Implantation promoting drug and screening system thereof

Abstract

The present invention provides: a drug which can improve the implantation rate of an embryo and can be used in conjunction with conventional drugs for infertility treatment; and a screening method and the like with which it is possible to reliably evaluate such a drug. Specifically, the integrin activator comprises: (1) a peptide composed of KFEERMRCKWMT, and (2) a peptide composed of KFEERMRCKWMT. (2) a peptide which is composed of KFEEERSRCKWMT; (3) a peptide comprising an amino acid sequence obtained by deleting, substituting and / or adding 1-3 amino acids in (1) or (2); or (4) a peptide obtained by binding any one of (1) to (3) to a membrane-permeable molecule. Provided are an artificial uterus and a screening method based on an implantation reproduction test system using a fertilized egg (for example, an embryo developed to an implanted blastocyst), the artificial uterus comprising: a hydrogel containing endometrial stromal cells and having an extracellular matrix as a main component; and a uterine epithelial organoid configured to be surrounded by the hydrogel.

Description

Technical Field

[0001] The present invention relates to: an integrin activator containing a specific peptide; an artificial uterus including a hydrogel containing endometrial stromal cells and mainly composed of an extracellular matrix and uterine epithelial organoids configured to be surrounded by the hydrogel; and a screening method for an infertility treatment agent using the artificial uterus, etc. Background Art

[0002] In developed countries, late marriage has become more common due to social conditions, leading to an increasing number of patients receiving infertility treatment. In Japan, where countermeasures against the declining birthrate are urgent, against the backdrop of this late marriage, the number of infertility treatments has exceeded 300,000 cases. In infertility treatment, three stages of treatment are carried out: hormone treatment to improve the uterine state, artificial insemination in which sperm and eggs are artificially introduced into the uterus, and highly reproductive assisted medical treatments such as in vitro fertilization or microinsemination. In particular, infertility treatments focusing on gametes such as artificial insemination and microinsemination are emphasized. In existing infertility treatments, although the probability of sperm and eggs meeting can be significantly increased, implantation that occurs later is left to nature.

[0003] Although implantation is left to nature in this way, in the case of advanced age, not only does the quality of gametes decline, but the efficiency of fertilized eggs implanting in the uterus also significantly decreases. Therefore, for example, the implantation rate of embryos in artificial insemination is 35% in the first half of the 30s, 25% in the second half of the 30s, and less than 10% for those over 40 years old, showing a decreasing trend with age.

[0004] However, although research has been conducted on treatment methods directly acting on implantation, methods showing effective improvement are still unknown. Therefore, there is a demand for the development of a medicament that can increase the implantation rate of embryos and can be used in combination with existing medicaments for infertility treatment (Patent Document 1). In addition, there is also a desire to establish a screening system that can reliably evaluate such a medicament.

[0005] Prior Art Documents

[0006] Patent Documents

[0007] Patent Document 1: International Publication No. 2019 / 101368. Summary of the Invention

[0008] Problems to be Solved by the Invention

[0009] Therefore, the problems of the present invention are to provide: a medicament that can increase the implantation rate of embryos and can be used in combination with existing medicaments for infertility treatment; and a screening method that can reliably evaluate such a medicament, etc.

[0010] Means for Solving the Problems

[0011] The present inventor has studied the existing infertility treatment, focusing on the following aspects: although the probability of sperm and egg meeting can be significantly increased in this treatment, the implantation that occurs afterwards is left to nature. In the case of attempting to screen for substances that change the implantation rate, first, the present inventor thought of simplifying the event of implantation that actually occurs in the organism to establish a screening system, which is a complex mechanism in which multiple mechanisms of both the endometrium and the embryo act to make the embryo adhere and infiltrate the endometrial stroma. By establishing this screening system, multiple samples can be evaluated quickly and reliably, and as a result, it is believed that it is related to the acquisition of substances that can improve the implantation rate. Therefore, the present inventor thought of controlling the existence environment of uterine epithelial organoids and the polarity of uterine epithelial organoids, and implanting naturally only by contacting the embryo (blastocyst) with the uterine epithelial organoids. When conducting in-depth research, the screening method of the present invention, etc., was successfully established.

[0012] In addition, in an attempt to improve the embryo implantation rate, further research was conducted on which substance to target, focusing on the adhesion between endometrial epithelial cells and embryonic trophoblast cells, and focusing on integrins associated with the adhesion. The inventors came up with the idea of ​​whether the embryo implantation rate could be improved by using a substance that activates the targeted integrin.

[0013] In order to realize the above concept, the screening method of the present invention has been completed, and it has been confirmed that substances that activate integrins can actually improve embryo implantation rates. Therefore, the structure of substances that can change the activity of integrins was predicted from the three-dimensional structure of integrins, and more than 60,000 substances were evaluated. Based on these insights, the inventors further conducted repeated research and completed the present invention.

