Agent for improving implantation rate and method for producing agent for improving implantation rate

Abstract

This agent is for improving the implantation rate, and comprises a culture supernatant of trophoblast stem cells. The present invention also pertains to a pharmaceutical composition that is for treating or preventing implantation failure, and that comprises the agent for improving the implantation rate. The present invention also pertains to a method for producing the agent for improving the implantation rate, the method comprising: (i) a step for culturing trophoblast stem cells; and (ii) a step for obtaining a culture supernatant from a cultured product obtained by the culturing.

Description

Implantation rate improver and method for producing the same

[0001] The present invention relates to an implantation rate improver and a method for producing the same. This application claims priority based on Japanese Patent Application No. 2023-121523, filed on July 26, 2023, the contents of which are incorporated herein by reference.

[0002] Although assisted reproductive technology is increasing year by year, the average success rate of in vitro fertilization and intracytoplasmic sperm injection (ICS) is approximately 20-25%. Among patients who do not achieve pregnancy, the number of patients whose pregnancy is presumed to be due to implantation failure remains high. Therefore, there is a need for the development of technology to improve implantation rates. A method known as the SEET method (Stimulation of Endometrium-Embryo Transfer) has been reported as a method for improving implantation rates (see, for example, Patent Document 1). The SEET method involves injecting the supernatant of a culture medium in which embryos have been cultured to the blastocyst stage into the uterus 2-3 days before embryo transfer.

[0003] The main component of placental tissue is trophoblast tissue. The present inventors have developed a method for establishing trophoblast stem cells (TS cells) by recovering CD49f antibody-positive cells from a cell suspension obtained from mammalian placental tissue (Patent Document 2). The present inventors have also developed a method for inducing TS cells from pluripotent stem cells (Patent Document 3) and a method for inducing TS cells from placenta-derived trophoblast cells from mid-pregnancy or later (Patent Document 4).

[0004] Japanese Patent No. 5139294 Japanese Patent No. 6400832 International Publication No. 2020 / 250438 International Publication No. 2022 / 014028

[0005] In order to increase the success rate of pregnancy in assisted reproductive technology, it is necessary to develop technology that improves the implantation rate. The SEET method has a limited amount of culture supernatant obtained from blastocyst culture, and the amount that can be injected into the uterus is extremely small. Therefore, there are technical and procedural issues, and the method is currently only used in a few clinics.

[0006] Therefore, an object of the present invention is to provide an implantation rate improver that can be easily produced, a pharmaceutical composition containing the implantation rate improver, and a method for producing the implantation rate improver.

[0007] The present invention includes the following aspects. [1] An implantation rate improver comprising a culture supernatant of trophoblast stem cells. [2] The implantation rate improver according to [1], which is used to treat the endometrium before embryo transfer. [3] The implantation rate improver according to [1] or [2], wherein the culture supernatant is obtained from a culture of trophoblast stem cells cultured in a serum-free medium. [4] The implantation rate improver according to any one of [1] to [3], wherein the culture supernatant is obtained from a culture of trophoblast stem cells cultured for 1 to 10 days. [5] A pharmaceutical composition for treating or preventing implantation failure, comprising the implantation rate improver according to any one of [1] to [4]. [6] A method for producing an implantation rate improver, comprising: (i) culturing trophoblast stem cells; and (ii) obtaining a culture supernatant from the culture obtained by the culturing. [7] A method for producing an implantation rate improver according to [6], wherein the culturing in step (i) is performed in a serum-free medium. [8] The method for producing an implantation rate improver according to [6] or [7], wherein the culture in step (i) is carried out for 1 to 10 days.

[0008] According to the present invention, there are provided an implantation rate improver that can be easily produced, a pharmaceutical composition containing the implantation rate improver, and a method for producing the implantation rate improver.

