-yl]-1H-indole-5-carbonitrile) and / or AR-A014418; the R-Spondins include R-Spondin 1 and / or R-Spondin 2; the vitamin C derivative includes 2-Phospho-L-ascorbic acid trisodium salt), L-Ascorbic acid sodium salt, Magnesium Ascorbyl Phosphate, Sodium Ascorbyl Phosphate, Calcium Ascorbate, Ascorbyl Palmitate and / or L-Ascorbic Acid.

[0039] The present invention also provides a method for constructing the above-mentioned organoid that can simulate uterine spiral artery remodeling, the method comprising: inducing and culturing pluripotent stem cells to obtain trophoblast stem cells (TSCs); inducing and culturing pluripotent stem cells to obtain early vascular cell spheres (EVCS); on the day when the early vascular cell spheres are formed, co-culturing single cells of the trophoblast stem cells and the early vascular cell spheres using a uterine spiral artery remodeling model culture medium to obtain organoids that can simulate uterine spiral artery remodeling.

[0040] In one embodiment of the present invention, the method includes: inducing and culturing pluripotent stem cells to obtain trophoblast stem cells (TSCs); inducing and culturing pluripotent stem cells to obtain early vascular cell spheres (EVCS); on the day when the early vascular cell spheres are formed, dispersing the trophoblast stem cells into single cells, and inoculating them together with the early vascular cell spheres into a culture container to obtain a mixture of single cells and cell spheres; culturing the mixture of single cells and cell spheres using a uterine spiral artery remodeling model culture medium until the trophoblast cells and the early vascular cell spheres form aggregates; mixing matrix gel with collagen to obtain a dual system mixture; mixing the dual system mixture and aggregates to obtain a dual system mixture containing aggregates; after the dual system mixture containing aggregates is condensed and formed, culturing the dual system mixture containing aggregates using a uterine spiral artery remodeling model culture medium until the trophoblast cells invade the early vascular cell spheres to obtain organoids; after the organoids are released from the dual system mixture, culturing the organoids using a uterine spiral artery remodeling model culture medium until vascular remodeling occurs in the vascular cell spheres to obtain organoids that can simulate uterine spiral artery remodeling.

[0041] In one embodiment of the present invention, the time for the trophoblast cells to form aggregates with early vascular cell spheres is 5 to 6 days; the time for the trophoblast cells to invade early vascular cell spheres is 5 to 6 days; and the time for vascular remodeling to occur in the organoids is 3 to 4 days.

[0042] In one embodiment of the present invention, the pluripotent stem cells include embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPSCs).

[0043] In one embodiment of the present invention, in the mixture, the ratio of the number of trophoblast stem cell single cells to the number of early vascular cell spheres is 500-3000:1.

[0044] In one embodiment of the present invention, the mixing volume ratio of the matrix gel to the collagen is 0.5-2:1.

[0045] In one embodiment of the present invention, the collagen comprises type I collagen.

[0046] In one embodiment of the present invention, the components of the uterine spiral artery remodeling model culture medium include trophoblast organoid culture medium and SFM complete culture medium additionally supplemented with vascular endothelial growth factor (VEGF); the concentration of the vascular endothelial growth factor in the trophoblast organoid culture medium is 20ng / mL~100ng / mL; the volume ratio of the trophoblast organoid culture medium and the SFM complete culture medium is 0.5~1.5:1.

[0047] In one embodiment of the present invention, the components of the trophoblast organoid culture medium include a TGF-β type I receptor inhibitor, a WNT signaling pathway activator and hepatocyte growth factor (HGF).

[0048] In one embodiment of the present invention, the concentration of the TGF-β type I receptor inhibitor in the trophoblast organoid culture medium is 0.1 μM~10 μM; the concentration of the WNT signaling pathway activator in the trophoblast organoid culture medium is 1 μM~6 μM; the concentration of the hepatocyte growth factor in the trophoblast organoid culture medium is 30 ng / mL~100 ng / mL.

[0049] In one embodiment of the present invention, the components of the trophoblast organoid culture medium further include a matrix; the matrix includes N2B27 basal culture medium.

[0050] In one embodiment of the present invention, the method for preparing early vascular cell spheres comprises: inoculating embryonic stem cells into a culture container, and then culturing them in sequence using an enrichment medium, a mesoderm induction medium and a vascular induction medium to obtain early vascular cell spheres.

[0051] In one embodiment of the present invention, the culture time using enriched medium is 20 hours to 26 hours; the culture time using mesoderm induction medium is 3 days to 4 days; and the culture time using vascular induction medium is 2 days to 3 days.

[0052] In one embodiment of the present invention, the components of the enriched medium include serum replacement, L-glutamine substitute Glutamax, non-essential amino acids (NEAA), penicillin-streptomycin and ROCK-I / II inhibitor.

[0053] In one embodiment of the present invention, the concentration of the serum substitute in the enriched medium is 10% to 30% by volume; the concentration of the L-glutamine substitute Glutamax in the enriched medium is 0.5% to 2% by volume; the concentration of the non-essential amino acids in the enriched medium is 0.5% to 2% by volume; the concentration of penicillin-streptomycin in the enriched medium is 0.5% to 2% by volume; the concentration of the ROCK-I / II inhibitor in the enriched medium is 10 μM to 100 μM.

[0054] In one embodiment of the present invention, the components of the enriched medium further include a matrix; the matrix includes DMEM / F12 medium.

[0055] In one embodiment of the present invention, the components of the mesoderm induction medium include a WNT signaling pathway activator and bone morphogenetic protein 4 (BMP4).

[0056] In one embodiment of the present invention, the concentration of the WNT signaling pathway activator in the mesoderm induction medium is 6 μM to 20 μM; the concentration of the bone morphogenetic protein 4 in the mesoderm induction medium is 20 ng / mL to 50 ng / mL.

[0057] In one embodiment of the present invention, the components of the mesoderm induction medium further include a matrix; the matrix includes N2B27 basal medium.

[0058] In one embodiment of the present invention, the components of the vascular induction medium include an adenylate cyclase activator and vascular endothelial growth factor (VEGF).

[0059] In one embodiment of the present invention, the concentration of the adenylate cyclase activator in the vascular induction medium is 1 μM to 3 μM; the concentration of the vascular endothelial growth factor in the vascular induction medium is 50 ng / mL to 100 ng / mL.

[0060] In one embodiment of the present invention, the components of the vascular induction medium further include a matrix; the matrix includes N2B27 basal medium.

[0061] In one embodiment of the present invention, the preparation method of the trophoblast stem cells comprises: after inoculating induced pluripotent stem cells into a culture container, first using E8 complete medium and trophoblast stem cell medium additionally supplemented with bone morphogenetic protein 4 (BMP4) for induction culture in sequence, and then using trophoblast stem cell medium without bone morphogenetic protein 4 for maintenance culture to obtain trophoblast stem cells.

[0062] In one embodiment of the present invention, the induction culture time is 3 to 5 days; the maintenance culture time is 7 to 12 days.

[0063] In one embodiment of the present invention, the concentration of bone morphogenetic protein 4 in the trophoblast stem cell culture medium is 4 ng / mL to 30 ng / mL.

