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

Abstract

To provide an implantation rate improving agent that is easily produced, a pharmaceutical composition containing the implantation rate improving agent, and a method for producing the implantation rate improving agent.SOLUTION: Provided is an implantation rate improving agent comprising a culture supernatant of trophoblastic stem cells. Also provided is a pharmaceutical composition for treating or preventing implantation failure, which comprises the implantation rate improving agent. Also provided is a method for producing an implantation rate improving agent, comprising the steps of (i) culturing trophoblastic stem cells, and (ii) obtaining a culture supernatant from the culture obtained by the culturing.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an implantation rate improver and a method for producing the same. [Background technology]

[0002] Although assisted reproductive technology is increasing year by year, the average success rate of in vitro fertilization and intracytoplasmic sperm injection is about 20-25%. Among patients who are unsuccessful in pregnancy, the number of patients who are suspected to be due to implantation failure remains high. Therefore, there is a demand for the development of technology to improve the implantation rate. As a method for improving the implantation rate, a method called the SEET method (Stimulation of Endometrium-Embryo Transfer) has been reported (for example, Patent Document 1). The SEET method is a method in which the culture medium in which an embryo has been cultured to the blastocyst stage is injected into the uterus two to three days before the embryo transfer.

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

[0004] [Patent Document 1] Patent No. 5139294 [Patent Document 2] Patent No. 6400832 [Patent Document 3] International Publication No. 2020 / 250438 [Patent Document 4] International Publication No. 2022 / 014028 Summary of the Invention [Problem to be solved by the invention]

[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 by culturing blastocysts, and the amount that can be injected into the uterus is extremely small. Therefore, there are technical and procedural problems, and the current situation is that it is 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. [Means for solving the problem]

[0007] The present invention includes the following aspects. [1] An agent for improving implantation rate, containing culture supernatant of trophoblast stem cells. [2] The implantation rate enhancer described in [1], which is used to treat the endometrium prior to embryo transfer. [3] The implantation rate improver according to [1] or [2], wherein the culture supernatant is a culture supernatant obtained from a culture of the trophoblast stem cells in a serum-free medium. [4] The implantation rate improver according to any one of [1] to [3], 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 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] The method for producing an implantation rate improver according to [6], wherein the culture in the step (i) is carried out in a serum-free medium. [8] The method for producing an implantation rate improver according to [6] or [7], wherein the culture in the step (i) is carried out for 1 to 10 days. Effect of the Invention

[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. [Brief description of the drawings]

[0009] [Figure 1] These are images of human TS cells (GFP-TS) that constitutively express EGFP and endometrial epithelial organoids (EMOs) co-cultured in Matrigel in three dimensions. The left image is a bright field image. The right image is a fluorescence microscope image detecting EGFP fluorescence. Arrows indicate the thickened epithelial cell layer. Scale bar, 100 μm. [Diagram 2] Immunostaining images of human TS cells (GFP-TS) that constitutively express EGFP and endometrial epithelial organoids (EMOs) in three-dimensional co-culture in Matrigel. Arrows: human TS cells. Scale bar, 100 μm. [Diagram 3] 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 (lower figure). The upper figure shows an outline of the test method. Control: Untreated group, Hormone: EPCP (E2 (10 nM), MPA (1 μM), 8-Bromo-cAMP (50 μM) and prolactin (50 mg / mL)) added group. [Figure 4A] 1 shows the results of gene expression analysis of genes included in the Endometrial Receptivity Analysis (ERA) in endometrial models treated with TS-CM, ST-CM, or Hormone. [Figure 4B] 1 shows the results of GO term analysis of genes whose expression was increased in the TS-CM-treated endometrial model. [Figure 5A]Phase contrast images showing the adhesion of blastoids to TS-CM-treated endometrial epithelial cells (Ishikawa cells). Scale bar, 200 μm. [Figure 5B] The results show the quantification of the adhesion rate of blastoids after co-culturing TS-CM-treated endometrial epithelial cells (Ishikawa cells) with blastoids for 4 hours. N=12 blastoids / group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] [Implantation rate improver] A first aspect of the present disclosure is an implantation rate improver, comprising a culture supernatant of trophoblast stem cells.