[0009] These images show the results of a three-dimensional co-culture of human TS cells (GFP-TS) that constitutively express EGFP and endometrial epithelial organoids (EMOs) in Matrigel. The left image is a bright-field image. The right image is a fluorescence microscope image detecting EGFP fluorescence. Arrows indicate thickened epithelial cell layers. Scale bar, 100 μm. These images show the results of a three-dimensional co-culture of human TS cells (GFP-TS) that constitutively express EGFP and endometrial epithelial organoids (EMOs) in Matrigel. Arrows indicate human TS cells. Scale bar, 100 μm. The results of gene expression analysis of implantation marker genes (PAEP, SPP1) in endometrial models treated with human TS cell culture supernatant (TS-CM), human ST cell culture supernatant (ST-CM), or hormone (EPCP) are shown (bottom panel). The diagram above shows an outline of the test method. Control: Untreated group, Hormone: EPCP (E ２ (10 nM), MPA (1 μM), 8-Bromo-cAMP (50 μM), and prolactin (50 mg / mL)) added group. Results of gene expression analysis of endometrial receptivity analysis (ERA) genes in TS-CM-treated, ST-CM-treated, or Hormones-treated endometrial models are shown. Results of GO term analysis of genes whose expression increased in the TS-CM-treated endometrial model are shown. Phase contrast images showing the adhesion of blastoid to TS-CM-treated endometrial epithelial cells (Ishikawa cells). Scale bar, 200 μm. The graph shows the results of quantifying the adhesion rate of blastoid after 4 hours of co-culture of TS-CM-treated endometrial epithelial cells (Ishikawa cells) with blastoid, N=12 blastoids / group.

[0010] A numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0011] 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 would exert a special function (such as a manner that would completely lose the effects of the invention). In this specification, when "comprise" is used, it includes both "consist of" and "consist essentially of" embodiments.

[0012] The cells may be isolated. "Isolated" means in a natural state or a state separated from other components. "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 cells described herein may be isolated cells.

[0013] [Implantation Rate Improver] A first aspect of the present disclosure is an implantation rate improver, which comprises a culture supernatant of trophoblast stem cells.

[0014] <Trophyblast stem cells (TS cells)> TS cells are cells that have the ability to differentiate into cells that constitute the placenta. More specifically, TS cells have the ability to differentiate into extravillous cytotrophoblast cells (EVT) and syncytiotrophoblast cells (ST). Differentiation induction tests into EVT and ST can be performed by known methods (e.g., Japanese Patent No. 6400832, International Publication No. 2020 / 250438, International Publication No. 2022 / 014028, etc.).

[0015] Positive markers for TS cells include GATA2, GATA3, TFAP2, ELF5, ZNF750, etc. Negative markers for TS cells include CDX2, etc. TS cells can be said to be cells that have the ability to differentiate into EVT and ST and are positive for at least one (preferably two or more, more preferably three or more, even more preferably four or more, and particularly preferably five) selected from the group consisting of GATA2, GATA3, TFAP2, ELF5, and ZNF750. Alternatively, TS cells can be said to be cells that are negative for CDX2 in addition to the above. TS cells can also be said to be cells induced from blastocysts, CT cells, or pluripotent stem cells, that have the ability to differentiate into EVT and ST, are positive for at least one selected from the group consisting of GATA2, GATA3, TFAP2, ELF5, and ZNF750, and are negative for CDX2.

[0016] TS cells can be produced by known methods. TS cells may be induced from blastocysts, cytotrophoblast cells (CT cells), or pluripotent stem cells. Methods for inducing TS cells from blastocysts include the following: For example, placental tissue is subjected to appropriate mechanical and / or enzymatic treatment to separate cells. The cells are then cultured in a TS cell-inducing medium (e.g., Okae et al., Derivation of Human Trophoblast Stem Cells. Cell Stem Cell (2018), 22(1), 50-63.e6.), and TS cells can be established using the expression of TS cell markers (e.g., GATA2-positive, GATA3-positive, TFAP2-positive, ELF5-positive, ZNF750-positive, CDX2-negative, etc.) as an indicator. Methods for inducing TS cells from CT cells include, for example, the method described in Japanese Patent No. 6400832. Methods for inducing TS cells from pluripotent stem cells include, for example, the method described in International Publication No. WO 2020 / 250438.

[0017] The biological species from which the TS cells are derived is preferably the same as the biological species to which the implantation improver is to be applied. For example, when the implantation improver is to be applied to humans, the TS cells are preferably human TS cells.