[0064] In one embodiment of the present invention, the components of the E8 complete medium include E8 basal medium (Essential 8 TM Basal Medium) and E8 supplement (Essential 8 TM Supplement (50X)); the E8 basal medium (Essential 8 TM Basal Medium) and E8 supplement (Essential 8 TM The volume ratio of Supplement (50X) is 30~80:1.

[0065] In one embodiment of the present invention, the components of the trophoblast stem cell culture medium include bovine serum albumin (BSA), insulin-transferrin-selenium-aminoethanol (Insulin-Transferrin-Selenium-X, ITS-X), 2-mercaptoethanol (2-mercaptoethanol), penicillin-streptomycin (penicillin-streptomycin), fetal bovine serum, antioxidants, TGF-β type I receptor inhibitor, valproic acid and ROCK-I / II inhibitor.

[0066] In one embodiment of the present invention, the concentration of bovine serum albumin in the trophoblast stem cell culture medium is 0.1% to 0.8% by volume; the concentration of insulin-transferrin-selenium-aminoethanol in the trophoblast stem cell culture medium is 0.5% to 2% by volume; the concentration of penicillin-streptomycin in the trophoblast stem cell culture medium is 0.3% to 0.8% by volume; the concentration of fetal bovine serum in the trophoblast stem cell culture medium is 0.05% to 0.5% by volume; The concentration of the 2-mercaptoethanol in the trophoblast stem cell culture medium is 0.05mM~0.2mM; the concentration of the antioxidant in the trophoblast stem cell culture medium is 0.5μg / mL~10μg / mL; the concentration of the TGF-βI type receptor inhibitor in the trophoblast stem cell culture medium is 0.5μM~10μM; the concentration of the valproic acid in the trophoblast stem cell culture medium is 0.5mM~1.5mM; the concentration of the ROCK-I / II inhibitor in the trophoblast stem cell culture medium is 2μM~10μM.

[0067] In one embodiment of the present invention, the components of the trophoblast stem cell culture medium further include a matrix; the matrix includes DMEM / F12 culture medium.

[0068] In one embodiment of the present invention, the TGF-β type I receptor inhibitor includes SB431542 (a selective inhibitor of activin receptor-like kinase 5), A83-01 (an inhibitor of activin receptor-like kinase 4 / 5 / 7), galensertib (LY2157299, also known as Galunisertib) and / or Repsox (E-616452); the WNT signaling pathway activator includes a GSK-3α / β inhibitor, WNT3a and / or R-Spondins; the ROCK-I / II inhibitor includes Y27632, Thiazovivin (N-benzyl-2-(pyrimidin-4-ylamino)thiazole-4-carboxamide) and / or GSK429286A; the adenylate cyclase activator includes Forskolin and / or Colforsin; the antioxidant includes vitamin C and / or vitamin C derivatives.

[0069] In one embodiment of the present invention, the GSK-3α / β inhibitor includes CHIR99021, BIO (6-bromoindirubin-3'-oxime, a specific inhibitor of bisindole and glycogen synthase kinase 3), lithium chloride (LiCl), SB216763, SB415286 (2-(1H-reduced)-3-cyano-4-(dimethylamino)cholic acid), AZD1080 (2-hydroxy-3-[5-(morpholine-4-methyl)pyridin-2-yl]-1H-indole-5-carbonitrile) and / or AR-A014418; the R-Spondins include R-Spondin 1 and / or R-Spondin 2; the vitamin C derivative includes 2-Phospho-L-ascorbic acid trisodium salt), L-Ascorbic acid sodium salt, Magnesium Ascorbyl Phosphate, Sodium Ascorbyl Phosphate, Calcium Ascorbate, Ascorbyl Palmitate and / or L-Ascorbic Acid.

[0070] The present invention also provides a uterine spiral artery remodeling model culture medium, the components of which include trophoblast organoid culture medium and SFM complete culture medium additionally supplemented with vascular endothelial growth factor (VEGF); the concentration of the vascular endothelial growth factor in the trophoblast organoid culture medium is 20 ng / mL~100 ng / mL; the volume ratio of the trophoblast organoid culture medium to the SFM complete culture medium is 0.5~1.5:1.

[0071] In one embodiment of the present invention, the components of the trophoblast organoid culture medium include a TGF-β type I receptor inhibitor, a WNT signaling pathway activator and hepatocyte growth factor (HGF).

[0072] In one embodiment of the present invention, the concentration of the TGF-β type I receptor inhibitor in the trophoblast organoid culture medium is 0.1 μM~10 μM; the concentration of the WNT signaling pathway activator in the trophoblast organoid culture medium is 1 μM~6 μM; the concentration of the hepatocyte growth factor in the trophoblast organoid culture medium is 30 ng / mL~100 ng / mL.

[0073] In one embodiment of the present invention, the components of the trophoblast organoid culture medium further include a matrix; the matrix includes N2B27 basal culture medium.

[0074] In one embodiment of the present invention, the TGF-β type I receptor inhibitors include SB431542 (a selective inhibitor of activin receptor-like kinase 5), A83-01 (an inhibitor of activin receptor-like kinase 4 / 5 / 7), galensertib (LY2157299, also known as Galunisertib) and / or Repsox (E-616452); the WNT signaling pathway activators include GSK-3α / β inhibitors, WNT3a and / or R-Spondins.

[0075] In one embodiment of the present invention, the GSK-3α / β inhibitor includes raglucib (CHIR99021), BIO (6-bromoindirubin-3'-oxime, a specific inhibitor of bisindole and glycogen synthase kinase 3), lithium chloride (LiCl, Lithium Chloride), SB216763, SB415286 (2-(1H-reduced)-3-cyano-4-(dimethylamino)cholic acid), AZD1080 (2-hydroxy-3-[5-(morpholine-4-methyl)pyridin-2-yl]-1H-indole-5-carbonitrile) and / or AR-A014418.

[0076] The present invention also provides a method for screening drugs for preventing and / or treating adverse pregnancy caused by abnormal uterine spiral artery remodeling process, the method comprising: first modeling the above-mentioned organoids that can simulate uterine spiral artery remodeling to obtain an in vitro disease model, and then using the in vitro disease model to screen drugs for preventing and / or treating adverse pregnancy caused by abnormal uterine spiral artery remodeling process.

[0077] In one embodiment of the present invention, the abnormal uterine spiral artery remodeling process leads to adverse pregnancy including pre-eclampsia, eclampsia, intrauterine growth restriction, placental abruption, premature birth and / or miscarriage.

[0078] The present invention also provides a method for evaluating the safety of a drug for pregnant patients, the method comprising: administering the drug to the above-mentioned organoid that can simulate uterine spiral artery remodeling, and evaluating the safety of the drug for pregnant patients by observing changes in indicators of the organoid that can simulate uterine spiral artery remodeling.

[0079] In one embodiment of the present invention, the safety of the drug to pregnant patients includes whether it is embryonic lethal, whether it is embryonic teratogenic and / or whether it will cause adverse pregnancy.

[0080] In one embodiment of the present invention, the adverse pregnancy includes pre-eclampsia, eclampsia, intrauterine growth restriction, placental abruption, premature birth and / or miscarriage.

[0081] The present invention also provides the use of the above-mentioned organoid that can simulate uterine spiral artery remodeling, or the above-mentioned method, or the above-mentioned uterine spiral artery remodeling model culture medium in screening drugs for preventing and / or treating adverse pregnancies caused by abnormal uterine spiral artery remodeling process or evaluating the safety of drugs for pregnant patients.

