Method for producing hypoblast stem cells, medium for inducing hypoblast stem cells, hypoblast stem cells, method for producing yolk sac organoid, and yolk sac organoid

Abstract

This method for producing hypoblast stem cells includes a step of culturing cells isolated from the yolk sac of a mammal in a culture medium containing an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor. This medium for inducing hypoblast stem cells contains an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor.

Description

Method for producing hypoblast stem cells, medium for inducing hypoblast stem cells, method for producing hypoblast stem cells, yolk sac organoids, and yolk sac organoids

[0001] The present invention relates to a method for producing hypoblast stem cells, a medium for inducing hypoblast stem cells, hypoblast stem cells, a method for producing yolk sac organoids, and yolk sac organoids. This application claims priority based on Japanese Patent Application No. 2024-086184, filed on May 28, 2024, the contents of which are incorporated herein by reference.

[0002] Human preimplantation embryos are composed of three types of cells: epiblasts, which contribute to the future fetus; trophectoderm, which contributes to the placenta; and hypoblasts, which contribute to the yolk sac. Embryonic stem cells (ES cells) have been established from the epiblast. Trophoblast stem cells (TS cells) have been established from the trophectoderm (Patent Document 1). These cells are being increasingly utilized in a wide range of fields, from basic research to applied research.

[0003] Meanwhile, in mice, primitive endodermal stem cells (PrES cells) have been reported as cells capable of differentiating into yolk sac cells (Non-Patent Document 1). Also, it has been reported that hypoblast-like cells have been induced from human pluripotent stem cells (Non-Patent Document 2).

[0004] Patent No. 6400832

[0005] Yasuhide Ohinata et al., Establishment of mouse stem cells that can recapitulate the developmental potential of primitive endoderm. Science. 2022 Feb 4;375(6580):574-578.Takumi Okubo et al., Hypoblast from human pluripotent stem cells regulates epiblast development. Nature. 2024 Feb;626(7998):357-366.

[0006] In addition to ES cells and TS cells, if hypoblast stem cells with the ability to differentiate into yolk sac cells can be established, they are expected to be useful in basic research into early mammalian development, testing the effects of drugs on fetuses in early pregnancy, and assisted reproductive technology.

[0007] In Non-Patent Document 1, mouse PrES cells are induced by culturing mouse blastocysts in the presence of fibroblast growth factor 4 (FGF4), platelet-derived growth factor (PDGF), and a GSK3β inhibitor. However, the conditions described in Non-Patent Document 1 do not allow for the induction of human hypoblast stem cells.

[0008] In Non-Patent Document 2, hypoblast-like cells are induced by culturing human pluripotent stem cells in the presence of FGF4, bone morphogenetic protein 4 (BMP4), interleukin 6 (IL-6), PDGF, a Wnt inhibitor, and a TGFβ / SMAD inhibitor. However, the hypoblast-like cells described in Non-Patent Document 2 cannot be maintained in culture for a long period of time (e.g., six months or more).

[0009] The objective of the present disclosure is to provide a method for producing hypoblast stem cells that is applicable to humans and capable of producing hypoblast stem cells that can be maintained by long-term culture, a culture medium for inducing hypoblast stem cells that can be used in the production method, hypoblast stem cells produced by the production method, a method for producing yolk sac organoids using the hypoblast stem cells, and yolk sac organoids containing the hypoblast stem cells.

[0010] The present disclosure includes the following aspects: (1) A method for producing hypoblast stem cells, comprising a step of culturing cells isolated from mammalian yolk sac in a medium containing an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor. (2) The method for producing hypoblast stem cells according to (1), wherein the medium further contains a TGFβ / SMAD inhibitor. (3) The method for producing hypoblast stem cells according to (1) or (2), wherein the medium further contains at least one inhibitor selected from the group consisting of an HDAC inhibitor and a p38 inhibitor. (4) A medium for inducing hypoblast stem cells, comprising an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor. (5) A medium for inducing hypoblast stem cells according to (4), further containing a TGFβ / SMAD inhibitor. (6) A medium for inducing hypoblast stem cells according to (4) or (5), further containing at least one inhibitor selected from the group consisting of an HDAC inhibitor and a p38 inhibitor. (7) A culture medium for inducing hypoblast stem cells according to any one of (4) to (6), which is used to induce hypoblast stem cells from cells isolated from a mammalian yolk sac. (8) Hypoblast stem cells having the following characteristics (a) to (e): (a) cells induced from cells isolated from a mammalian yolk sac; (b) a lower level of α-fetoprotein compared to cells isolated from the mammalian yolk sac; (c) the ability to differentiate into yolk sac organoids containing cells expressing at least one selected from the group consisting of α-fetoprotein and albumin; (d) the ability to proliferate; and (e) the ability to form cell masses. (9) The hypoblast stem cells according to (8), which can be maintained in culture for six months or more by subculturing in the culture medium for inducing hypoblast stem cells according to any one of (4) to (7). (10) The hypoblast stem cells according to (8) or (9), which are cells induced from cells isolated from a human yolk sac. (11) A method for producing a yolk sac organoid, comprising a step of culturing the hypoblast stem cells according to (8) or (9) in a hydrogel. (12) A yolk sac organoid comprising the hypoblast stem cells according to any one of (8) to (10).

[0011] According to the present disclosure, there are provided a method for producing hypoblast stem cells that is applicable to humans and capable of producing hypoblast stem cells that can be maintained by long-term culture, a culture medium for inducing hypoblast stem cells that can be used in the production method, hypoblast stem cells produced by the production method, a method for producing yolk sac organoids using the hypoblast stem cells, and yolk sac organoids containing the hypoblast stem cells.