[0018] <Culture Medium> The culture medium for culturing TS cells may be any medium capable of maintaining the culture of TS cells. Examples of the culture medium include basal media commonly used for culturing animal cells. The basal medium may contain components necessary for cell survival, such as amino acids, vitamins, and inorganic salts. Examples of amino acids include glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. These amino acids may be in the form of a salt, such as a hydrochloride, or in the form of a salt hydrate. Examples of vitamins include biotin, choline chloride, calcium pantothenate, folic acid, nicotinamide, para-aminobenzoic acid, pyridoxine, riboflavin, thiamine, vitamin B12, and inositol. These vitamins may be in the form of a salt, such as a hydrochloride, or in the form of a salt hydrate. Inorganic salts include alkali metal salts such as potassium and sodium; alkaline earth metal salts such as calcium and magnesium; and transition metal salts such as iron, copper, and zinc. These salts include hydrochlorides, nitrates, sulfates, phosphates, hydrogen phosphates, dihydrogen phosphates, carbonates, and bicarbonates. Specific examples of inorganic salts include, but are not limited to, calcium chloride, copper sulfate, ferrous nitrate, ferrous sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, and zinc sulfate. These inorganic salts may also be in the form of hydrates. Other components contained in the basal medium include, but are not limited to, sugars such as glucose; peptides such as glutathione; purine derivatives such as hypoxanthine; fatty acids such as linoleic acid; pH indicators such as phenol red; lipoic acid; putrescine; pyruvic acid; and thymidine.

[0019] Specific 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 thereof. A preferred basal medium is, for example, DMEM / F12.

[0020] The medium may be a basal medium to which appropriate components have been added. Examples of components that can be added to the basal medium include serum and serum substitutes. The medium is preferably a serum-free medium that does not contain serum. The medium is preferably a basal medium to which a serum substitute has been added. A serum substitute is a substance or composition that is added to a medium as a serum substitute. The serum substitute is preferably composed of known components. The serum substitute preferably does not contain components derived from a xenogeneic animal. A xenogeneic animal refers to an animal of a different species from the animal species to which the implantation rate enhancer is applied. 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, 3'-thiolglycerol, L-ascorbic acid, albumin, and the like. One type of serum substitute may be used alone, or two or more types may be used in combination. Preferred examples of serum substitutes include ITS-X supplement, KSR, L-ascorbic acid, and albumin (bovine serum albumin (BSA) and the like).

[0021] The medium is preferably a basal medium supplemented with a serum substitute, more preferably a basal medium supplemented with at least one serum substitute selected from the group consisting of ITS-X supplement, KSR, L-ascorbic acid, and albumin (BSA, etc.), and even more preferably a basal medium supplemented with ITS-X supplement, KSR, L-ascorbic acid, and albumin (BSA, etc.). As the basal medium, DMEM / F12 medium is preferably used.

[0022] In addition to the above-mentioned components, the medium may contain components such as lipids, Glutamax, growth factors (EGF, FGF, BMP, etc.), kinase inhibitors, signal transduction inhibitors, signal transduction activators, antibiotics, antioxidants, pyruvic acid, and buffers. The medium may be one that can maintain TS cells as TS cells without differentiating them (TS cell maintenance medium). In this case, the medium preferably does not contain components that induce TS cell differentiation (e.g., growth factors, kinase inhibitors, signal transduction inhibitors, signal transduction activators, differentiation inducers, etc.). The medium may also be one that induces TS cell differentiation (TS cell differentiation induction medium). In this case, the medium may contain components that induce the differentiation of TS cells into EVT or ST (e.g., growth factors, kinase inhibitors, signal transduction inhibitors, signal transduction activators, differentiation inducers, etc.). The medium for inducing differentiation of TS cells into EVT may contain a growth factor such as neuregulin (NRG)1; a TGFβ inhibitor such as A83-01; and a ROCK inhibitor such as Y27632. The medium for inducing differentiation of TS cells into ST may contain 2-mercaptoethanol; a ROCK inhibitor such as Y27632; and forskolin. The medium preferably does not contain components derived from xenogeneic animals, and is preferably xeno-free.

[0023] Specific examples of preferred media include DMEM / F12 medium and the "basal medium for TS cells" used in the Examples described below.

[0024] <Culturing> TS cells can be cultured by known methods. Culture conditions can be those generally used for culturing animal cells. The culture temperature can be 30 to 40°C, preferably 35 to 38°C, and typically 37°C. ２ The concentration may be 2 to 5%, typically 5%.

[0025] The culture vessel is not particularly limited, and any culture vessel commonly used for culturing animal cells can be used. Examples of the culture vessel include a culture flask, a culture dish, and a well plate. The culture vessel can be appropriately selected depending on the volume of the medium.