[0082] In one embodiment of the present invention, the abnormal uterine spiral artery remodeling process leads to adverse pregnancy including pre-eclampsia, eclampsia, intrauterine growth restriction, placental abruption, premature birth and / or miscarriage.

[0083] In one embodiment of the present invention, the safety of the drug to pregnant patients includes whether it is embryonic lethal, whether it is embryonic teratogenic and / or whether it will cause adverse pregnancy.

[0084] In one embodiment of the present invention, the adverse pregnancy includes pre-eclampsia, eclampsia, intrauterine growth restriction, placental abruption, premature birth and / or miscarriage.

[0085] The technical solution of the present invention has the following advantages:

[0086] The present invention provides an organoid capable of simulating uterine spiral artery remodeling, wherein the organoid capable of simulating uterine spiral artery remodeling is obtained by co-culturing trophoblast stem cells and early vascular cell spheres; the trophoblast stem cells and the early vascular cell spheres are both induced by pluripotent stem cells. The present invention constructs an organoid capable of simulating uterine spiral artery remodeling by co-culturing trophoblast stem cells and early vascular cell spheres. Studies have shown that the cellular composition of this organoid includes a variety of cells and extracellular matrix involved in the process of uterine spiral artery remodeling in vivo, including extravillous trophoblasts (EVT), syncytiotrophoblasts (STB), endothelial cells (EC), smooth muscle cells (SMC) and wall cells; and studies have shown that this organoid simulates the biological events and key structures of early uterine spiral artery remodeling, such as trophoblast invasion, partial loss of endothelial cells, phenotypic transformation of smooth muscle cells and partial degradation of extracellular matrix, and can better reproduce the key characteristics of trophoblasts and blood vessels in the uterine artery remodeling stage; at the same time, studies have shown that this organoid can also reveal the molecular characteristics of trophoblast- and vascular-related cells and their interactions. Therefore, this organoid can be used as an in vitro model to simulate uterine spiral artery remodeling, to analyze the causes of adverse pregnancies caused by abnormalities in this process, and to be used to induce a research model for pregnancy-related diseases in batches, thereby providing safety testing for the use of drugs in certain clinical pregnant patients. In addition, the preparation method of this organoid uses trophoblast stem cells obtained by transformation of pluripotent stem cells to replace conventional human placental tissue, which is easy to repeat and has easy access to cell sources, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 : Schematic diagram of the process for preparing trophoblast stem cells derived from human pluripotent stem cells.

[0088] Figure 2 : Schematic diagram of a microscope showing trophoblast stem cells derived from human pluripotent stem cells.

[0089] Figure 3 : Immunofluorescence images of trophoblast stem cells expressing trophoblast marker proteins KRT7 (keratin 7), GATA3 (GATA binding protein 3), TP63 (tumor protein P63), and TEAD4 (TEA domain transcription factor 4).

[0090] Figure 4 :Schematic diagram of the process for preparing early vascular cell spheres (EVCs) derived from human pluripotent stem cells.

[0091] Figure 5 : Schematic diagram of microscopy of early vascular cell spheres (EVCs) derived from human pluripotent stem cells.

[0092] Figure 6 : Immunofluorescence images of early vascular glomeruli expressing vascular-associated proteins CD31 (platelet endothelial cell adhesion factor), PDGFR-β (platelet-derived growth factor receptor-β), and α-SMA (α-smooth muscle actin).

[0093] Figure 7 : Immunofluorescence images of the expression of vascular-related proteins CD31 (platelet endothelial cell adhesion factor), HLA-G (human leukocyte antigen-G), and KRT7 (keratin 7) in the uterine spiral artery remodeling model at different days of formation. Figure 7 In the figure, A: day 5; B: day 13.

[0094] Figure 8 : Immunofluorescence images of vascular-related proteins CD31 (platelet endothelial cell adhesion factor), COLIV (type IV collagen), and PDGFR-β (platelet-derived growth factor receptor-β) in the uterine spiral artery remodeling model (UASRM) and the monocultured vascular organoid model (moVO) without co-culture with trophoblast stem cells.

[0095] Fig. 9 :Bioinformatic analysis of the uterine spiral artery remodeling model. Fig. 9 In the figure, A: UMAP clustering diagram of single-cell sequencing data; B: relevant clustering basis (EC is endothelial cell, TC is trophoblast cell, and MC is mural cell).

[0096] Fig.10 : UAMP diagram of the similarity between the constructed uterine spiral artery remodeling model and the in vivo uterine spiral artery.

[0097] Fig.11 :The uterine spiral artery remodeling model (USARM) can predict the cell communication between trophoblast cells and vascular cells. Fig.11 Figure 3, A: Schematic diagram of the interaction between TC ligands and EC receptors in USARM; B: Heat map of the expression levels of EC receptor-encoding genes in USARM on days 1 and 5; C: Upregulated target genes regulated by TC ligand-EC receptor interaction in USARM on day 5 compared with day 1; D: Downregulated target genes regulated by TC ligand-EC receptor interaction in USARM on day 5 compared with day 1.

[0098] Fig.12:The uterine spiral artery remodeling model (USARM) can predict the cell communication between trophoblast cells and vascular cells. Fig.12 Figure 3, A: Schematic diagram of the interaction between TC ligands and MC receptors in USARM; B: Heat map of the expression levels of MC receptor-encoding genes in USARM on days 1 and 5; C: Upregulated target genes regulated by TC ligand-MC receptor interaction in USARM on day 5 compared with day 1; D: Downregulated target genes regulated by TC ligand-MC receptor interaction in USARM on day 5 compared with day 1.

[0099] Fig.13 : The co-culture process of TSC single cell + EVCS single cell co-culture model in TSV medium or TOMV medium and the bright field morphology under the microscope after 5 days of co-culture.

[0100] Fig.14 : The co-culture process of TSC sphere + EVCS sphere co-culture model in TSV medium or TOMV medium and the bright field morphology under the microscope after 5 days of co-culture.

[0101] Fig.15 : Immunofluorescence results of TSC sphere + EVCS sphere co-culture model after 5 days of co-culture in TSV medium or TOMV medium.

[0102] Fig.16 : The co-culture process of TSC single cell + EVC sphere co-culture model in TSV medium or TOMV medium and the bright field morphology under the microscope after 5 days of co-culture.

[0103] Fig.17 : Immunofluorescence results of TSC single cell + EVC sphere co-culture model in TOMV medium for 5 days.

[0104] Fig.18 : Immunofluorescence results of TSC single cell + EVC sphere co-culture model in TOMV medium for 13 days.

[0105] Fig.19 : The co-culture process of TSC single cell + EVCS sphere co-culture model in the uterine spiral artery remodeling model culture medium and the bright field morphology under the microscope at each time point. DETAILED DESCRIPTION

[0106] 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.

[0107] 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.