[0014] That is, the present invention is as follows.

[0015] [1] An integrin activator comprising:

[0016] (1) Peptide consisting of KFEEERMRCKWMT;

[0017] (2) a peptide consisting of KFEEERSRCKWMT;

[0018] (3) a peptide consisting of an amino acid sequence in which 1 to 3 amino acids in (1) or (2) are deleted, substituted and / or added; or

[0019] (4) A peptide obtained by binding any one of (1) to (3) to a membrane-permeable molecule.

[0020] [2] The agent described in [1], wherein the membrane permeable molecule is myristic acid.

[0021] [3] The agent described in [1] or [2], which is used for promoting embryo implantation.

[0022] [4]An infertility treatment agent, comprising the agent according to any one of [1] to [3] and a hormonal agent.

[0023] [5]An artificial uterus, comprising a hydrogel and uterine epithelial organoids configured to be surrounded by the hydrogel, the hydrogel containing endometrial stromal cells and having an extracellular matrix as a main component.

[0024] [6]The artificial uterus according to [5], wherein an opening is provided in the upward direction of the hydrogel configured to surround the uterine epithelial organoids.

[0025] [7]The artificial uterus according to [5] or [6], wherein the extracellular matrix contains laminin and / or a fragment thereof.

[0026] [8]The artificial uterus according to any one of [5] to [7], wherein for the uterine epithelial organoids, the outside of the organoids is the apical side.

[0027] [9]A kit for manufacturing an artificial uterus, comprising: a device having a support body with an object surface and at least one protrusion protruding from the object surface; and a solution having an extracellular matrix as a main component.

[0028]

[10] A method for manufacturing an artificial uterus, comprising the following steps:

[0029] Step (1), filling a culture vessel with a solution containing endometrial stromal cells and having an extracellular matrix as a main component;

[0030] Step (2), pressing a device having a support body with an object surface and at least one protrusion protruding from the object surface into the filled solution; and

[0031] Step (3), after the filled solution gels, removing the pressed device.

[0032]

[11] A method for screening an infertility treatment agent, comprising the following steps:

[0033] Step (1), contacting the artificial uterus according to any one of [5] to [8] with a fertilized egg in the presence or absence of a test substance;

[0034] Step (2), measuring the implantation rate of the uterine epithelial organoids and the fertilized egg; and

[0035] Step (3), when the implantation rate is higher in the presence of a candidate substance than in the absence of the test substance in Step (2), screening the test substance as a candidate substance for a therapeutic or prophylactic drug for infertility.

[0036]

[12] A method for promoting embryo implantation in an object, comprising: administering the agent described in [1] or [2].

[0037]

[13] A method for treating infertility in an object, comprising: administering a therapeutically effective amount of the agent described in [1] or [2].

[0038]

[14] The method described in

[13] , wherein the agent further comprises a hormonal agent.

[0039] [I]An embryo implantation promoter or an infertility treatment agent, comprising:

[0040] (1) A peptide consisting of KFEEERMRCKWMT;

[0041] (2) A peptide consisting of KFEEERSRCKWMT;

[0042] (3) A peptide consisting of an amino acid sequence obtained by deleting, substituting and / or adding 1 to 3 amino acids in (1) or (2); or

[0043] (4) A peptide obtained by conjugating a membrane-permeable molecule to any one of (1) to (3).

[0044] [II]The agent described in [I], wherein the peptide conjugated with the membrane-permeable molecule is a myristoylated peptide.

[0045] [III]The agent described in [I] or [II], which comprises a hormonal agent.

[0046] [IV]An integrin activator, comprising:

[0047] (1) A peptide consisting of KFEEERMRCKWMT;

[0048] (2) A peptide consisting of KFEEERSRCKWMT;

[0049] (3) A peptide consisting of an amino acid sequence obtained by deleting, substituting and / or adding 1 to 3 amino acids in (1) or (2); or

[0050] (4) A peptide obtained by conjugating a membrane-permeable molecule to any one of (1) to (3).

[0051] [V]An artificial uterus, comprising a hydrogel and uterine epithelial organoids configured to be surrounded by the hydrogel, the hydrogel comprising endometrial stromal cells and having an extracellular matrix as a main component, and having an opening in an upward direction of the hydrogel configured to surround the uterine epithelial organoids.

[0052] [VI]The artificial uterus described in [V], wherein the extracellular matrix comprises laminin and / or a fragment thereof.

[0053] [VII][V] The artificial uterus described above, wherein, for uterine epithelial organoids, the outer side of the organoid is the apical side.

[0054] [VIII] A kit for manufacturing an artificial uterus, comprising: a device having a support body with a substance-bearing surface and at least one protrusion protruding from the substance-bearing surface; and a solution mainly composed of an extracellular matrix.