[0026] The culture can be initiated by seeding TS cells in a medium placed in an appropriate culture vessel. The cell density at the time of seeding TS cells is, for example, 10 ３ ~10 １０ cells / mL, and 10 ４ ~10 ８ cells / mL is preferred.

[0027] The culture may be either adhesion culture or suspension culture, with adhesion culture being preferred. When adhesion culture is performed, it is preferable to use a culture vessel having a cell-adhesive surface. The surface (e.g., bottom surface) of the culture vessel may be coated with a cell-supporting substrate such as an extracellular matrix (ECM) in order to improve adhesion to TS cells. Examples of cell-supporting substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin. Alternatively, commercially available extracellular matrix products such as Matrigel (registered trademark) (Corning Life Science) and iMAtrix (registered trademark) 511 may be used.

[0028] The culture period is preferably 1 day or more, more preferably 2 days or more. There is no particular upper limit to the culture period, and the cells may be cultured until confluent. The culture period may be, for example, 1 to 10 days, or may be 2 to 10 days, or may be 3 to 10 days. A preferred example of the culture period is 2 to 3 days. During the culture period, the medium may be replaced at intervals of about 1 to 3 days (preferably about 2 to 3 days). The medium used for medium replacement may be the same as or different from the medium used before the replacement, but is preferably the same. The medium obtained by medium replacement before the medium replacement may be used as a culture supernatant or may be discarded.

[0029] <Culture Supernatant> The TS cell culture supernatant is the liquid portion of the culture obtained by culturing TS cells. The TS cell culture supernatant does not need to be completely free of TS cells, and may contain TS cells. The cell concentration of TS cells contained in the TS cell culture supernatant is, for example, 0 to 10 ３ cells / mL, and ２ From the viewpoints of quality maintenance and safety, the TS cell culture supernatant preferably contains no TS cells (0 cells / mL).

[0030] The TS cell culture supernatant can be obtained from a culture of TS cells. The TS cell culture may be, for example, a culture of TS cells cultured in a serum-free medium, or a culture of TS cells cultured for 1 to 10 days. A preferred example of a TS cell culture is a culture of TS cells cultured in a serum-free medium for 2 to 3 days. A preferred example of a TS cell culture supernatant is a culture supernatant obtained from a culture of TS cells cultured in a serum-free medium for 2 to 3 days.

[0031] When TS cells are cultured in an adherent manner, the culture supernatant can be obtained by collecting the liquid portion (supernatant portion) of the culture from which the adherent cells have been removed. The collected culture supernatant may be used as is, or may be used after removing the TS cells by centrifugation and / or filtration, etc. When centrifugation is performed, for example, centrifugation may be performed at a centrifugal acceleration of about 300 to 1000 g for about 3 to 15 minutes. Centrifugation is preferably performed at 4°C. When filtration is performed, a 0.22 μm filter can be used. The culture supernatant obtained from a TS cell culture is preferably filtered after centrifugation. When TS cells are cultured in suspension, the culture supernatant can be obtained by centrifuging the culture. After centrifugation, filtration is preferably performed. Centrifugation and filtration conditions can be the same as those described above.

[0032] The culture supernatant of TS cells is preferably stored frozen until use at a temperature of -80°C to -20°C, preferably -80°C.

[0033] <Method of Use> The TS cell culture supernatant is used as an implantation enhancer. An implantation enhancer is a drug that has the effect of improving the implantation rate of an embryo transferred into the uterus in assisted reproductive technology. The implantation enhancer is used to treat the endometrium before embryo transfer. Treatment of the endometrium with the implantation enhancer can be carried out by injecting the implantation enhancer into the uterine cavity.

[0034] If the TS cell culture supernatant has been frozen and stored, it is thawed before use. The TS cell culture supernatant may be used as is, or may be diluted with a pharmaceutically inactive diluent (e.g., sterilized purified water, physiological saline, PBS, the medium used for culturing TS cells, etc.).

[0035] The subject for which the implantation rate improver is used is not particularly limited as long as it is a placental animal that forms a placenta during pregnancy. Examples of placental animals include primates (humans, chimpanzees, rhesus monkeys, marmosets, etc.), rodents (mice, rats, hamsters, guinea pigs, etc.), and carnivores (dogs, cats, weasels, etc.). The subject for which the implantation rate improver is used is preferably a primate, and more preferably a human. The subject for which the implantation rate improver is used may be the same as or different from the donor of cells from which the TS cells used to prepare the TS cell culture supernatant are derived.