[0108] The Matrigel matrix gel involved in the following examples was purchased from Corning, model 354230, Collagen type I collagen was purchased from Corning, model 354236, DMEM / F12 culture medium was purchased from Thermo Fisher Scientific, model 11320033, and E8 basal medium (Essential 8TM Basal Medium) was purchased from Thermo Fisher Scientific, model A1517001 (containing E8 supplement (Essential 8TM Supplement (50X))), EDTA was purchased from Thermo Fisher, model 15575020, GSK-3α / β inhibitor raglucilib (CHIR99021) was purchased from Tocris, model 4423, TGF-β type I receptor inhibitor A83-01 (inhibitor of activin receptor-like kinase 4 / 5 / 7) was purchased from Selleck, model S7692, TGF-β type I receptor inhibitor SB431542 (selective activin receptor-like kinase 5 inhibitor) was purchased from Selleck, model S1067; ROCK-I / II inhibitor Y27632 was purchased from APExBIO, model B1421; adenylate cyclase activator Forskolin was purchased from APExBIO, model A3008; vascular endothelial growth factor (VEGF) was purchased from StemImmune, model HVG-VF5-0100; hepatocyte growth factor (HGF) was purchased from Sino Biological biologica), model 10463-HNAS, bone morphogenetic protein 4 (BMP4) was purchased from StemImmune, model HST-B4-0100, SFM complete medium was purchased from Thermo Fisher, model 10639011, bovine serum albumin (BSA) was purchased from Sigma, model A1933, insulin-transferrin-selenium-X (ITS-X) was purchased from Thermo Fisher, model 51500056, penicillin-streptomycin was purchased from Thermo Fisher, model 15140122, fetal bovine serum was purchased from Thermo Fisher, model 10270106, 2-Phospho-L-ascorbic acid trisodium salt was purchased from Sigma-Aldrich, model 49752, serum replacement (Serum L-glutamine replacement (Glutamax) was purchased from Thermo Fisher Scientific, model 10828028, L-glutamine replacement (Glutamax) was purchased from Thermo Fisher Scientific, model 35050061, and non-essential amino acids (NEAA) were purchased from Thermo Fisher Scientific, model 11140050;The Matrigel-coated culture dishes, Matrigel-coated six-well plates and Matrigel-coated twelve-well plates involved in the following examples were purchased from NEST, with models 705001, 714011 and 712001, respectively; the low-adsorption six-well plates involved in the following examples were purchased from Corning, model 3471, and the low-adsorption 96-well plates were purchased from BIOFIL, model TCP-002-096. ;

[0109] Example 1: A method for preparing in vitro organoids that can simulate uterine spiral artery remodeling

[0110] This embodiment provides an organoid that can simulate uterine spiral artery remodeling, wherein the organoid that can simulate uterine spiral artery remodeling is obtained by co-culturing trophoblast stem cells (TSCs) and early vascular cell spheres (EVCS); the trophoblast stem cells are induced by induced pluripotent stem cells (iPSCs); the early vascular cell spheres are induced by embryonic stem cells (ESCs). The preparation process of the organoid that can simulate uterine spiral artery remodeling is as follows:

[0111] 1. Establishment of trophoblast stem cells derived from human iPSCs

[0112] Step S1: Human iPSCs were prepared using an iPSC reprogramming kit (CytoTune™-iPS 2.0 Sendai Reprogramming Kit, purchased from Thermo Fisher Scientific, Catalog No. A16517); human iPSCs were cultured at 1×10 5 Pieces / cm 2 The cells were seeded at a density of 1.50 μg / mL in a Matrigel-coated dish supplemented with 2 mL of E8 complete medium and cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2.

[0113] Step S2: After culturing for 24 hours (this time is marked as day 0), the culture medium is discarded, 2 mL of trophoblast stem cell culture medium containing 10 ng / mL bone morphogenetic protein 4 (BMP4) is added to the culture dish, and the cells are continued to be cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2;

[0114] Step S3: When the cell density reaches 90% (here, culture is required for 72 hours, which is the third day), add 0.5 mM ethylenediaminetetraacetic acid (EDTA) to the culture dish, incubate in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2 for 5 minutes to digest the cells, then use DMEM / F12 medium to wash the digested cells in the culture dish, and then use DMEM / F12 medium to blow off the digested cells in the culture dish to obtain a cell suspension; transfer the cell suspension to a 15 mL centrifuge The tube was centrifuged at 800 rpm for 3 minutes. After the centrifugation, the supernatant was discarded and the cell pellet was taken; all the cell pellets were resuspended in 1 mL of trophoblast stem cell culture medium without bone morphogenetic protein 4 (BMP4) to obtain a cell resuspension; the cell resuspension was divided into three parts, one of which was inoculated into a culture dish, and the trophoblast stem cell culture medium without bone morphogenetic protein 4 (BMP4) was added to the culture dish to make up the culture system to 2 mL, and then cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2;

[0115] Step S4: When the cell density reaches 90% (here, culture is required for 48 hours, which is the 5th day), first add 0.5mM ethylenediaminetetraacetic acid (EDTA) to the culture dish, incubate in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2 for 5 minutes to digest the cells, then use DMEM / F12 medium to wash the digested cells in the culture dish, and then use DMEM / F12 medium to blow off the digested cells in the culture dish to obtain a cell suspension; transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 800rpm for 3 minutes, and after the centrifugation, discard the supernatant and take the cell pellet; use 1mL trophoblast stem cell culture medium to resuspend all the cell pellets to obtain a cell resuspension; the cell resuspension is diluted with 1×10 5 The cells were inoculated into Matrigel-coated six-well plates at a volume of 100 cells / well, and trophoblast stem cell culture medium without bone morphogenetic protein 4 (BMP4) was added to the six-well plates to make up the culture system of each well to 2 mL, and then cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2.

[0116] Step S5: On the 7th to 8th day of culture, the emergence of trophoblast stem cell-like cell clones can be observed; on the 10th to 12th day of culture, the trophoblast stem cell-like cell clones can be observed to grow larger and occupy most of the positions in the culture wells of the six-well plate; on the 12th day of culture, the cell clones in the six-well plate are first washed with PBS buffer, and then trophoblast stem cell culture medium is added to the six-well plate at an addition amount of 1 mL / well, and then under a stereomicroscope, cells are scraped along the edge of the cell clones with a cell needle to collect these trophoblast stem cell-like cell clones (pay attention to remove non-trophoblast stem cell-like cell clones during the collection process) to obtain trophoblast stem cells derived from human iPSCs (see the specific process). Figure 1 );

[0117] Step S6: After the trophoblast stem cells are successfully constructed, the trophoblast stem cells are subcultured every 3 days at a subculture ratio of 1:3. The subculture process is the same as step S4, except that the digestion reagent TrypLE™ Express Enzyme is used for digestion and subculture instead of EDTA;

[0118] The E8 complete medium is composed of the E8 basal medium (Essential 8 TM BasalMedium) and E8 supplements (Essential 8 TM Supplement (50X)) mixed;

[0119] The trophoblast stem cell culture medium was DMEM / F12 medium containing 0.3% (v / v) bovine serum albumin (BSA), 1% (v / v) insulin-transferrin-selenium-X (ITS-X), 0.1 mM 2-mercaptoethanol, 0.5% (v / v) penicillin-streptomycin, 0.2% (v / v) fetal bovine serum, 1.5 μg / mL 2-Phospho-L-ascorbic acid trisodium salt, 1 μM TGF-β type I receptor inhibitor SB431542 (selective activin receptor-like kinase 5 inhibitor), 0.8 mM valproic acid, and 5 μM ROCK-I / II inhibitor Y27632.