[0012] 1 shows a phase-contrast microscope image of hypoblast stem cells obtained in Experimental Example 1. 2 shows a phase-contrast microscope image (left) and an immunofluorescence stained image (right) of yolk sac organoids obtained in Experimental Example 2. 3 shows the expression level of alpha-fetoprotein (AFP) obtained from RNA-seq analysis of hypoblast stem cells (HS cells) and yolk sac organoids (HS organoids) in Experimental Example 3. 4 shows the expression level of albumin obtained from RNA-seq analysis of hypoblast stem cells (HS cells) and yolk sac organoids (HS organoids) in Experimental Example 3. 5 shows the results of UMAP analysis of gene expression data obtained by RNA-seq analysis of hypoblast stem cells (HS cells) and yolk sac organoids (HS organoids) in Experimental Example 3. The cell classification was obtained from a previous study (Issac Goh et al., Science. 2023 Aug 18;381(6659):eadd7564. doi: 10.1126 / science.add7564.). In Experimental Example 6, the results of UMAP analysis of gene expression data obtained by single-cell RNA-seq analysis of hypoblast stem cells (HS cells) and yolk sac organoids (HS organoids) are shown. The cell classification was obtained from a previous study (Issac Goh et al., Science. 2023 Aug 18;381(6659):eadd7564. doi: 10.1126 / science.add7564.). In Experimental Example 6, the results of comparing gene expression between HS cells and HS organoids based on gene expression data obtained by single-cell RNA-seq analysis are shown.

[0013] [Definitions] The term "comprise" means that it may contain components other than the target component. The term "consist of" means that it does not contain components other than the target component. The term "consist essentially of" means that it does not contain components other than the target component in a manner that exerts a special function (such as a manner that completely loses the effect of the invention). In this specification, when "comprise" is used, it includes an embodiment that "consists of" and an embodiment that "consists essentially of."

[0014] A numerical range expressed using "to" means a range that includes the numerical values ​​written before and after "to" as the upper and lower limits. When multiple upper and lower limit values ​​are written for a specific parameter, any upper and lower limit values ​​can be combined to form a suitable numerical range.

[0015] Proteins, cells, and organoids may be isolated. "Isolated" refers to a state separated from other components. An "isolated" component may be separated from its natural state. "Isolated" may be substantially free of other components. "Substantially free of other components" means that the content of other components contained in the isolated component is negligible. The content of other components contained in the isolated component may be, for example, 10% by mass or less, 5% by mass or less, 4% by mass or less, 3% by mass or less, 2% by mass or less, 1% by mass or less, 0.5% by mass or less, or 0.1% by mass or less. The proteins, cells, and organoids described herein may be isolated proteins, isolated cells, and isolated organoids, respectively.

[0016] [Method for Producing Hypoblast Stem Cells] A first aspect of the present disclosure is a method for producing hypoblast stem cells, which includes a step of culturing cells isolated from a mammalian yolk sac in a medium containing an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor (hereinafter also referred to as the "culturing step").

[0017] (Hypoblast stem cells) "Hypoblast stem cells" (hereinafter also referred to as "HS cells") refer to cells having the following characteristics (a) to (e). HS cells may also be cells having the following characteristics (b) to (e): (a) being cells derived from cells isolated from a mammalian yolk sac; (b) expressing a lower amount of α-fetoprotein compared to cells isolated from the mammalian yolk sac; (c) having the ability to differentiate into yolk sac organoids containing cells expressing at least one selected from the group consisting of α-fetoprotein and albumin; (d) having the ability to proliferate; and (e) forming cell clusters.

[0018] Regarding (a): HS cells are cells induced from cells isolated from the yolk sac (hereinafter also referred to as "yolk sac cells"). HS cells can be induced by culturing cells isolated from the yolk sac in a medium containing epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor. Specific examples of the culture method will be described later.

[0019] Regarding (b): HS cells express a lower amount of alpha-fetoprotein (AFP) than yolk sac cells. The yolk sac cells used for comparison are the yolk sac cells from which HS cells are derived. AFP is a yolk sac marker that is highly expressed in the yolk sac. When HS cells are induced from yolk sac cells, the expression level of AFP decreases. The decrease in the expression level of AFP in HS cells is thought to indicate that the HS cells have transitioned to a more undifferentiated state than yolk sac cells.

[0020] The expression level of AFP in yolk sac cells and HS cells can be measured by known methods. Examples of such methods include RNA-seq analysis, quantitative reverse transcription PCR (RT-qPCR), Northern blotting analysis, fluorescent immunostaining, Western blotting analysis, and ELISA. AFP sequence information that can be used to measure the expression level of AFP can be obtained from known sequence databases such as GeneBank. For example, the sequence information of human AFP registered in GeneBank under accession numbers NM_001134.3 and NM_001354717.2 can be used. Commercially available anti-AFP antibodies can be used to measure the expression level of AFP.

[0021] Regarding (c): HS cells have the ability to differentiate into yolk sac organoids. "Yolk sac organoids" are organoids containing cells expressing at least one selected from the group consisting of AFP and albumin (hereinafter also referred to as "AFP / Alb-expressing cells"). AFP / Alb-expressing cells may be cells expressing only AFP, cells expressing only albumin, or cells expressing both AFP and albumin. It is preferable that yolk sac organoids are mainly composed of AFP / Alb-expressing cells. For example, it is preferable that 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the total cells of the yolk sac organoids are composed of AFP / Alb-expressing cells.

[0022] The expression of AFP and albumin in yolk sac organoids can be measured by known methods as described above. AFP sequence information that can be used to measure the expression levels of AFP and albumin can be obtained from known sequence databases such as GeneBank. For example, the sequence information of human albumin can be used that registered in GeneBank under accession number NM_000477.7. Commercially available anti-albumin antibodies can be used to measure the expression level of albumin.

[0023] The expression level of AFP and albumin in yolk sac organoid is preferably the same as that of the yolk sac from which HS cells are derived.For example, when the expression level of AFP in yolk sac is 1, the relative expression level of AFP in yolk sac organoid is preferably 0.1 or more, 0.2 or more, 0.5 or more, 0.6 or more, or 0.7 or more.When the expression level of AFP in yolk sac is 1, the upper limit of the relative expression level of AFP in yolk sac organoid is, for example, 10.0 or less, 9.0 or less, 8.0 or less, 7.0 or less, 6.0 or less, 5.0 or less, 4.0 or less, 3.0 or less, or 2.0 or less.For example, when the expression level of albumin in yolk sac is 1, the relative expression level of albumin in yolk sac organoid is preferably 0.1 or more, 0.2 or more, 0.5 or more, 0.6 or more, or 0.7 or more. When the expression level of albumin in the yolk sac is set to 1, the upper limit of the relative expression level of albumin in the yolk sac organoid is, for example, 10.0 or less, 9.0 or less, 8.0 or less, 7.0 or less, 6.0 or less, 5.0 or less, 4.0 or less, 3.0 or less, or 2.0 or less.