[0036] In order to increase the implantation rate during embryo transfer, the implantation rate enhancer is injected into the uterine cavity of a subject prior to implantation of an embryo into the uterus of the subject. This allows the implantation rate enhancer to come into contact with the subject's endometrium, and the subject's endometrium can be treated with the implantation rate enhancer. The implantation rate enhancer is preferably injected into the uterine cavity of the subject 1 to 5 days, more preferably 2 to 4 days, before implantation of the embryo into the uterine cavity of the subject. Injection may be performed once (e.g., once 3 days before embryo transfer), or multiple times, every other day or every day. The volume of the implantation rate enhancer injected into the uterine cavity may be sufficient to treat the endometrium. The volume of the implantation rate enhancer may be, for example, 0.01 to 1 mL, with 0.05 to 0.5 mL being preferred.

[0037] The embryo used for embryo transfer may be any of a fertilized egg, a 2-cell stage embryo, a 4-cell stage embryo, an 8-cell stage embryo, a morula, and a blastocyst. The embryo is preferably a blastocyst.

[0038] According to the implantation rate improver of this embodiment, by injecting it into the uterine cavity of a subject prior to embryo transfer, the endometrial epithelium can be treated with the implantation rate improver. This improves the embryo receptivity of the endometrial epithelium, thereby improving the embryo implantation rate during embryo transfer. This increases the pregnancy success rate of subjects who have undergone embryo transfer.

[0039] [Pharmaceutical Composition] A second aspect of the present disclosure is a pharmaceutical composition for treating or preventing implantation failure, which comprises the implantation rate improver according to the first aspect as an active ingredient.

[0040] The pharmaceutical composition of this embodiment may contain components other than the implantation rate enhancer according to the first aspect (TS cell culture supernatant). Examples of other components include pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of the active ingredient and is not substantially toxic to the recipient. The term "not substantially toxic" refers to a carrier that is not toxic to the recipient at a commonly used dose. In the pharmaceutical composition of this embodiment, the pharmaceutically acceptable carrier is a carrier that does not inhibit the implantation rate enhancer activity of the implantation rate enhancer according to the first aspect and is not substantially toxic to the recipient. Pharmaceutically acceptable carriers include any known pharmaceutically acceptable component that is typically considered an inactive component. Examples of pharmaceutically acceptable carriers include, but are not limited to, diluents, buffers, vehicles, etc. Pharmaceutically acceptable carriers may be used alone or in combination.

[0041] The pharmaceutical composition may contain other ingredients in addition to the above-mentioned ingredients. The other ingredients are not particularly limited, and those commonly used in the pharmaceutical field can be used without particular limitation. Examples of other ingredients include pharmaceutical additives other than those mentioned above.

[0042] The dosage form of the pharmaceutical composition is not particularly limited and may be any dosage form commonly used for pharmaceutical preparations. The pharmaceutical composition of this embodiment is a parenteral formulation that is injected into the uterine cavity. The dosage form of the pharmaceutical composition is preferably a liquid.

[0043] The pharmaceutical composition can be administered in a therapeutically effective amount of the implantation rate improver according to the first aspect. "Therapeutically effective amount" refers to the amount of a drug that is effective for treating or preventing a target disease. For example, the therapeutically effective amount of the implantation rate improver can be an amount that is effective for treating or preventing implantation failure. The therapeutically effective amount can be, for example, 0.01 to 1 mL / individual, or 0.05 to 0.5 mL / individual, as the amount of TS cell culture supernatant administered.

[0044] The pharmaceutical composition may be administered in a single dose or multiple doses. For example, the pharmaceutical composition may be administered only once, 1 to 5 days, more preferably 2 to 4 days, prior to embryo transfer. Alternatively, the pharmaceutical composition may be administered every other day or every day, starting 2 to 5 days prior to embryo transfer.

[0045] The pharmaceutical composition of this embodiment is used to treat or prevent implantation failure. Implantation failure refers to a state in which pregnancy does not occur even after multiple embryo transfers. Since the pharmaceutical composition of this embodiment contains the implantation rate improver according to the first aspect as an active ingredient, it can improve the implantation rate by injecting it into the uterine cavity prior to embryo transfer. Therefore, it can treat or prevent implantation failure.