[0120] 2. Establishment of Early Vascular Spheroids from Human ESCs

[0121] Step S1: Human embryonic stem cells (ESCs, purchased from WiCell) were cultured at 1×105 Pieces / cm 2 The inoculum volume was re-inoculated into a culture dish supplemented with 1 mL of E8 complete medium and cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2;

[0122] Step S2: When the cell density reaches 80% and the clones are dense (culture for 3 days), the culture medium is discarded, 0.5 mL of Accutase digestion solution is added to the culture dish, and the cells are incubated in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2 to digest the cells. During the digestion process, the cell state is continuously observed under a microscope. When the cells are digested into a single cell state (incubation for 5 minutes), the cells are blown to detach from the bottom of the culture dish to obtain a cell suspension; the cells in the cell suspension are aspirated from the liquid and transferred to a 15 mL centrifuge tube, and centrifuged at 300 g for 5 minutes. After the centrifugation is completed, the supernatant is discarded and the cell pellet is taken; all the cell pellets are resuspended in 3 mL of enrichment medium (aggregation medium) to obtain a cell resuspension; the cell resuspension is concentrated to 3 × 10 5 Inoculate the cells / well into a low-adsorption six-well plate, add enriched medium to the six-well plate to make up the culture system in each well to 3 mL, and then culture in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2;

[0123] Step S3: When the human embryonic stem cells are cultured until they form cell aggregates of uniform size with smooth boundaries (here, culture is required for 24 hours, which is recorded as day 0), the medium in the low-adsorption six-well plate is replaced with mesoderm induction medium (mesoderm induction medium dosage 3 mL / well), and the culture is continued in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2;

[0124] Step S4: After culturing for 3 days (i.e., day 3), the culture medium in the low-adsorption six-well plate was replaced with vascular induction medium (3 mL / well of vascular induction medium), and the cells were continued to be cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2;

[0125] Step S5: After 5 days of culture (i.e., day 5), human ESCs-derived early vascular cell spheres were formed, and a culture system containing early vascular cell spheres was obtained (see the detailed process). Figure 4 );

[0126] The enriched medium was DMEM / F12 medium containing 20% ​​(v / v) serum replacement, 1% (v / v) L-glutamine substitute Glutamax, 1% (v / v) non-essential amino acids (NEAA), 1% (v / v) penicillin-streptomycin, and 50 μM ROCK-I / II inhibitor Y27632.

[0127] Mesoderm induction medium was N2B27 basal medium containing 12 µM GSK-3α / β inhibitor raglucillib (CHIR99021) and 30 ng / mL bone morphogenetic protein 4 (BMP4);

[0128] The vascular induction medium was N2B27 basal medium containing 2 µM adenylate cyclase activator Forskolin and 100 ng / mL vascular endothelial growth factor (VEGF);

[0129] N2B27 basal medium is a mixed medium containing 1% (v / v) N2 supplement, 2% (v / v) B27 supplement, 1% (v / v) L-glutamine substitute 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 medium in a volume ratio of 1:1.

[0130] 3. Establishment of uterine spiral artery remodeling model

[0131] Step S1: On the day of early vascular cell sphere formation, the early vascular cell sphere in the culture system was aspirated from the liquid and transferred to a 15 mL centrifuge tube, and centrifuged at 800 rpm for 3 minutes. After the centrifugation, the supernatant was discarded to obtain a cell pellet of the early vascular cell sphere;

[0132] Take a six-well plate in which trophoblast stem cells derived from human pluripotent stem cells are cultured. When the cell density reaches 90% and the clones are dense, first aspirate the culture medium, then wash the cells in the six-well plate with PBS buffer, then add 0.5mL / well Accutase digestion solution to the six-well plate, and incubate in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2 to digest the cells. During the digestion process, the cell state is continuously observed under a microscope. When the digestion degree reaches 90% (incubation for 8 minutes), first use DMEM / F12 culture medium to wash the digested cells in the six-well plate, and then use DMEM / F12 culture medium to blow off the digested cells in the six-well plate to obtain a cell suspension; transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 800rpm for 3 minutes, discard the supernatant after centrifugation, and take the cell pellet; resuspend all cell pellets with uterine spiral artery remodeling model culture medium to a cell concentration of 5×10 4 / mL, and obtain a cell resuspension of trophoblast stem cells;

[0133] The cell pellet of early vascular cell spheres was added at a volume of 50 cells / mL to a cell concentration of 5×10 4 / mL of trophoblast stem cell cell resuspension to obtain a cell mixed suspension; 2mL of the cell mixed suspension was transferred to a 15mL centrifuge tube, centrifuged at 1300rpm for 4 minutes, and after the centrifugation, the supernatant was discarded and the mixed precipitate was taken; 10mL of uterine spiral artery remodeling model culture medium was used to resuspend all the mixed precipitates to obtain a mixed resuspension; the mixed resuspension was inoculated into a low-adsorption 96-well plate at an inoculation volume of 0.2mL / well, and cultured in a 37℃ cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2 (the day of inoculation was recorded as day 0);

[0134] Step S2: After 5 days of culture (i.e., day 5), cell aggregates of trophoblast cells and early vascular cell spheres are formed to obtain a culture system containing cell aggregates; the cell aggregates in the culture system are aspirated from the liquid and transferred to a 15 mL centrifuge tube, and centrifuged at 1300 rpm for 4 minutes. After the centrifugation, the supernatant is discarded to obtain a cell precipitate of the cell aggregates; Matrigel matrix glue and collagen are mixed. Type I collagen was mixed in a volume ratio of 1:1 to obtain a dual system mixture of matrix gel and collagen; 60 cell aggregates were mixed with 0.5 mL of the dual system mixture to obtain a dual system mixture containing cell aggregates; the dual system mixture containing cell aggregates was placed in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2; after the dual system mixture containing cell aggregates was solidified, it was placed in a twelve-well plate with 1 mL of uterine spiral artery remodeling model culture medium added to each well (60 cell aggregates correspond to one well), and continued to be cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2;

[0135] Step S3: After another 5 days of culture (i.e., day 10), trophoblast cells invaded early vascular cell spheres to obtain a dual-system mixture containing organoids; excess glue at the edge of the dual-system mixture containing organoids was cut off to release the organoids; the organoids were added at a rate of 1 / well to a low-adsorption 96-well plate with 0.2 mL of uterine spiral artery remodeling model culture medium added to each well, and cultured in a 37°C cell culture incubator with 5% (v / v) CO2 and 5% (v / v) O2;

[0136] Step S4: After another 3 days of culture (i.e., day 13), organoids that can simulate uterine spiral artery remodeling are successfully constructed;

[0137] The uterine spiral artery remodeling model culture medium was prepared by mixing trophoblast organoid culture medium supplemented with 50 ng / mL vascular endothelial growth factor (VEGF) and SFM complete culture medium at a volume ratio of 1:1;

[0138] The trophoblast organoid culture medium was N2B27 basal medium containing 0.5 μM TGF-β type I receptor inhibitor A83-01 (an inhibitor of activin receptor-like kinase 4 / 5 / 7), 1.5 μM GSK-3α / β inhibitor raglucirib (CHIR99021), and 50 ng / mL hepatocyte growth factor (HGF).

[0139] Experimental Example 1: Verification of organoid performance

[0140] This experimental example provides a validation experiment of organoid performance. The experimental process is as follows:

[0141] 1. Experimental methods

[0142] Organoids were constructed using the method of Example 1.