[0024] The yolk sac organoids are preferably composed of cells with gene expression patterns similar to those of the yolk sac. For example, when UMAP analysis of gene expression data obtained by RNA-seq analysis or single RNA-seq analysis is performed, the cells are preferably located in the same cluster as the yolk sac.

[0025] Whether or not a cell has the ability to differentiate into a yolk sac organoid can be confirmed by culturing the cells to be evaluated in a hydrogel. If the cells to be evaluated have the ability to differentiate into an organoid, organoids are formed by culturing them in a hydrogel for about 5 to 20 days. If the formed organoids contain AFP / Alb cells, the organoids can be determined to be yolk sac organoids. If the formed organoids are yolk sac organoids, the cells can be determined to have the ability to differentiate into a yolk sac organoid. Details of the method for forming yolk sac organoids from HS cells will be described later.

[0026] Regarding (d): HS cells have the ability to proliferate. Examples of media in which HS cells can proliferate include media containing epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor. Specific examples of such media include the media for inducing hypoblast stem cells described below.

[0027] The conditions for culturing HS cells can be the same as those typically used for culturing animal cells. The culture temperature is 32 to 40°C (preferably 35 to 38°C, typically 37°C). ２ The concentration can be 2 to 5% (preferably 5%). HS cells can be cultured as an adhesion culture using a culture substrate such as laminin. The term "culture substrate" refers to a substance that serves as a scaffold for cells to adhere to during cell culture. Examples of culture substrates include extracellular matrices.

[0028] HS cells were cultured in a medium containing epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor in the presence of a culture substrate at 37°C, 5% CO ２ These cells can also be said to be capable of proliferation when cultured under these conditions. As HS cells proliferate, the size of the cell clusters described in (e) below increases. Therefore, proliferation of HS cells can be confirmed by observing the increase in the size of the cell clusters.

[0029] Regarding (e): HS cells form cell clusters. More specifically, HS cells form spherical cell clusters as shown in FIG. 1 by culturing them in a medium containing an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor. The size of the cell cluster is, for example, about 20 to 200 μm in minimum diameter. The maximum diameter of the cell cluster is, for example, about 20 to 200 μm. When cell clusters formed by HS cells are observed at 20x magnification using a phase-contrast microscope, the boundaries between the cells cannot usually be clearly observed. This is thought to be because the cells are densely packed into cell clusters with high cell density.

[0030] HS cells were cultured in a medium containing epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor in the presence of a culture substrate at 37°C, 5% CO ２It can also be said that these cells are capable of forming cell masses when cultured under certain conditions.

[0031] Other Characteristics: In addition to the above characteristics (a) to (e), the HS cells preferably have the following characteristic (f): (f) Maintenance culture for 6 months or more is possible by subculturing in a medium containing an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor.

[0032] Examples of media containing epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor include the media for inducing hypoblast stem cells described below. The culture conditions for subculture can be those typically used for culturing animal cells. The culture temperature can be the same as the culture conditions listed in (d) above.

[0033] Passaging may be performed, for example, every 3 to 10 days, or when the culture vessel becomes confluent. HS cells can be passaged by methods commonly used for passage of animal cells. For example, a proteolytic enzyme (e.g., trypsin, trypsin substitute, etc.) is added, and the cells are detached from the culture vessel by pipetting or the like. At this time, cell clumps may be dispersed into free cells. The detached cells are recovered by centrifugation or the like. The recovered cells can be passaged by seeding them in new medium.

[0034] HS cells were cultured in a medium containing epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor in the presence of a culture substrate at 37°C, 5% CO ２ Preferably, the cells can be maintained in culture for six months or more by subculturing under the conditions.

[0035] "Maintenance culture" refers to culturing cells while maintaining the properties of the cells. During maintenance culture, HS cells maintain the above characteristics (a) to (e).

[0036] The size of HS cells varies depending on the organism from which the yolk sac cells are derived. For example, HS cells induced from human yolk sac cells have a cell diameter of approximately 10 to 50 μm.

[0037] (Culturing Step) In the culturing step, cells isolated from a mammalian yolk sac (yolk sac cells) are cultured in a medium containing an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor.

[0038] <Yolk sac cells> Mammals from which yolk sac cells are derived are mammals that have a yolk sac during pregnancy. Examples of mammals include humans and non-human mammals. Examples of non-human mammals include, but are not limited to, non-human primates (monkeys, chimpanzees, gorillas, marmosets, etc.), rodents (mice, hamsters, rats, guinea pigs, etc.), rabbits, dogs, cats, cows, pigs, goats, sheep, and horses. Mammals from which yolk sac cells are derived are preferably primates, and more preferably humans.

[0039] The yolk sac cells are cells isolated from the yolk sac. The yolk sac cells may be a cultured line of cells isolated from the yolk sac. The yolk sac may be extracted from a mammal in the early stages of pregnancy, for example. When the mammal is a human, the yolk sac may be extracted by surgical abortion.

[0040] Known methods can be used to isolate cells from the yolk sac. Yolk sac cells can be isolated, for example, by separating the cells through mechanical and / or enzymatic treatment of the yolk sac. Examples of mechanical treatment include cutting with scissors or a scalpel. Examples of enzymatic treatment include treatment with proteolytic enzymes (trypsin, etc.) or proteases with extracellular matrix degrading activity (e.g., dispase, collagenase, etc.). After mechanical and / or enzymatic treatment of the endometrial epithelial tissue, yolk sac cells can be recovered using centrifugation, a strainer, a filter, or the like. The recovered yolk sac cells may be washed with a medium (e.g., TS medium), a buffer solution (phosphate-buffered saline (PBS), etc.), or the like, as appropriate.

[0041] <Culture Medium> Examples of the culture medium include a basal medium supplemented with an epidermal growth factor, a GSK3β inhibitor, a ROCK inhibitor, and the like.

[0042] <Basal medium> Examples of basal media include media commonly used for culturing animal cells. Examples of basal media include Doulbecco's modified Eagle's Medium (DMEM) medium, DMEM / F12 medium, Advanced DMEM / F12 medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media of two or more of these. A preferred basal medium is, for example, DMEM / F12.