[0046] [Method for producing an implantation rate improver] A third aspect of the present disclosure is a method for producing an implantation rate improver. The method includes (i) culturing trophoblast stem cells, and (ii) obtaining a culture supernatant from the culture obtained by the culturing. The method of this embodiment can produce the implantation rate improver of the first aspect.

[0047] <Step (i)> Step (i) can be carried out as described above in the section "Implantation rate improver." Step (i) is preferably carried out in a serum-free medium. The culture period in step (i) can be 1 to 10 days.

[0048] <Step (ii)> Step (ii) can be carried out as described above in the section on “Implantation Rate Enhancer.” For example, a culture supernatant can be obtained from a culture of TS cells by centrifugation, filtration, or the like.

[0049] <Optional Steps> The production method of this embodiment may include optional steps in addition to the steps described above. Examples of optional steps include a step of cryopreservation. The cryopreservation temperature is, for example, −80° C. to −20° C., and −80° C. is preferred.

[0050] [Other Embodiments] In one embodiment, the present disclosure provides a method for improving implantation rate, comprising the step of injecting a TS cell culture supernatant into the uterine cavity of a subject receiving the embryo transfer prior to the embryo transfer. In one embodiment, the present disclosure provides an embryo transfer method, comprising the steps of injecting a TS cell culture supernatant into the uterine cavity of a subject and transferring an embryo into the uterine cavity of the subject. In the method, the TS cell culture supernatant is preferably injected into the uterine cavity 1 to 5 days prior to the embryo transfer, and more preferably 2 to 4 days prior to the embryo transfer.

[0051] In one embodiment, the present disclosure provides use of a TS cell culture supernatant in the manufacture of an implantation rate improver. In one embodiment, the present disclosure provides use of a TS cell culture supernatant in the manufacture of a pharmaceutical composition for treating or preventing implantation failure. In one embodiment, the present disclosure provides a TS cell culture supernatant for use in improving the implantation rate of transplanted embryos. In one embodiment, the present disclosure provides a TS cell culture supernatant for use in treating or preventing implantation failure.

[0052] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0053] <Methods> 1. Culture of human TS cells Maintenance culture of human TS cells and differentiation induction into ST cells were performed according to a previously reported method (Okae et al. Derivation of Human Trophoblast Stem Cells. Cell Stem Cell (2018), 22(1), 50-63.e6.). In addition, a cell line constitutively expressing EGFP (Enhanced Green Fluorescent Protein) was used to track the cells during co-culture with endometrial epithelial organoids.

[0054] 2. Obtaining human TS cell culture supernatant (TS-CM) 1 μL / well of i-Matrix® 511 (Nippi Corporation) was placed in the wells of a 6-well flat-bottom well plate (IWAKI Microplate, AGC Technoglass Co., Ltd.) and allowed to stand for 10 minutes. This allowed the inner walls of the wells to be coated with i-Matrix. 2 mL / well of basal TS cell medium or DMEM / F-12 medium was placed in the coated well plate. 1 x 10 human TS cells were cultured. ５ The cells were seeded at a cell density of 1000 cells / well and incubated at 37°C, 5% CO ２ After 2 or 3 days of adherent culture, the cells were washed with PBS. Then, the medium was replaced with basal TS cell medium or DMEM / F-12 medium, and incubated at 37°C, 5% CO ２ The cells were cultured at a concentration of 0.1% for 2 days. The supernatant was then collected and centrifuged at 500 g for 5 minutes. The supernatant was then passed through a 0.22 μm filter and stored at -80°C until use. To obtain ST-CM, the TS cells were induced to differentiate into ST cells using a previously reported method (Okae et al. Derivation of Human Trophoblast Stem Cells. Cell Stem Cell (2018), 22(1), 50-63.e6.), and the supernatant was collected in the same manner as above.

[0055] The composition of the basal medium for TS cell culture (TS basal medium) is as follows: DMEM / F-12 (Life Technologies) supplemented with 1% ITS-X supplement (FUJIFILM Wako), 0.15% bovine serum albumin (BSA) (FUJIFILM Wako), 1% Knockout Serum Replacement (KSR, Thermo Fisher Scientific), 200 mM L-ascorbic acid (FUJIFILM Wako), 5,000 units / mL penicillin, and 5,000 μg / mL streptomycin.