[0011] <Trophyll 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 (EVT) and syncytiotrophoblast (ST). Differentiation induction tests into EVT and ST can be performed by known methods (e.g., Japanese Patent Publication No. 6400832, International Publication No. 2020 / 250438, International Publication No. 2022 / 014028, etc.).

[0012] 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, 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.

[0013] TS cells can be prepared 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 appropriately treated mechanically and / or enzymatically to separate cells. After that, the cells are cultured in a medium for inducing TS cells (e.g., Okae et al. Derivation of Human Trophoblast Stem Cells. Cell Stem Cell (2018), 22(1), 50-63.e6.), and then TS cells can be established using the expression of TS cell markers (GATA2 positive, GATA3 positive, TFAP2 positive, ELF5 positive, ZNF750 positive, CDX2 negative, etc.) as an indicator. An example of a method for inducing TS cells from CT cells is the method described in Japanese Patent No. 6400832. Methods for inducing TS cells from pluripotent stem cells include, for example, the methods described in International Publication No. 2020 / 250438.

[0014] The species from which the TS cells are derived is preferably the same as the species to which the implantation rate improver is to be applied. For example, when the implantation rate improver is to be applied to humans, the TS cells are preferably human TS cells.

[0015] <Culture medium> The medium for culturing TS cells may be any medium capable of maintaining and culturing TS cells. Examples of the medium include basal media generally 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, valine, etc. 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, inositol, etc. These vitamins may be in the form of a salt such as a hydrochloride, or in the form of a salt hydrate. Examples of 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. Examples of these salts include hydrochlorides, nitrates, sulfates, phosphates, hydrogen phosphates, dihydrogen phosphates, carbonates, and hydrogen carbonates. Specific examples of inorganic salts include, but are not limited to, calcium chloride, copper sulfate, iron nitrate, iron sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium hydrogen carbonate, sodium chloride, sodium hydrogen phosphate, sodium dihydrogen phosphate, and zinc sulfate. These inorganic salts may 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; thymidine, etc.

[0016] Specific examples of the basal medium include Doulbecco's modified Eagle's Medium (DMEM) medium, DMEM / F12 medium, Advanced DMEM / F12 medium, IMDM medium, Medium199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Ham's F12 medium, RPMI1640 medium, Fischer's medium, and mixed media thereof. A preferred basal medium is, for example, DMEM / F12.

[0017] The medium may be a basal medium to which components have been appropriately 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 substitute for serum. The serum substitute is preferably composed of known components. The serum substitute is preferably free of components derived from a different animal species. 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, etc. 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, albumin (bovine serum albumin (BSA), etc.).

[0018] The medium is preferably a medium obtained by adding a serum substitute to a basal medium, more preferably a medium obtained by adding at least one selected from the group consisting of ITS-X supplement, KSR, L-ascorbic acid, and albumin (BSA, etc.) as a serum substitute to a basal medium, and even more preferably a medium obtained by adding ITS-X supplement, KSR, L-ascorbic acid, and albumin (BSA, etc.) to a basal medium. As the basal medium, it is preferable to use DMEM / F12 medium.

[0019] 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 they are without differentiating them (TS cell maintenance medium). In this case, the medium preferably does not contain components that induce the differentiation of TS cells (e.g., growth factors, kinase inhibitors, signal transduction inhibitors, signal transduction activators, differentiation inducers, etc.). The medium may be one that induces the differentiation of TS cells (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 differentiation induction medium for TS cells into EVT may contain growth factors such as neuregulin (NRG)1; TGFβ inhibitors such as A83-01; and ROCK inhibitors such as Y27632. The medium for inducing differentiation of TS cells into ST cells may contain 2-mercaptoethanol, a ROCK inhibitor such as Y27632, and forskolin, etc. The medium preferably does not contain components derived from different animals, and is preferably xeno-free.

[0020] Specific examples of preferred media include DMEM / F12 medium and the "basal medium for TS cells" used in the Examples described below.