[0043] The basal medium may contain serum (such as fetal bovine serum (FBS)) and / or serum substitutes as needed. Examples of serum substitutes include albumin, transferrin, sodium selenite, ITS-X (Invitrogen), Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, and 3'-thiolglycerol. The basal medium may contain components such as lipids, amino acids, L-glutamine, Glutamax, non-essential amino acids, vitamins, growth factors, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts as needed. These components may be used in appropriate combinations.

[0044] The basal medium may be, for example, a medium obtained by adding bovine serum albumin (BSA), ITS-X, a serum substitute (KSR, etc.), and an antibiotic (penicillin, streptomycin, etc.) to the above-mentioned basal medium (e.g., DMEM / F12). A specific example of the basal medium is the TS basal medium used in the Examples described below.

[0045] <Epidermal Growth Factor> Epidermal growth factor (EGF) binds to EGF receptors (EGFR) present on the cell surface to induce EGF signal transduction and act as a mitogen. The organism from which EGF is derived is not particularly limited, but is preferably the same as the organism from which yolk sac cells are derived. When human yolk sac cells are used, human EGF is preferred. Human EGF may be a recombinant EGF produced by non-human cells. Commercially available EGF can be used. The concentration of EGF in the medium is not particularly limited, but can be, for example, 5 to 200 ng / mL. The concentration of EGF in the medium is preferably 10 to 150 ng / mL, more preferably 10 to 100 ng / mL, even more preferably 10 to 80 ng / mL, and particularly preferably 10 to 60 ng / mL.

[0046] GSK3β Inhibitors GSK (Glycogen Synthase Kinase) 3β inhibitors are substances that inhibit the function of GSK3β, for example, its kinase activity (for example, its ability to phosphorylate β-catenin). Examples of GSK3β inhibitors include 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile, Kenpaullone, 1-Azakenpaullone, CHIR98014, AR-A014418, and CT9902. 1, CT20026, SB216763, AR-A014418, lithium, SB415286, TDZD-8, BIO, BIO-acetoxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridocarbazole-cyclopentadienylruthenium complex, TDZD-8 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, OTDZT, alpha-4-dibromoacetophenone, AR-AO 144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione; TWS1 19 pyrrolopyrimidine compound, L803 Examples of suitable GSK3β inhibitors include small molecule inhibitors such as H-KEAPPAPPQSpP-NH2 or its myristoylated form; 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone, SB216763, and SB415286. GSK3β inhibitors may be antisense nucleic acids, siRNA, dominant-negative mutants, and expression vectors thereof against GSK3β. Commercially available 6-[[2-[[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]nicotinonitrile includes CHIR99021. GSK3β inhibitors may be used singly or in combination of two or more. It is preferable to use CHIR99021 as the GSK3β inhibitor.The concentration of the GSK3β inhibitor in the medium is not particularly limited, but can be, for example, 0.1 to 50 μM, preferably 0.2 to 30 μM, more preferably 0.5 to 20 μM, and even more preferably 1 to 10 μM.

[0047] <ROCK Inhibitors> ROCK (Rho associated coiled-coil containing protein kinase: Rho-associated kinase) inhibitors are substances that inhibit the function of Rho-associated kinase. Examples of ROCK inhibitors include trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 1-(5-isoquinolinylsulfonyl)homopiperazine, and salts thereof. Examples of ROCK inhibitors also include small molecule inhibitors such as Fasudil / HA1077, H-1152, and Wf-536, as well as derivatives thereof. The ROCK inhibitor may be an antisense nucleic acid, siRNA, a dominant-negative mutant of ROCK, or an expression vector thereof. Commercially available products of trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide or a salt thereof include Y27632 ((R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide.2HCl.H ２ O) and the like. One type of ROCK inhibitor may be used alone, or two or more types may be used in combination. It is preferable to use Y27632 as the ROCK inhibitor. The concentration of the ROCK inhibitor in the medium is not particularly limited, but can be, for example, 0.1 to 50 μM, preferably 0.5 to 20 μM, more preferably 1 to 15 μM, and even more preferably 1 to 10 μM.

[0048] <<Other Factors>> The medium may contain other factors in addition to the epidermal growth factor, the GSK3β inhibitor, and the ROCK inhibitor. Examples of other factors include a TGFβ / SMAD inhibitor, an HDAC inhibitor, a p38 inhibitor, and nicotinamide.

[0049] TGFβ / SMAD inhibitors TGFβ (Transforming Growth Factor β) / SMAD inhibitors are substances that inhibit the binding of TGFβ to the ALK (Activin Receptor-Like Kinase) family, or substances that inhibit the phosphorylation of SMAD by the ALK family. Examples of TGFβ / SMAD inhibitors include substances that inhibit the kinase activity of ALK4, ALK5, ALK7, etc. Examples of TGFβ / SMAD inhibitors include Lefty-1 (NCBI accession numbers: mouse: NP_034224.1, human: NP_066277.1), SB431542, SB202190 (R.K. Lindemann et al., Mol. Cancer, 2003, 2:20), SB505124 (GlaxoSmithKline), NPC30345, SD093, SD908, SD208 (Scios), LY2109761, LY364947, LY580276 (Lilly Research Laboratories), A83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, WO2009146408), ALK5 inhibitor II (2-[3-[6-methylpyridin-2-yl]-1H-pyrazol-4-yl]-1,5-naphthyridine), TGFβRI kinase inhibitor VIII (6-[2-tert-butyl-5-[6-methyl-pyridin-2-yl]-1H-imidazol-4-yl]-quinoxaline) and derivatives thereof. The TGFβ / SMAD inhibitor may be an antisense nucleic acid against the ALK family, an RNA interference-inducing nucleic acid (e.g., siRNA), a dominant-negative mutant, an expression vector thereof, or the like. The TGFβ / SMAD inhibitor may be used alone or in combination of two or more. It is preferable to use A83-01 as the TGFβ / SMAD inhibitor. The concentration of the TGFβ / SMAD inhibitor in the medium is not particularly limited, but may be, for example, 0.1 to 50 μM, preferably 0.5 to 20 μM, more preferably 1 to 15 μM, and even more preferably 1 to 10 μM.