[0056] 2. Culture of Human Endometrial Epithelial Organoids Endometrial epithelial organoids were established as previously reported (Turco et al. Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium. Nature Cell Biology (2017) 19(5), 568-577.). Decidual tissue from aborted women 6-8 weeks pregnant was minced, immersed in an enzyme solution (collagenase V / dispase II), and incubated at 37°C for 1 hour with shaking. The cell fraction trapped in a cell strainer (100 μm) was then embedded in Matrigel and cultured. The medium was changed every 2-3 days. For subculture, the organoids were detached from the culture plate using basal TS cell medium and fragmented by pipetting. The fragmented organoids were diluted, embedded in Matrigel, and passaged.

[0057] 3. Preparation of a human endometrial model Fragmented human endometrial epithelial organoids were suspended in a matrix containing a mixture of collagen gel and Matrigel (registered trademark) (Corning) (collagen gel:Matrigel = 7:3 (volume ratio)). Droplets of the suspension were left to solidify in a 48-well plate for 30 minutes, and then cultured in ESCY medium for 7-8 days. Subsequently, estradiol (E ２ ) (10 nM) was added and cultured for another 2 days, and then E ２ (10 nM), medroxyprogesterone acetate (MPA) (1 μM), and 8-Bromo-cAMP (50 μM) were added to carry out maturation.

[0058] The composition of ESCY medium is as follows: DMEM / F-12 (Life Technologies) supplemented with 1% ITS-X supplement, 0.15% BSA (FUJIFILM Wako), 1% Knockout Serum Replacement (KSR, Thermo Fisher Scientific), 200 mM L-ascorbic acid (FUJIFILM Wako), 50 ng / ml EGF, 2 μM CHIR99021, 2 μM SB202190, and 10 μM Y27632.

[0059] 4. Immunostaining Cells and organoids were fixed with 4% paraformaldehyde (PFA), washed with PBS, and then blocked for 1 hour with 5% normal goat serum and 0.3% Triton X-100 in PBS. Primary antibodies were then added and incubated overnight at 4°C. Then, cells were stained with fluorescently labeled secondary antibodies (Cell Signaling) and Hoechst 33342 (Dojindo). Observation was performed using a BZ-X810 (Keyence) and an LSM 710 (Carl Zeiss).

[0060] 5. Quantitative real-time RT-PCR. RNA was extracted and purified from cells and organoids using the RNeasy Mini Kit (QIAGEN). cDNA was generated using PrimeScript II (Takara Bio). Real-time PCR reactions were performed using the StepOnePlus Real-Time PCR System (Applied Biosystems) and TB Green Premix Ex Taq™ II (Takara Bio).

[0061] 6. RNA was extracted and purified from cells and organoids using the RNA-sequencing RNeasy Mini Kit (QIAGEN), and genomic DNA was digested with DNase I (QIAGEN). The RNA library was prepared using NEBNext® Ultra ＴＭThe RNA was prepared using the II Directional RNA Library Prep Kit (New England Biolabs) and sequenced using the Illumina NovaSeq 6000 platform (Illumina) using 150-bp paired-end reads. The sequence data was trimmed using TrimGalore and aligned to the reference genome (UCSC hg38) using STAR. Gene expression levels (TPM) were calculated using RSEM. Differentially expressed genes were extracted using DESeq2 software. GO term analysis was performed using clusterProfiler.

[0062] 7. Induction of blastoids from human ES cells According to a previous report (Yu et al. Blastocyst-like structures generated from human pluripotent stem cells. Nature (2021) 591, 620-626.), blastocyst-like structures (blastoids) were produced from naive ES cells.

[0063] 8. In Vitro Implantation Assay Ishikawa cells (endometrial epithelial cells) were seeded in 96-well plates and cultured in 10% FBS-containing DMEM medium (Life Technologies) at 37°C until confluent. In the treated group, the medium was replaced with TS-CM and cultured for 48 hours. In the untreated group, culture was continued for the same period without changing the medium. Subsequently, blastocyst-like structures (blastoids) prepared from naive pluripotent stem cell-type ES cells were added to the treated and untreated groups and co-cultured at 37°C. The adhesion rate of blastocyst-like structures (blastoids) prepared from naive ES cells was quantified 5 hours after co-culture in the treated and untreated (control) groups.