[0021] <Culture> TS cells can be cultured by known methods. The culture conditions can be those generally used for culturing animal cells. The culture temperature is, for example, 30 to 40°C, preferably 35 to 38°C, and typically 37°C. The CO2 concentration may be 2 to 5%, and is typically 5%.

[0022] The culture vessel is not particularly limited, and any culture vessel generally 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.

[0023] The culture can be started 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, 10 4 ~10 8 cells / mL is preferred.

[0024] The culture may be adhesion culture or suspension culture, but adhesion culture is 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) for the purpose of improving adhesion to TS cells. Examples of cell-supporting substrates include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, etc. In addition, commercially available extracellular matrix products such as Matrigel (registered trademark) (Corning Life Science) and iMAtrix (registered trademark) 511 may be used.

[0025] The culture period is preferably 1 day or more, and more preferably 2 days or more. The upper limit of the culture period is not particularly limited, and the culture may be continued until the cells become confluent. The culture period may be, for example, 1 to 10 days, 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 the medium before replacement or may be different, but is preferably the same. The medium before medium replacement obtained by medium replacement may be used as a culture supernatant or may be discarded.

[0026] <Culture supernatant> The TS cell culture supernatant is a liquid portion of a 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, 0 to 10 2 From the viewpoints of quality maintenance and safety, it is preferable that the TS cell culture supernatant does not contain TS cells (0 cells / mL).

[0027] The culture supernatant of TS cells can be obtained from a culture in which TS cells are cultured. The culture of TS cells may be, for example, a culture in which TS cells are cultured in a serum-free medium, or a culture in which TS cells are cultured for 1 to 10 days. A preferred example of the culture of TS cells is a culture in which TS cells are cultured in a serum-free medium for 2 to 3 days. A preferred example of the culture supernatant of TS cells is a culture supernatant obtained from a culture in which TS cells are cultured in a serum-free medium for 2 to 3 days.

[0028] 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 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 the culture of TS cells is preferably filtered after centrifugation. When TS cells are cultured in suspension, the culture can be centrifuged to obtain a culture supernatant. After centrifugation, filtration is preferably performed. The conditions for centrifugation and filtration may be the same as those described above.

[0029] The culture supernatant of TS cells is preferably frozen and stored until use at a temperature of, for example, -80°C to -20°C, with -80°C being preferred.

[0030] <How to use> The culture supernatant of TS cells is used as an implantation rate enhancer. The implantation rate 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 rate enhancer is used to treat the endometrium before embryo transfer. Treatment of the endometrium with the implantation rate enhancer can be performed by injecting the implantation rate enhancer into the uterine cavity.

[0031] 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 pharma- ceutical inactive diluent (e.g., sterilized purified water, physiological saline, PBS, the medium used for culturing TS cells, etc.).

[0032] The subject of use of the implantation rate enhancer 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 of use is preferably a primate, and more preferably a human. The subject for which the implantation rate enhancer 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.

[0033] In order to increase the implantation rate during embryo transfer, the implantation rate improver is injected into the uterine cavity of the subject prior to the implantation of the embryo into the uterus of the subject. This allows the implantation rate improver to come into contact with the endometrium of the subject, and the endometrium can be treated with the implantation rate improver. The implantation rate improver is preferably injected into the uterine cavity of the subject 1 to 5 days, more preferably 2 to 4 days, before the embryo is transferred into the uterine cavity of the subject. The injection may be performed once (for example, once 3 days before), or multiple times every other day or every day. The volume of the implantation rate improver injected into the uterine cavity may be sufficient to treat the endometrium. The volume of the implantation rate improver may be, for example, 0.01 to 1 mL, and preferably 0.05 to 0.5 mL.

[0034] 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.

[0035] According to the implantation rate improver of the present embodiment, by injecting it into the uterine cavity of the subject prior to embryo transfer, the endometrial epithelium can be treated with the implantation rate improver. This improves the embryo receptive capacity of the endometrial epithelium, and improves the implantation rate of the embryo in the embryo transfer. This increases the pregnancy success rate of the subject who received the embryo transfer.