[0050] HDAC Inhibitors HDAC (Histone Deacetylase) inhibitors are substances that inhibit the function of histone deacetylase. HDAC inhibitors are not particularly limited as long as they can suppress the function of HDAC. Examples of HDAC inhibitors include small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC1293, and M344. HDAC inhibitors may be antisense nucleic acids, siRNA, dominant-negative mutants, and expression vectors thereof against HDAC. One HDAC inhibitor may be used alone, or two or more may be used in combination. VPA is preferably used as the HDAC inhibitor. The concentration of the HDAC inhibitor in the medium is not particularly limited, but can be, for example, 0.01 to 10 μM, preferably 0.05 to 5 μM, more preferably 0.1 to 2 μM, and even more preferably 0.5 to 1 μM.

[0051] p38 inhibitors are substances that inhibit the function of p38 MAPK (P38 mitogen-activated protein kinase). Examples of p38 inhibitors include SB202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole), SB203580 (4-[4-(4-fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-1H-imidazol-5-yl]pyridine), VX702 (6-(N-carbamoyl-2,6-difluoroanilino)-2-(2,4-difluorophenyl)pyridine-3-carboxamide), and VX745 (5-(2,6-dichlorophenyl)-2-[2,4-difluorophenyl)thio]-6H-pyrimido[ 1,6-b]pyridazine-6-one), PD169316 (4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole), RO4402257 (6-(2,4-difluorophenoxy)-2-{[3-hydroxy-1-(2-hydroxyethyl)propyl]amino}-8-methylpyrido[2,3-D]pyrimidine-7(8h)-one), BIRB796 (1-[5-tert-butyl-2-(4-methylphenyl)pyrazol-3-yl]-3-[4-(2-morpholin-4-ylethoxy)naphthalen-1-yl]urea), and the like. The p38 inhibitor may be an antisense nucleic acid, siRNA, dominant-negative mutant, or expression vector thereof against p38 MAPK. One type of p38 inhibitor may be used alone, or two or more types may be used in combination. SB202190 is preferably used as the p38 inhibitor. The concentration of the p38 inhibitor in the medium is not particularly limited, but can be, for example, 0.01 to 50 μM, preferably 0.1 to 20 μM, more preferably 0.5 to 10 μM, and even more preferably 0.5 to 5 μM.

[0052] Nicotinamide The medium may contain nicotinamide in addition to the above components. The concentration of nicotinamide in the medium is not particularly limited, but may be, for example, 0.01 to 500 nM, preferably 0.1 to 100 nM, more preferably 0.5 to 50 nM, and even more preferably 1 to 30 nM.

[0053] Examples of media include a medium containing an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor; a medium containing an epidermal growth factor, a GSK3β inhibitor, a ROCK inhibitor, and a TGFβ / SMAD inhibitor; a medium containing an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor, as well as at least one selected from the group consisting of an HDAC inhibitor and a p38 inhibitor; a medium supplemented with an epidermal growth factor, a GSK3β inhibitor, a ROCK inhibitor, and a TGFβ / SMAD inhibitor, as well as at least one selected from the group consisting of an HDAC inhibitor and a p38 inhibitor; and an epidermal growth factor. , a GSK3β inhibitor, a ROCK inhibitor, and an HDAC inhibitor; a medium containing an epidermal growth factor, a GSK3β inhibitor, a ROCK inhibitor, and a p38 inhibitor; a medium containing an epidermal growth factor, a GSK3β inhibitor, a ROCK inhibitor, a TGFβ / SMAD inhibitor, and an HDAC inhibitor; a medium containing an epidermal growth factor, a GSK3β inhibitor, a ROCK inhibitor, a TGFβ / SMAD inhibitor, and a p38 inhibitor; a medium containing an epidermal growth factor, a GSK3β inhibitor, a ROCK inhibitor, a TGFβ / SMAD inhibitor, and a p38 inhibitor; and the like. The medium may be any of the above media, further containing nicotinamide. The medium may be a basal medium (e.g., TS basal medium) containing the above factors. Specific examples of the medium include YS-2D medium, YS medium, YS-2D-3 medium, YS-2D-5 medium, etc., which are used in the examples described below. The medium is preferably a liquid medium.

[0054] <Culture Conditions> The culture conditions can be those generally used for culturing animal cells. For example, the culture temperature is 32 to 40°C (preferably 35 to 38°C, typically 37°C), CO ２ The concentration can be 2 to 5% (preferably 5%).

[0055] The cell seeding density at the start of culture is, for example, 10 ３ ~10 ７ / ml, or 10 ４ ~10 ６ Examples include cells / ml.

[0056] The culture can be, for example, adhesion culture using a culture substrate. Examples of the culture substrate include extracellular matrices. Examples of the extracellular matrix include basement membrane-based extracellular matrices such as laminin, type IV collagen, heparan sulfate proteoglycan, and entactin; cartilage-based extracellular matrices such as hyaluronic acid, type II collagen, and link protein; and interstitial extracellular matrices such as type I collagen, proteoglycans (versican, decorin, etc.), and fibronectin. Commercially available products such as Matrigel (CORNING) and iMatrix (registered trademark) (FUJIFILM Wako) may also be used as the culture substrate. For example, iMatrix 511 is preferred.

[0057] The culture vessel may be any culture vessel commonly used for culturing animal cells, such as a well plate, a culture dish, or a culture flask.

[0058] The culture period can be, for example, about 2 to 60 days, about 4 to 50 days, or about 5 to 30 days. Culture may be continued until the expression levels of yolk sac markers such as AFP and albumin in the cells decrease. For example, culture may be continued until the expression levels of yolk sac markers such as AFP and albumin become 1 / 100 or less, 1 / 300 or less, 1 / 500 or less, 1 / 800 or less, or 1 / 1000 or less compared to yolk sac cells.

[0059] The culture may be carried out, for example, until the cells form cell clusters (for example, cell clusters as shown in FIG. 1).

[0060] The culture may be subcultured at appropriate intervals. Subculture may be performed, for example, when the cells reach a confluent state. Subculture may be performed, for example, every 2 to 10 days, every 2 to 8 days, every 2 to 5 days, or every 2 to 4 days. During subculture, the cells may be detached by pipetting or the like. A protein enzyme such as trypsin may be used to detach the cells.

[0061] The medium used for subculture may be a medium containing an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor. The medium used for subculture may be the same as the medium described above in the section <Culture Medium>.