[0143] After the construction of trophoblast stem cells derived from human pluripotent stem cells was completed, the trophoblast stem cells in the six-well plate were observed under a microscope. The observation results are shown in Figure 2 After the construction of early vascular cell spheres derived from human pluripotent stem cells was completed, the early vascular cell spheres in the six-well plate were observed under a microscope. The observation results are shown in Figure 5 .

[0144] Human pluripotent stem cell-derived trophoblast stem cells, human pluripotent stem cell-derived early vascular cell spheres, organoids on day 5, and organoids on day 13 were used as test samples for immunofluorescence staining. The staining results are shown in Figure 3 , Figure 6~Figure 7 ; Among them, the immunofluorescence staining method is as follows:

[0145] Take the sample to be tested, immerse it in 4% (w / v, g / 100mL) paraformaldehyde aqueous solution at room temperature (25℃) for 3h for fixation; after fixation, rinse it with PBS buffer for 3 times, each rinse for 5 minutes; after rinsing, incubate it with PBS buffer containing 0.2% (v / v) Triton X-100 at room temperature (25℃) for 10min to break the membrane; after breaking the membrane, incubate it with PBS buffer containing 10% (v / v) donkey serum at room temperature (25℃) for 1 hour for blocking; after blocking, incubate it with PBS buffer containing 5% (v / v) donkey serum and 0.1% (v / v) Triton The primary antibody was diluted with PBS buffer containing X-100, and the organoids were incubated with the diluted primary antibody (incubated in a 4°C refrigerator for 16 hours); after the incubation, the organoids were rinsed with PBS buffer for 3 times, each time for 10 minutes; after the rinsing, the secondary antibody was diluted with PBS buffer containing 5% (v / v) donkey serum, and the organoids were incubated with the diluted secondary antibody (incubated at room temperature for 1 hour); after the incubation, the organoids were rinsed with PBS buffer for 3 times, each time for 10 minutes; after the rinsing, the organoids on the 13th day were observed and photographed using a Nikon upright microscope or a Zeiss laser confocal microscope (Zeiss700b) to obtain the immunofluorescence staining results;

[0146] During incubation, the primary antibody used for KRT7 protein was Rabbit anti-KRT7, purchased from Proteintech, and used after dilution of 500 times; the secondary antibody used for KRT7 protein was Secondary ab-Donkey anti-rabbit 488, purchased from Thermo Fisher, and used after dilution of 1000 times; the primary antibody used for GATA3 protein was Mouse anti-GATA3, purchased from Proteintech, and used after dilution of 500 times; the secondary antibody used for GATA3 protein was Secondary ab-Donkey anti-mouse 555, purchased from Thermo Fisher, and used after dilution of 1000 times; the primary antibody used for TP63 protein was Rabbit anti-TP63, purchased from Proteintech, and used after dilution of 500 times; the secondary antibody used for TP63 protein was Secondary ab-Donkey anti-rabbit 488, purchased from Thermo Fisher, and used after dilution of 1000 times; the primary antibody used for TEAD4 protein was Mouse anti-GATA3, purchased from Proteintech, and used after dilution of 500 times; Anti-TEAD4 was purchased from Abcam and diluted 500 times before use. The secondary antibody used for TEAD4 protein was Secondary ab-Donkey anti-mouse 555, purchased from Thermo Fisher, and diluted 1000 times before use. The primary antibody used for CD31 protein was Sheep anti-CD31, purchased from R&D System, and diluted 1000 times before use. The secondary antibody used for CD31 protein was Secondary ab-Donkey anti-sheep 488, purchased from Thermo Fisher, and diluted 1000 times before use. The primary antibody used for PDGFR-β protein was Rabbit anti-PDGFR-β, purchased from CST, and diluted 1000 times before use. The secondary antibody used for PDGFR-β protein was Secondary ab-Donkey anti-rabbit 647, purchased from Thermo Fisher, and diluted 1000 times before use. The primary antibody used for α-SMA protein was Mouse anti-CD31, purchased from R&D System, and diluted 1000 times before use. Anti-ɑ-SMA was purchased from Sigma and used after dilution 500 times. The secondary antibody used for α-SMA protein was Secondary ab-Donkey anti-mouse 555, purchased from Thermo Fisher, and used after dilution 1000 times.

[0147] The monocultured vascular organoid (moVO) model without co-culture with trophoblast stem cells (moVO was prepared by culturing early vascular cell spheres in uterine spiral artery remodeling model culture medium for 13 days) was used as a control to compare the immunofluorescence staining results of the uterine spiral artery remodeling model (UASRM) (i.e., organoids on day 13). The comparison results are shown in Figure 8.

[0148] The uterine spiral artery remodeling model (UASRM) (i.e., organoids on day 13) was library-built and sequenced using single-cell transcriptomics technology. The raw data were processed using CeleScope v1.5.2 (Singleron Biotechnologies) software with default parameters, and the Seurat (v4.3.0.1) R package was used to perform filtering, normalization, dimensionality reduction, and clustering analysis. The analysis results are shown in Fig. 9 . Fig. 9 A total of 19 cell clusters were defined, and differential gene expression analysis was used to divide the 19 cell clusters into trophoblast cells (TC), endothelial cells (EC), and mural cells (MC).

[0149] Through bioinformatics analysis, a UAMP map similar to the uterine spiral artery remodeling model and the in vivo uterine spiral artery was constructed. The construction results are shown in Fig.10 The cell-cell interaction analysis was performed using the multiichenetr (version 1.0.3) package to compare the interaction between EC and TC. The results are shown in Fig.11 The cell-cell interaction analysis was performed using the multiichenetr (version 1.0.3) package to compare the interaction between MC and TC. The results are shown in Fig.12 .

[0150] 2. Experimental results

[0151] Depend on Figure 2~Figure 3 It can be seen that human pluripotent stem cells can induce trophoblast stem cells that can be stably propagated and have stable trophoblast cell characteristics during continuous culture.

[0152] Depend on Figure 5~Figure 6 It can be seen that human pluripotent stem cells can efficiently induce the formation of cell aggregates within 24 hours, and further form early glomeruli with multicellular structures. Early glomeruli express vascular cell-related marker proteins CD31, PDGFR-β, and α-SMA, indicating that they already have vascular endothelial cells, perivascular cells, and vascular smooth muscle cells.

[0153] Depend on Figure 7 It can be seen that KRT7 and HLA-G positive trophoblast cells surrounding CD31 positive early glomeruli can be observed on the 5th day of co-culture with early glomeruli, and KRT7 and HLA-G positive trophoblast cells and CD31 positive endothelial cells can be seen interlaced on the 13th day of culture. It can be seen that trophoblast stem cells can further spontaneously differentiate and produce a large number of extravillous trophoblasts during co-culture with early glomeruli for subsequent migration and invasion of blood vessels.

[0154] Depend on Figure 8 It can be seen that compared with vascular organoids (moVO) without KRT7 and HLA-G positive trophoblast cells, the vascular network of co-cultured uterine spiral artery organoids was significantly damaged and remodeled. It can be seen that the vascular glomeruli undergo vascular remodeling with the invasion of the extravillous trophoblast, and some CD31 positive endothelial cells are lost and replaced by HLA-G positive extravillous trophoblast cells. In addition, the extracellular matrix of the co-cultured uterine spiral artery organoids showed a certain degree of degradation. This result also shows that the blood vessels have undergone significant remodeling, which is very close to the uterine spiral artery remodeling process in vivo.