[0036] [Pharmaceutical composition] A second aspect of the present disclosure is a pharmaceutical composition for treating or preventing implantation failure, the pharmaceutical composition comprising the implantation rate improver according to the first aspect as an active ingredient.

[0037] The pharmaceutical composition of this embodiment may contain other components other than the implantation rate improver according to the first aspect (culture supernatant of TS cells). Examples of other components include pharma- ceutically acceptable carriers. The term "pharma-ceutically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of an active ingredient and does not show substantial toxicity to the subject of administration. The term "not substantially toxic" refers to a carrier that does not show substantial toxicity to the subject of administration at a dose that is normally used. In the pharmaceutical composition of this embodiment, the pharma-ceutically acceptable carrier is a carrier that does not inhibit the implantation rate improver activity of the implantation rate improver according to the first aspect and does not show substantial toxicity to the subject of administration. The pharma-ceutically acceptable carrier includes any known pharma-ceutically acceptable component that is typically considered to be a non-active component. The pharma-ceutically acceptable carrier is not particularly limited, and examples thereof include a diluent, a buffer, a vehicle, and the like. The pharma-ceutically acceptable carrier may be used alone or in combination of two or more types.

[0038] The pharmaceutical composition may contain other ingredients in addition to the above 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.

[0039] The dosage form of the pharmaceutical composition is not particularly limited, and can be a dosage form generally used as a pharmaceutical preparation. The pharmaceutical composition of the present embodiment is a parenteral preparation, which is injected into the uterine cavity. The dosage form of the pharmaceutical composition is preferably a liquid.

[0040] The pharmaceutical composition can be administered in a therapeutically effective amount of the implantation rate improver according to the first aspect. The term "therapeutically effective amount" refers to the amount of a drug effective for treating or preventing a target disease. For example, the therapeutically effective amount of the implantation rate improver can be an amount 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 culture supernatant of TS cells administered.

[0041] The pharmaceutical composition may be administered once or repeatedly. For example, the pharmaceutical composition may be administered only once, 1 to 5 days, more preferably 2 to 4 days, before the embryo transfer. Alternatively, the pharmaceutical composition may be administered every other day or every day, starting 2 to 5 days before the embryo transfer.

[0042] 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, the implantation rate can be improved by injecting the pharmaceutical composition into the uterine cavity prior to embryo transfer. Therefore, implantation failure can be treated or prevented.

[0043] [Method of manufacturing the implantation rate enhancer] A third aspect of the present disclosure is a method for producing an implantation rate improver. The method includes the steps of (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 according to the first aspect.

[0044] <Process (i)> Step (i) can be carried out as described above in the section entitled "Agent for improving implantation rate." Step (i) is preferably carried out in a serum-free medium. The culture period in step (i) may be 1 to 10 days.

[0045] <Process (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 the culture of TS cells by centrifugation, filtration, or the like.

[0046] <Optional process> The manufacturing method of this embodiment may include an optional step in addition to the steps described above. An example of the optional step is a step of cryopreservation. The cryopreservation temperature is, for example, −80° C. to −20° C., and −80° C. is preferred.

[0047] [Other embodiments] In one embodiment, the present disclosure provides a method for improving implantation rate, comprising the step of injecting a culture supernatant of TS cells into the uterine cavity of a subject receiving said embryo transfer prior to said embryo transfer. In one embodiment, the present disclosure provides a method of embryo transfer comprising the steps of injecting a culture supernatant of TS cells into the uterine cavity of a subject, and transferring an embryo into the uterine cavity of the subject. In the above-mentioned method, the culture supernatant of TS cells is preferably injected into the uterine cavity 1 to 5 days prior to embryo transfer, and more preferably 2 to 4 days prior to embryo transfer.