[0064] <Experimental Results> Toward the development of infertility treatments utilizing human TS cells, we investigated the effects of TS cells on endometrial epithelial cells. When TS cells constitutively expressing EGFP (GFP-TS) were co-cultured with endometrial epithelial organoids (EMOs) in Matrigel, the epithelial cell layer in contact with the TS cells thickened (Figure 1). Immunostaining confirmed that endometrial epithelial cells in contact with TS cells were positive for the proliferative cell marker Ki67, the hormone-responsive PGR, and the glandular epithelial marker FOXA2 (Figure 2). This suggests that proximity to TS cells induces cell proliferation, hormone response, and glandular epithelial differentiation in endometrial epithelial cells.

[0065] Next, we confirmed whether this effect was due to direct contact or to humoral factors. 250 μL of TS cell culture supernatant (TS-CM) was added to the endometrial model prepared in "3. Preparation of a human endometrial model" above, and gene expression analysis was performed. The endometrial model treated with TS-CM showed increased expression of both PAEP and SPP1, which are markers of the implantation stage (Figure 3). In Figure 3, "ST-CM" represents the endometrial model to which 250 μL of ST cell culture supernatant (ST-CM) was added instead of TS-CM. "Hormone" represents the endometrial model to which EPCP (E ２ (10 nM), MPA (1 μM), 8-Bromo-cAMP (50 μM), and prolactin (50 mg / mL) were added. The control was a group that was not treated with any of the above (untreated). In the endometrial model treated with ST-CM, PAEP and SPP1 expression was also elevated compared to the control, but the rate of increase in expression was lower than in the endometrial model treated with TS-CM. In the endometrial model treated with hormone, PAEP and SPP1 expression was also elevated compared to the control, but the rate of increase in SPP1 expression was lower than in the endometrial models treated with TS-CM and ST-CM. From these results, it was inferred that TS-CM is highly effective in transitioning the endometrium to the implantation stage.

[0066] RNA was extracted from endometrial models treated with TS-CM, ST-CM, or Hormones (EPCP) and subjected to RNA-seq analysis. RNA-seq analysis examined the expression of the top 20 genes (ERA-UP(TO20)) that increase during the implantation period, as used in the Endometrial Receptivity Analysis (ERA). TS-CM treatment increased the expression of most genes (13 / 20). GO term analysis revealed enrichment of terms related to signals that increase in the endometrial epithelium during the early stages of implantation, such as "regulation of inflammatory response" (Figures 4A and 4B).

[0067] Next, we examined the implantation-promoting effect of TS-CM using an implantation assay (see "8. In vitro implantation assay" above) that measures blastoid adhesion to the apical surface of plate-cultured endometrial epithelial (Ishikawa) cells. Pretreatment of endometrial epithelial (Ishikawa) cells with TS-CM increased the blastoid adhesion rate 1.6-fold after 4 hours of co-culture compared to the untreated control group (Figure 5).

[0068] These results confirmed that TS-CM has the effect of improving the embryo receptive capacity of endometrial epithelium, suggesting that factors secreted by TS cells improve the embryo receptive capacity of endometrial epithelium.

[0069] According to the present invention, there are provided an implantation rate improver that can be easily produced, a pharmaceutical composition containing the implantation rate improver, and a method for producing the implantation rate improver.

[0070] 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. An agent that improves implantation rate, containing culture supernatant of trophoblast stem cells.

2. The implantation rate enhancer according to claim 1, which is used to treat the endometrium prior to embryo transfer.

3. The implantation rate improver according to claim 1 or 2, wherein the culture supernatant is a culture supernatant obtained from a culture of the trophoblast stem cells cultured in a serum-free medium.

4. The implantation rate improver according to claim 1 or 2, wherein the culture supernatant is a culture supernatant obtained from a culture of trophoblast stem cells cultured for 1 to 10 days.

5. A pharmaceutical composition for treating or preventing implantation failure, comprising the implantation rate improver according to claim 1 or 2.

6. A method for producing an implantation rate improver, comprising: (i) a step of culturing trophoblast stem cells; and (ii) a step of obtaining a culture supernatant from the culture obtained by the culturing.

7. The method for producing an implantation rate improver according to claim 6, wherein the culture in step (i) is carried out in a serum-free medium.

8. A method for producing an implantation rate improver according to claim 6 or 7, wherein the culture in step (i) is carried out for 1 to 10 days.

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