[0155] Depend on Fig. 9 It can be seen that the co-cultured uterine spiral artery organoids contain trophoblast cells, endothelial cells and parietal cells. It can be seen that this type of organ contains the main cell types for uterine spiral artery remodeling.

[0156] Depend on Fig.10 It can be seen that the co-cultured uterine spiral artery organoids contain cell types similar to those of the uterine spiral arteries in vivo. It can be seen that this type of organ is close to the in vivo in terms of cell types.

[0157] Depend on Figure 11~Figure 12 It can be seen that the co-cultured uterine spiral artery organoids can explore the cell communication between trophoblast cells and endothelial cells and between trophoblast cells and parietal cells, as well as their downstream regulatory genes. It can be seen that this type of organ model can be used to further study the relevant mechanisms in the process of uterine spiral artery remodeling.

[0158] In the prior art, there have been studies that have attempted to co-culture vascular endothelial cells with trophoblast cells to explore the interaction between the two. For example, immortalized and primary tissue-derived trophoblast cells (including BeWo cells, JEG-3 cells, and HTR8 cells, etc.) and endothelial cells (including HUVEC cells and HEMEC cells, etc.) are co-cultured with the aid of a specific device to simulate the interaction between the trophoblast and endothelial cells. However, this co-culture model has obvious defects. First, the starting cells used are difficult to obtain. Second, the starting cells used are mostly derived from immortalized or cancerous cell lines, which are quite different from the cells of in vivo tissues in terms of physiological characteristics. Third, it only contains endothelial cells, no other vascular-related cells, and lacks a variety of cells involved in the physiological process of uterine spiral artery remodeling. Fourth, its culture mode is mostly 2D culture, and the physiological structure of the model obtained by 2D culture is quite different from the physiological structure of in vivo tissues. Fifth, it cannot reproduce the key events of extravillous trophoblast (EVT) invasion and vascular remodeling in vivo. The above five defects make this co-culture model unable to effectively simulate the process of uterine spiral artery remodeling. In addition, this co-culture model also needs to rely on a specific culture device, which is costly.

[0159] And by Figure 2 to Figure 12The results show that Example 1 uses trophoblast stem cells and early vascular cell spheres to co-culture to construct an organoid that can simulate uterine spiral artery remodeling. Studies have shown that the cell composition of the organoid includes a variety of cells and extracellular matrix that participate in the process of uterine spiral artery remodeling in vivo, among which the various cells involved in the process of uterine spiral artery remodeling in vivo include extravillous trophoblast cells (EVT), syncytiotrophoblasts (STB), endothelial cells (EC), smooth muscle cells (SMC) and wall cells, etc.; and the study shows that the organoid simulates the biological events and key structures of early uterine spiral artery remodeling, such as trophoblast invasion, partial loss of endothelial cells, phenotypic transformation of smooth muscle cells and partial degradation of extracellular matrix, etc., and can better reproduce the key characteristics of trophoblast and blood vessels in the uterine artery remodeling stage; at the same time, the study shows that the organoid can also reveal the molecular characteristics of trophoblast and vascular-related cells and their interactions. Therefore, this organoid can be used as an in vitro model to simulate uterine spiral artery remodeling, to analyze the causes of adverse pregnancies caused by abnormalities in this process, and to be used to induce a research model for pregnancy-related diseases in batches, thereby providing safety testing for the use of drugs in certain clinical pregnant patients. In addition, the preparation method of this organoid uses trophoblast stem cells obtained by transformation of pluripotent stem cells to replace conventional human placental tissue, which is easy to repeat and has easy access to cell sources, and has great application prospects.

[0160] Experimental Example 2: Exploration of Organoid Construction Conditions

[0161] This experimental example provides an exploratory experiment on the conditions for organoid construction. The experimental process is as follows:

[0162] 1. Experimental process

[0163] 1.1. Exploration of co-cultivation model

[0164] Explore two co-culture modes: co-culture mode of single cells of trophoblast stem cells and single cells of early vascular spheres (i.e., TSC single cell + EVCS single cell co-culture mode), and co-culture mode of spheroids of trophoblast stem cells and spheroids of early vascular spheres (i.e., TSC sphere + EVCS sphere co-culture mode), and mark the start of co-culture as day 0;

[0165] Among them, the TSC single cell + EVCS single cell co-cultivation mode is: on the basis of Example 1, the early vascular cell spheres derived from human pluripotent stem cells are digested into single cells and then co-inoculated with single cells of trophoblast stem cells in a 1:1 ratio into the culture medium for co-culture, and during the co-culture process, the uterine spiral artery remodeling model culture medium is replaced with TSV culture medium (trophoblast stem cell culture medium + 50ng / mL VEGF) or TOMV culture medium (trophoblast organoid culture medium + 50ng / mL VEGF) respectively;

[0166] The TSC sphere + EVCS sphere co-cultivation mode is: based on Example 1, early vascular cell spheres derived from human pluripotent stem cells and trophoblast stem cell spheres are co-inoculated into the culture medium in a 1:1 ratio for co-culture, and during the co-culture process, the uterine spiral artery remodeling model culture medium is replaced with TSV culture medium (trophoblast stem cell culture medium + 50ng / mL VEGF) or TOMV culture medium (trophoblast organoid culture medium + 50ng / mL VEGF), respectively.

[0167] After the organoids were constructed, the organoids in the 96-well plate were observed under a microscope. The results are shown in Figure 13~Figure 14 . The organoids constructed in the TSC sphere + EVCS sphere co-culture mode were subjected to immunofluorescence staining according to the method of Experimental Example 1. The results of immunofluorescence staining are shown in Fig.15 .

[0168] 1.2. Exploration of culture medium

[0169] Three culture media were explored: TSV (trophoblast stem cell culture medium + 50 ng / mL VEGF), TOMV (trophoblast organoid culture medium + 50 ng / mL VEGF) and uterine spiral artery remodeling model culture medium. In the experiment, based on Experimental Example 1, TSV culture medium, TOMV culture medium and uterine spiral artery remodeling model culture medium were used in the co-culture process respectively. Among them, the uterine spiral artery remodeling model culture medium was obtained by mixing trophoblast organoid culture medium additionally supplemented with 50 ng / mL vascular endothelial growth factor (VEGF) and SFM complete culture medium in a volume ratio of 1:1.

[0170] After the organoids were constructed, the organoids in the 96-well plate were observed under a microscope. The results are shown in Fig.16 and Fig.19 The constructed organoids were subjected to immunofluorescence staining according to the method of Experimental Example 1. The results of immunofluorescence staining are shown in Figure 17-18 .

[0171] 2. Experimental results

[0172] 2.1. Exploration results of co-cultivation model

[0173] Depend on Fig.13 It can be seen that in the TSC single cell + EVCS single cell co-culture model, whether in the trophoblast stem cell culture medium (TSV) containing VEGF or the trophoblast organoid culture medium (TOMV) containing VEGF, the cells were still dispersed and unable to aggregate on the 5th day.

[0174] Depend on Fig.14 It can be seen that in the TSC sphere + EVC sphere co-culture model, the two original spheres contacted and aggregated, which seemed more promising on day 5. However, Fig.15 Immunostaining results showed that although CD31+ cells formed a vascular-like network, KRT7+ / HLA-G+ TC cells rarely appeared in the cell aggregates in TSV or TOMV culture medium.