[0048] In one embodiment, the present disclosure provides use of a culture supernatant of TS cells in the manufacture of an implantation rate improver. In one embodiment, the present disclosure provides the use of a culture supernatant of TS cells in the manufacture of a pharmaceutical composition for treating or preventing implantation failure. In one embodiment, the present disclosure provides a culture supernatant of TS cells for use in improving the implantation rate of transplanted embryos. In one embodiment, the present disclosure provides a culture supernatant of TS cells for use in treating or preventing implantation failure. EXAMPLES

[0049] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0050] <Method> 1. Culturing Human TS Cells Maintenance culture of human TS cells and differentiation induction into ST cells were performed as previously reported (Okae et al. Derivation of Human Trophoblast Stem Cells. Cell Stem Cell (2018), 22(1), 50-63.e6.). In addition, a cell line that constitutively expresses EGFP (Enhanced Green Fluorescent Protein) was used to track the cells during co-culture with endometrial epithelial organoids.

[0051] 2. Obtaining human TS cell culture supernatant (TS-CM) i-Matrix (registered trademark) 511 (Nippi Corporation) was placed at 1 μL / well into the wells of a 6-well flat-bottom well plate (IWAKI Microplate, AGC Technoglass Corporation) 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 medium for TS cell culture or DMEM / F-12 medium was placed into the coated well plate. 1x10 human TS cells were cultured at 1x10 5 The cells were seeded at a cell density of 10000 cells / well, cultured at 37°C and 5% CO2 for 2 or 3 days, and then washed with PBS. The medium was then replaced with TS cell basal medium or DMEM / F-12 medium, and cultured at 37°C and 5% CO2 for 2 days. The supernatant was then collected and centrifuged at 500g for 5 minutes, filtered through a 0.22μm filter, and stored at -80°C until use. When obtaining ST-CM, ST cells were induced from the above TS cells by the differentiation induction method according to a previous report (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.

[0052] 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.

[0053] 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 who provided consent was cut into small pieces, soaked in an enzyme solution (collagenase V / dispase II), and reacted with shaking at 37°C for 1 hour. The cell fraction trapped in a cell strainer (100 μm) was then embedded in Matrigel and cultured. The medium was changed once every 2-3 days. When subculturing, the cells were detached from the culture plate using basal TS cell medium and fragmented by pipetting. The fragmented organoids were diluted, embedded in Matrigel, and subcultured.

[0054] 3. Preparation of human endometrial model The fragmented human endometrial epithelial organoids were suspended in a matrix of collagen gel and Matrigel (registered trademark) (Corning) mixed together (collagen gel: Matrigel = 7: 3 (volume ratio)). Droplets of the suspension were left to stand in a 48-well plate for 30 minutes to solidify, and then cultured in ESCY medium for 7 to 8 days. Estradiol (E2) (10 nM) was then added and cultured for another 2 days, after which E2 (10 nM), medroxyprogesterone acetate (MPA) (1 μM) and 8-Bromo-cAMP (50 μM) were added to perform maturation.

[0055] The composition of the 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),200mM L-ascorbic acid(FUJIFILM Wako),50ng / ml EGF,2μM CHIR99021,2μM SB202190, and 10μM Y27632

[0056] 4. Immunostaining Cells and organoids were fixed with 4% paraformaldehyde (PFA), washed with PBS, and then blocked for nonspecific reactions with 5% normal goat serum and 0.3% Triton X-100 in PBS for 1 hour. Primary antibodies were then added and incubated overnight at 4°C, followed by staining with fluorescently labeled secondary antibodies (Cell Signaling) and Hoechst 33342 (Dojindo). Observations were performed using BZ-X810 (Keyence) and LSM 710 (Carl Zeiss).

[0057] 5. Quantitative Real-time RT-PCR RNA was extracted and purified from cells and organoids using the RNeasy Mini Kit (QIAGEN). cDNA was prepared using PrimeScript II (Takara Bio), and real-time PCR was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems) and TB Green Premix Ex Taq II (Takara Bio).

[0058] 6.RNA-sequencing RNA was extracted and purified from cells and organoids using the RNeasy Mini Kit (QIAGEN), and genomic DNA was digested with DNase I (QIAGEN). RNA libraries were prepared using NEBNext® Ultra TM The RNA library was generated using the II Directional RNA Library Prep Kit (New England Biolabs) and sequenced using 150 bp paired-end reads on the Illumina NovaSeq 6000 platform (Illumina). 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.