[0062] HS cells can be obtained by culturing yolk sac cells as described above.

[0063] (Other Steps) The production method of this embodiment may include other steps in addition to the culture step described above. Examples of other steps include a step of recovering HS cells after the culture step, a step of measuring the expression level of a yolk sac marker in HS cells, and a step of observing HS cells using a phase-contrast microscope or the like.

[0064] HS cells can be recovered by detaching the cells by pipetting or the like, and then centrifuging the cell suspension. The recovered cells may be passaged in a new medium, or may be cryopreserved using a cell cryopreservation solution or the like. As a cell cryopreservation solution, a commercially available cryopreservation solution such as CELLBANKER (registered trademark) 1 (Xenogen Pharma) can be used. Alternatively, the cells may be maintained by subculture using the method described above.

[0065] The expression levels of yolk sac markers such as AFP and albumin may be measured for HS cells. If the expression levels of these yolk sac markers are reduced compared to yolk sac cells, it can be determined that HS cells have been induced. The expression levels of yolk sac markers can be measured by known methods such as those described above.

[0066] The formation of cell clusters of HS cells may be observed using a phase-contrast microscope, etc. If the cells form cell clusters, it can be determined that HS cells have been induced.

[0067] The HS cells obtained by the production method of this embodiment are considered to have the ability to differentiate into yolk sac cells. Therefore, in addition to forming yolk sac organoids (hereinafter also referred to as "HS organoids") described below, HS cells are expected to be used as model cells for drug evaluation tests in early pregnancy, in regenerative medicine, assisted reproductive technology, and the like.

[0068] [Culture Medium for Inducing Hypoblast Stem Cells] A second aspect of the present disclosure is a culture medium for inducing hypoblast stem cells (hereinafter also referred to as "HS cell induction medium"). The HS cell induction medium contains an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor.

[0069] The HS cell induction medium is the same as the medium described in the section <Culture Medium> of the (Culture Step) in the above-mentioned [Method for Producing Hypoblast Stem Cells]. The HS cell induction medium can be used to induce HS cells from yolk sac cells.

[0070] [Hypoblast Stem Cells] A third aspect of the present invention relates to hypoblast stem cells (HS cells). HS cells are the same as those described in the section (Hypoblast Stem Cells) in the above [Method for Producing Hypoblast Stem Cells]. In one embodiment, the HS cells are derived from cells isolated from human yolk sacs.

[0071] [Method for producing yolk sac organoids] A fourth aspect of the present invention is a method for producing yolk sac organoids, which includes a step of culturing the hypoblast stem cells according to the third aspect in a hydrogel (hereinafter also referred to as a "hydrogel culture step").

[0072] (Intra-hydrogel culture step) In the intra-hydrogel culture step, HS cells are cultured in a hydrogel.

[0073] Hydrogels are polymeric materials that gel with water. Examples of polymeric materials used for hydrogels include, but are not limited to, extracellular matrices such as collagen (type I, type II, type III, type V, type XI, etc.), gelatin, elastin, proteoglycans, glycosaminoglycans, fibronectin, vitronectin, laminin, pectin, hyaluronic acid, chitin, and chitosan; polysaccharides such as alginic acid and starch; amino acid polymers such as polylysine and polyarginine; fibrous proteins such as fibrin; and synthetic polymers such as polyethylene glycol. Commercially available hydrogels, such as Matrigel (registered trademark) (CORNING) and iMatrix (registered trademark) (FUJIFILM Wako), may also be used.

[0074] Culturing can be initiated by embedding HS cells in the hydrogel. The HS cells may be subcultured as described above, or cryopreserved cells. If cryopreserved, the HS cells are preferably cultured in a medium containing epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor before culturing in the hydrogel. Examples of the medium used in this process include the same medium as described in the section <Culture Medium> under (Culture Step) in the above-mentioned [Method for Producing Hypoblast Stem Cells].

[0075] Before embedding the HS cells in the hydrogel, the cells may be detached from the culture vessel by pipetting or the like. If the HS cells form cell clusters, the cell clusters may be separated by pipetting or the like to obtain free cells. A protein enzyme such as trypsin may be used to detach the cells and / or separate the cell clusters.

[0076] The number of HS cells to be embedded in the hydrogel is, for example, 10 ３ ~10 １０ It can be made into one.

[0077] The position at which HS cells are embedded in the hydrogel may be, for example, at a depth of about 0.1 to 5 cm, or about 0.5 to 3 cm from the surface of the hydrogel.

[0078] The hydrogel with embedded HS cells can be cultured by immersing it in an appropriate liquid medium. Examples of the medium include a medium containing epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor. Examples of the medium include the same medium as described in the section "Culture Medium" in the "Culture Step" of the "Method for Producing Hypoblast Stem Cells" above.

[0079] The culture conditions can be those generally used for culturing animal cells, such as a culture temperature of 32 to 40°C (preferably 35 to 38°C, typically 37°C), CO ２ The concentration can be 2 to 5% (preferably 5%).

[0080] The culture period may be any period sufficient for the formation of yolk sac organoids. The formation of yolk sac organoids can be confirmed by observation using a phase-contrast microscope, for example. The culture period may be, for example, 3 days or more, 5 days or more, 7 days or more, or 10 days or more. The range of the culture period may be, for example, about 3 to 50 days, about 5 to 50 days, about 7 to 50 days, or about 10 to 50 days.

[0081] The yolk sac organoids obtained by the manufacturing method of this embodiment are the same as the yolk sac organoids described in "Regarding (c)" in the (Hypoblast Stem Cells) section of the above [Method for Manufacturing Hypoblast Stem Cells].

[0082] The yolk sac organoids obtained by the production method of this embodiment are expected to have similar gene expression patterns and functions to those of the yolk sac, and therefore are expected to be used as a model for drug evaluation tests in early pregnancy, regenerative medicine, assisted reproductive technology, etc.

[0083] In another aspect, the present disclosure also provides yolk sac organoids obtained by the production method according to the fourth aspect, which contain HS cells as described in the section (hypoblast stem cells) in the above "Method for producing hypoblast stem cells."

[0084] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0085] [Reagents and Media] Table 1 shows the reagents used in the experiment.

[0086]

[0087] The reagents shown in Table 2 were used as the inhibitors.