[0175] comprehensive Figure 13~Figure 15 The results show that compared with the TSC single cell + EVCS sphere co-culture model in Example 1, these two models are not ideal.

[0176] 2.2 Exploration of culture medium

[0177] Depend on Fig.16 It can be seen that on day 5, it can be observed that the co-culture of trophoblast stem cells and early glomeruli in TSV presents a dispersed sphere pattern. On the contrary, in TOMV, trophoblast stem cells effectively encapsulate early glomeruli to form a large cell aggregate. At this time, Fig.17 Immunostaining results showed that although KRT7+ / HLA-G+ cells were surrounded by CD31+ cells, the number of CD31+ endothelial cells was still small. This is opposite to the in vivo situation, which may be due to insufficient vascular network formation. Fig.18 It can be seen that when the co-culture time was extended to day 13, CD31+ cells and KRT7+ / HLA-G+ cells were dispersed instead of forming a staggered arrangement.

[0178] Since SFM medium has shown its efficacy in promoting angiogenesis, it was added to TOMV medium at the beginning of co-culture, that is, co-culture was performed using the uterine spiral artery remodeling model medium. Fig.19 and Figure 7 It can be seen that at day 5, KRT7+ / HLA-G+ TCs were already embedded in CD31+ EVCs, and at day 13, the spatial distribution of different cell lineages changed significantly, with CD31+ cells interlaced between KRT7+ / HLA-G+ cells, presenting a long rope-like structure. This distribution is consistent with the phenomenon of EVT invading arterial vessels observed at the maternal-fetal interface in early pregnancy in monkeys and humans.

[0179] comprehensive Figure 16~Figure 19 as well as Figure 7 The results showed that a uterine spiral artery remodeling model (USARM) was successfully developed by co-culturing TSC single cells + EVCS spheres in a mixture of TOMV and SFM culture medium (i.e., uterine spiral artery remodeling model culture medium).

[0180] 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. An organoid that can simulate uterine spiral artery remodeling, characterized in that: The organoids that can simulate uterine spiral artery remodeling are obtained by co-culturing single cells of trophoblast stem cells and early vascular cell spheres using a uterine spiral artery remodeling model culture medium; the trophoblast stem cells and the early vascular cell spheres are both induced by pluripotent stem cells; the components of the uterine spiral artery remodeling model culture medium include SFM complete culture medium and trophoblast organoid culture medium supplemented with vascular endothelial growth factor.

2. The organoid capable of simulating uterine spiral artery remodeling according to claim 1, characterized in that: The pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells; The cell composition of the organoid that can simulate uterine spiral artery remodeling includes cells involved in the process of uterine spiral artery remodeling in vivo and extracellular matrix; the cells involved in the process of uterine spiral artery remodeling in vivo include extravillous trophoblast cells, syncytial trophoblast cells, endothelial cells, smooth muscle cells and mural cells; In the organoid that can simulate uterine spiral artery remodeling, cytokeratin 7 and human leukocyte antigen-G positive trophoblast cells are staggered with platelet endothelial cell adhesion factor positive endothelial cells, and trophoblast stem cells spontaneously differentiate to produce extravillous trophoblast; In the organoids that can simulate uterine spiral artery remodeling, part of the endothelial cells positive for platelet endothelial cell adhesion factor are lost and replaced by extravillous trophoblast cells positive for human leukocyte antigen-G, and the extracellular matrix is ​​degraded.

3. The organoid capable of simulating uterine spiral artery remodeling according to claim 1 or 2, characterized in that: The method for preparing an organoid capable of simulating uterine spiral artery remodeling comprises: inducing and culturing pluripotent stem cells to obtain trophoblast stem cells; inducing and culturing pluripotent stem cells to obtain early vascular cell spheres; on the day when the early vascular cell spheres are formed, co-culturing single cells of the trophoblast stem cells and the early vascular cell spheres using a uterine spiral artery remodeling model culture medium to obtain an organoid capable of simulating uterine spiral artery remodeling.

4. The organoid capable of simulating uterine spiral artery remodeling according to claim 3, characterized in that: The preparation method of the organoid capable of simulating uterine spiral artery remodeling comprises: inducing and culturing pluripotent stem cells to obtain trophoblast stem cells; inducing and culturing pluripotent stem cells to obtain early vascular cell spheres; on the day when the early vascular cell spheres are formed, dispersing the trophoblast stem cells into single cells, and inoculating them together with the early vascular cell spheres into a culture container to obtain a mixture of single cells and cell spheres; culturing the mixture of single cells and cell spheres using a uterine spiral artery remodeling model culture medium until the trophoblast cells and the early vascular cell spheres form aggregates; mixing matrix gel with collagen to obtain a dual-system mixture; mixing the dual-system mixture and aggregates to obtain a dual-system mixture containing aggregates; after the dual-system mixture containing aggregates is condensed and formed, culturing the dual-system mixture containing aggregates using a uterine spiral artery remodeling model culture medium until the trophoblast cells invade the early vascular cell spheres to obtain organoids; after releasing the organoids from the dual-system mixture, culturing the organoids using a uterine spiral artery remodeling model culture medium until vascular remodeling occurs in the vascular cell spheres to obtain organoids capable of simulating uterine spiral artery remodeling.

5. The organoid capable of simulating uterine spiral artery remodeling according to claim 4, characterized in that: In the mixture, the ratio of the number of trophoblast stem cell single cells to the number of early vascular cell spheres is 500-3000:1; The concentration of the vascular endothelial growth factor in the trophoblast organoid culture medium is 20 ng / mL to 100 ng / mL; the volume ratio of the trophoblast organoid culture medium to the SFM complete culture medium is 0.5 to 1.5:

1.

6. A method for constructing an organoid capable of simulating uterine spiral artery remodeling according to any one of claims 1 to 5, characterized in that: The method comprises: inducing and culturing pluripotent stem cells to obtain trophoblast stem cells; inducing and culturing pluripotent stem cells to obtain early vascular cell spheres; on the day when the early vascular cell spheres are formed, co-culturing single cells of the trophoblast stem cells and the early vascular cell spheres using a uterine spiral artery remodeling model culture medium to obtain organoids that can simulate uterine spiral artery remodeling.

7. A method for screening drugs for preventing and / or treating adverse pregnancy caused by abnormal uterine spiral artery remodeling, characterized in that: The method comprises: first modeling the organoid capable of simulating uterine spiral artery remodeling as described in any one of claims 1 to 5 to obtain an in vitro disease model, and then using the in vitro disease model to screen drugs for preventing and / or treating adverse pregnancy caused by abnormal uterine spiral artery remodeling.

8. A method for evaluating the safety of a drug for pregnant patients, characterized in that: The method comprises: administering a drug to the organoid capable of simulating uterine spiral artery remodeling as described in any one of claims 1 to 5, and evaluating the safety of the drug on pregnant patients by observing changes in indicators of the organoid capable of simulating uterine spiral artery remodeling.

9. Use of the organoid capable of simulating uterine spiral artery remodeling as described in any one of claims 1 to 5 or the method as described in claim 6 in screening drugs for preventing and / or treating adverse pregnancy caused by abnormal uterine spiral artery remodeling or evaluating the safety of drugs for pregnant patients.

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