[0059] 7. Induction of blastoids from human ES cells As previously reported (Yu et al. Blastocyst-like structures generated from human pluripotent stem cells. Nature (2021) 591, 620-626.), blastocyst-like structures (blastoids) were prepared from naive ES cells.

[0060] 8. In vitro implantation assay Ishikawa cells (endometrial epithelial cells) were seeded in 96-well plates and cultured at 37°C in 10% FBS-containing DMEM medium (Life Technologies) until confluent. In the treatment 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. After that, blastocyst-like structures (Blastoids) made from naive pluripotent stem cell-type ES cells were added to the treatment and untreated groups and co-cultured at 37°C. The adhesion rate of blastocyst-like structures (Blastoids) made from naive ES cells was quantified 5 hours after co-culture in the treatment and untreated (control) groups.

[0061] <Experimental Results> To develop an infertility treatment using human TS cells, we investigated the effect of TS cells on endometrial epithelial cells. When TS cells (GFP-TS) cells that constitutively express EGFP were co-cultured with endometrial epithelial organoids (EMOs) in Matrigel, the epithelial cell layer in contact with the TS cells thickened (Fig. 1). Furthermore, immunostaining confirmed that endometrial epithelial cells in contact with TS cells were positive for the proliferative cell marker Ki67, hormone-responsive PGR, and glandular epithelial marker FOXA2 (Fig. 2). This suggests that the proximity of TS cells induces cell proliferation, hormone response, and glandular epithelial differentiation in endometrial epithelial cells.

[0062] Next, to confirm whether this effect was due to direct contact or humoral factors, 250 μL of TS cell culture supernatant (TS-CM) was added to the endometrial model prepared in "3. Preparation of 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" indicates that 250 μL of ST cell culture supernatant (ST-CM) was added to the endometrial model instead of TS-CM. "Hormone" indicates that EPCP (E2 (10 nM), MPA (1 μM), 8-Bromo-cAMP (50 μM), and prolactin (50 mg / mL)) was added instead of TS-CM. The control is a sample that was not treated with any of the above (untreated). In the endometrial model treated with ST-CM, the expression of PAEP and SPP1 was increased compared to the control, but the increase in expression was lower than that in the model treated with TS-CM. In the endometrial model treated with hormone, the expression of PAEP and SPP1 was increased compared to the control, but the increase in SPP1 expression was lower than that 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.

[0063] In addition, RNA was extracted from endometrial models treated with TS-CM, ST-CM, or Hormone (EPCP) and subjected to RNA-seq analysis. In the RNA-seq analysis, the expression of the top 20 genes (ERA-UP(TO20)) that increase during the implantation stage, which are used in the endometrial receptivity analysis (ERA), was examined. The expression of most genes (13 / 20 genes) was increased by TS-CM treatment. In addition, GO term analysis showed that terms related to signals that increase in the endometrial epithelium during the early implantation stage, such as "regulation of inflammatory response," were enriched (Figures 4A and 4B).

[0064] Next, the implantation-promoting effect of TS-CM was verified using an implantation assay (see "8. In vitro implantation assay" above) using the adhesion of blastoids to the apical surface of plate-cultured endometrial epithelial cells (Ishikawa) as an indicator. By pretreating endometrial epithelial cells (Ishikawa) with TS-CM, the adhesion rate of blastoids after 4 hours of co-culture increased 1.6-fold compared to the untreated group (Control) (Figure 5).

[0065] These results confirmed that TS-CM has the effect of improving the embryo receptive capacity of endometrial epithelium, suggesting that the secretion of TS cells improves the embryo receptive capacity of endometrial epithelium. [Industrial Applicability]

[0066] 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.

Claims

1. An agent for improving implantation rate, containing culture supernatant of trophoblast stem cells.

2. The method of 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. 3. The implantation rate improver according to claim 1, 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. (i) culturing trophoblast stem cells; (ii) obtaining a culture supernatant from the culture obtained by the culture; A method for producing an implantation rate improver comprising the steps of:

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

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

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