[0088]

[0089] For the preparation of the medium, the reagent solutions shown in Table 3 were prepared.

[0090]

[0091] The composition of the TS basal medium is shown in Table 4. The TS basal medium was stored at 4°C and used within one month after preparation.

[0092]

[0093] The composition of YS-2D medium is shown in Table 5. YS-2D medium was stored at 4°C and used within 2 weeks after preparation.

[0094]

[0095] The composition of the YS medium is shown in Table 6. The YS medium was stored at 4°C and used within 2 weeks after preparation.

[0096]

[0097] [Experimental Example 1] (Induction of hypoblast stem cells) <Isolation of cells from human yolk sac> Human yolk sac tissue was minced with scissors and treated in TrypLE solution at 37°C for 10 minutes, after which the cells (yolk sac cells) were collected by centrifugation.

[0098] <Induction of Hypoblast Stem Cells> Hypoblast stem cells (hereinafter also referred to as "HS cells") were induced from human yolk sac cells according to the following procedure. (1) 1 ml of YS-2D medium and 1 μl of iMatrix511 were added to each well of a 12-well dish. (2) The 12-well dish was maintained at 37°C for at least 10 minutes. (3) Approximately 0.5 x 10 cells were added to the YS-2D medium in each well. ５ (4) The cells were suspended at 37°C, 5% CO ２ The cells were cultured under the conditions described above and subcultured every 3 days using YS-2D medium.

[0099] The cells prepared as described above could be cryopreserved using CELLBANKER® 1 (Xenogen Pharma).

[0100] The cells were passaged according to the following procedure: (1) The YS-2D medium in the wells was aspirated. (2) 0.25 ml of phosphate-buffered saline (PBS) and 0.25 ml of TrypLE were added to each well. (3) The 12-well dish was maintained at 37°C for approximately 10 minutes. (4) The cells were detached by pipetting. (5) The detached cells were transferred to a new tube containing 0.5 ml of TS basal medium. (6) The tube was centrifuged at approximately 380 g for 2 minutes. (7) YS-2D medium and iMatrix 511 were placed in the wells of a new 12-well dish and warmed to 37°C. The cell pellet obtained by centrifugation was seeded into the wells (split ratio = 1:6 to 1:8).

[0101] Figure 1 shows a phase-contrast microscope image of HS cells induced by the above method. The HS cells in Figure 1 were obtained by subculturing yolk sac cells in YS-2D medium for approximately one month. HS cells were used on the sixth day after the last passage. The HS cells formed cell clusters and had a different appearance from yolk sac cells. No clear boundaries between cells were observed in the HS cell clusters. This suggested that they were cell clusters with a high cell density. The appearance of the HS cells was also different from that of previously reported mouse PrES cells (Ohinata et al. Science. 2022 Feb 4;375(6580):574-578.) and hypoblast-like cells (Okubo et al. Nature. 2024 Feb;626(7998):357-366.).

[0102] Experimental Example 2 (Formation of Yolk Sac Organoids) Yolk sac organoids were formed using the HS cells obtained in Experimental Example 1 according to the following procedure. (1) The YS-2D medium in the wells was aspirated. (2) 0.25 ml of phosphate-buffered saline (PBS) and 0.25 ml of TrypLE were added to each well. (3) The 12-well dish was maintained at 37°C for approximately 10 minutes. (4) The cells were detached by pipetting. (5) The detached cells were transferred to a new tube containing 0.5 ml of TS basal medium. (6) The tube was centrifuged at approximately 380 g for 2 minutes. (7) The cell pellet obtained by centrifugation was embedded in Matrigel and placed in the wells of a new 12-well dish. (8) The Matrigel from (7) was incubated in YS medium at 37°C and 5% CO ２ The cells were cultured under the conditions.

[0103] Yolk sac organoids were passaged according to the following procedure: (1) The YS medium surrounding the Matrigel was aspirated. (2) PBS and TrypLE (PBS:TrypLE = 1:1; 1.5 ml per 35 cm dish) were added to a 35 cm dish. (3) Using a Pipetman P1000 (Gilson), the liquid in the dish was pipetted approximately five times. (4) The dish was maintained at 37°C for approximately 15 minutes. (5) Using a Pipetman P200 (Gilson), the liquid in the dish was pipetted approximately 10 times. (6) The cell clumps were transferred to a new 15 ml tube containing 5 ml of TS basal medium. (7) The tube was centrifuged at approximately 800 g for 2 minutes. (8) The cell pellet obtained by centrifugation was embedded in Matrigel (split ratio = 1:4 to 1:8) and placed in a well of a new 12-well dish. This was incubated in YS medium at 37°C and 5% CO ２ The cells were cultured under the conditions.

[0104] Figure 2 shows a phase-contrast microscope image (left) and an immunofluorescent staining image (right) of the organoids obtained by the above method. The organoids in Figure 2 were obtained by subculturing HS cells in Matrigel for approximately one month. Organoids were used on the 11th day after the last passage. Immunofluorescent staining was performed using an anti-AFP (alpha-fetoprotein) antibody. AFP is a yolk sac marker highly expressed in the yolk sac. Yolk sac organoids induced from HS cells (hereinafter also referred to as "HS organoids") highly expressed AFP.

[0105] [Experimental Example 3] (RNA-seq analysis) Gene expression of the HS cells obtained in Experimental Example 1 and the HS organoids obtained in Experimental Example 2 was examined by RNA-seq analysis.

[0106] Figure 3A shows the expression level of AFP obtained by RNA-seq analysis. HS cells showed almost no AFP expression. On the other hand, HS organoids showed AFP expression. Figure 3B shows the expression level of albumin obtained by RNA-seq analysis. HS cells showed almost no albumin expression. On the other hand, HS organoids showed albumin expression.

[0107] Figure 4 shows the results of UMAP (Uniform Manifold Approximation and Projection) analysis of gene expression data obtained by RNA-seq analysis. Yolk sac gene expression data were obtained from the data reported by Issac Goh et al. (Science. 2023 Aug 18;381(6659)). The gene expression patterns of HS cells and HS organoids were similar to those of yolk sac endoderm.

[0108] Experimental Example 4 (Investigation of HS Cell Induction Conditions) Media were prepared with the same composition as YS-2D medium, except that the combination of factors shown in Table 7 was used instead of the combination of Y27632, EGF, A83-0, CHIR99021, SB02190, and VPA in YS-2D medium (see Table 5).

[0109] HS cells were induced from yolk sac cells in the same manner as in Experimental Example 1, except that media containing the combinations of factors shown in Table 7 were used. After 5 days of culture in each of the media shown in Table 7, the number of cells was counted using an automatic cell counter (Olympus Cell Counter model R1) to evaluate cell proliferation. The results are also shown in Table 7. "+" indicates that cell proliferation was confirmed, and the more "+" there are, the greater the amount of cell proliferation. "-" indicates that cell proliferation was not confirmed.

[0110]

[0111] In the medium in which cell growth was confirmed, cell clusters of HS cells with the same shape as in Figure 1 were observed. In the medium in which cell growth was not confirmed, no cell clusters of HS cells were observed.

[0112] Cell proliferation was best in YS-2D medium containing all six factors. Cell proliferation was next best in YS medium (without VPA) and YS-2D-5 medium (without SB202190). In YS-2D-3 medium (without A83-01), cell clusters of HS cells were observed, but cell proliferation was low. In YS-2D-1 medium (without EGF), YS-2D-2 medium (without CHIR99021), and YS-2D-4 medium (without Y27632), neither cell proliferation nor cell clusters of HS cells were observed.

[0113] These results confirmed that EGF, a GSK3β inhibitor, and a ROCK inhibitor are essential factors for the induction of HS cells from yolk sac cells. It was suggested that the use of a TGFβ / SMAD inhibitor in combination with the above three factors could efficiently induce HS cells. Furthermore, it was suggested that the addition of an HDAC inhibitor and a p38 inhibitor to the culture medium could more efficiently induce HS cells.

[0114] [Experimental Example 5] (Long-term culture of HS cells) The HS cells obtained in Experimental Example 1 were cultured for 6 months or more at 37°C using YS-2D medium or YS medium. The HS cells were passaged every 3 days by the method described in Experimental Example 1.

[0115] During the culture period of 6 months or more, the HS cells formed cell masses similar to those shown in Figure 1 after subculture. HS cells cultured for 6 months or more were used to form HS organoids using the same method as in Experimental Example 2. As a result, it was confirmed that HS organoids similar to those shown in Figure 2 were formed.

[0116] The above results confirmed that HS cells can be maintained in long-term culture for six months or more.

[0117] [Experimental Example 6] (Single-cell RNA-seq analysis) HS organoids were obtained by the same method as in Experimental Example 2. Single-cell RNA-seq analysis was performed on the HS cells and HS organoids.

[0118] Figure 5 shows the results of UMAP (Uniform Manifold Approximation and Projection) analysis of gene expression data obtained by single-cell RNA-seq analysis. Yolk sac gene expression data was obtained from the data reported by Issac Goh et al. (Science. 2023 Aug 18;381(6659)). Similar to the results in Experimental Example 3, the gene expression pattern of HS organoids was similar to that of yolk sac endoderm.

[0119] Figure 6 shows the results of a comparison of gene expression between HS cells and HS organoids based on gene expression data obtained by single-cell RNA-seq analysis. The upper panel (A) in Figure 6 shows the expression of genes involved in cell proliferation, such as DNA replication and cell division. The lower panel (B) in Figure 6 shows the expression of genes involved in the uptake, metabolism, transport, and sulfate conjugation of biomolecules (lipids, carbohydrates, proteins), as well as MAPK signaling activity. Compared to HS cells, HS organoids showed decreased expression of genes involved in cell proliferation and increased expression of genes related to yolk sac function.

[0120] The present disclosure provides a method for producing hypoblast stem cells that can be applied to humans and can produce hypoblast stem cells that can be maintained by long-term culture, a medium for inducing hypoblast stem cells that can be used in the method, hypoblast stem cells produced by the method, a method for producing yolk sac organoids using the hypoblast stem cells, and yolk sac organoids containing the hypoblast stem cells.The hypoblast stem cells and yolk sac organoids are expected to be applied to drug evaluation tests targeting early pregnancy, regenerative medicine, assisted reproductive technology, and the like.

[0121] Although the preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments. Addition, omission, substitution, and other modifications of the configuration are possible within the scope of the spirit of the present invention. The present invention is not limited by the above description, but is limited only by the scope of the appended claims.

Claims

1. A method for producing hypoblast stem cells, comprising the step of culturing cells isolated from mammalian yolk sac in a medium containing epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor.

2. The method for producing hypoblast stem cells according to claim 1, wherein the culture medium further contains a TGFβ / SMAD inhibitor.

3. The method for producing hypoblast stem cells according to claim 1 or 2, wherein the culture medium further contains at least one selected from the group consisting of an HDAC inhibitor and a p38 inhibitor.

4. A medium for inducing hypoblast stem cells, comprising an epidermal growth factor, a GSK3β inhibitor, and a ROCK inhibitor.

5. The medium for inducing hypoblast stem cells according to claim 4, further containing a TGFβ / SMAD inhibitor.

6. The medium for inducing hypoblast stem cells according to claim 4, further comprising at least one selected from the group consisting of an HDAC inhibitor and a p38 inhibitor.

7. A medium for inducing hypoblast stem cells according to any one of claims 4 to 6, which is used to induce hypoblast stem cells from cells isolated from mammalian yolk sacs.

8. Hypoblast stem cells having the following characteristics (a) to (e): (a) being cells derived from cells isolated from a mammalian yolk sac; (b) expressing a lower amount of α-fetoprotein compared to cells isolated from the mammalian yolk sac; (c) having the ability to differentiate into a yolk sac organoid comprising cells expressing at least one selected from the group consisting of α-fetoprotein and albumin; (d) having the ability to proliferate; and (e) forming cell clusters.

9. The hypoblast stem cells according to claim 8, which can be maintained in culture for six months or more by subculturing in a medium for inducing hypoblast stem cells according to any one of claims 4 to 6.

10. The hypoblast stem cells of claim 8, which are derived from cells isolated from human yolk sacs.

11. A method for producing yolk sac organoids, comprising culturing the hypoblast stem cells described in claim 8 in a hydrogel.

12. A yolk sac organoid comprising the hypoblast stem cells of claim 8.

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