[0055] [IX] A method for manufacturing an artificial uterus, comprising the following steps:

[0056] Step (1), filling a culture vessel with a solution containing endometrial stromal cells and mainly composed of an extracellular matrix;

[0057] Step (2), pressing a device having a support body with a substance-bearing surface and at least one protrusion protruding from the substance-bearing surface into the filled solution; and

[0058] Step (3), after the filled solution gels, removing the pressed device.

[0059] [X] A method for screening an infertility therapeutic agent, comprising the following steps:

[0060] Step (1), contacting the artificial uterus described in [V] with a fertilized egg in the presence or absence of a test substance;

[0061] Step (2), measuring the implantation rate of uterine epithelial organoids and the fertilized egg; and

[0062] Step (3), when the implantation rate is higher in the presence of a candidate substance than in the absence of the test substance in Step (2), screening the test substance as a candidate substance for a therapeutic or prophylactic drug for infertility.

[0063] [XI] The following peptides for use in promoting embryo implantation or infertility treatment:

[0064] (1) A peptide composed of KFEEERMRCKWMT;

[0065] (2) A peptide composed of KFEEERSRCKWMT;

[0066] (3) A peptide composed of an amino acid sequence obtained by deleting, substituting, and / or adding 1 to 3 amino acids in (1) or (2); or

[0067] (4) A peptide obtained by conjugating a membrane-permeable molecule to any one of (1) to (3).

[0068] [XII] The following peptides for use in integrin activation:

[0069] (1) A peptide consisting of KFEEERMRCKWMT;

[0070] (2) A peptide consisting of KFEEERSRCKWMT;

[0071] (3) A peptide consisting of an amino acid sequence obtained by deleting, substituting and / or adding 1 to 3 amino acids in (1) or (2); or

[0072] (4) A peptide obtained by combining a membrane-permeable molecule with any one of (1) to (3).

[0073] The peptide described in [XIII], [XI] or [XII], wherein the peptide combined with the membrane-permeable molecule is a myristoylated peptide.

[0074] Use of the following peptide for producing an embryo implantation promoter or an infertility treatment agent:

[0075] (1) A peptide consisting of KFEEERMRCKWMT;

[0076] (2) A peptide consisting of KFEEERSRCKWMT;

[0077] (3) A peptide consisting of an amino acid sequence obtained by deleting, substituting and / or adding 1 to 3 amino acids in (1) or (2); or

[0078] (4) A peptide obtained by combining a membrane-permeable molecule with any one of (1) to (3).

[0079] Use of the following peptide for producing an integrin activator:

[0080] (1) A peptide consisting of KFEEERMRCKWMT;

[0081] (2) A peptide consisting of KFEEERSRCKWMT;

[0082] (3) A peptide consisting of an amino acid sequence obtained by deleting, substituting and / or adding 1 to 3 amino acids in (1) or (2); or

[0083] (4) A peptide obtained by combining a membrane-permeable molecule with any one of (1) to (3).

[0084] The use described in [XVI], [XIV] or [XV], wherein the peptide combined with the membrane-permeable molecule is a myristoylated peptide.

[0085] Advantages of the Invention

[0086] According to the present invention, by activating integrin, the embryo implantation rate after natural pregnancy or artificial insemination can be increased. In addition, the agent of the present invention can also be used in combination with existing agents for infertility treatment, so it can be one of the countermeasures for solving the problem of low birthrate. In addition, it also results in a reduction in infertility treatment costs or increased subsidies related to such treatment. Moreover, according to the present invention, the evaluation of substances that can increase the embryo implantation rate can be carried out quickly and reliably. Therefore, the present invention is also very useful for obtaining such substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 Figure 1 ( Figure 1 A to Figure 1 F) show an overview of the artificial uterus (in vitro implantation system) of the present invention. Figure 1 A shows that the uterine organoid is a spherical cell mass composed of a single-layer columnar epithelial cell layer in Matrigel. Figure 1 B shows the artificial uterus of the present invention in which a uterine organoid (endometrial epithelial organoid) and a zona pellucida-free blastocyst are arranged in a pore of a hydrogel containing endometrial stromal cells and having a main component of extracellular matrix (Matrigel TM ). Figure 1 C to E show that stromal cells are suspended in Matrigel, placed on a glass-bottomed culture dish, and a mold made by a 3D printer Figure 1 C) is placed and fixed to produce a structure having a pore with a diameter of 200 μm Figure 1 D, E). Figure 1 On the right side of F, it shows that in the uterine organoid cultured in Matrigel, the nucleus and actin fibers are oriented from the outside to the inside of the sphere. Figure 1 On the left side of F, it shows that in the uterine organoid cultured in an ultra-low adsorption incubator (ultra-low adsorption culture dish), the nucleus and actin fibers are oriented from the inside to the outside of the sphere.

[0088] Figure 2 Figure 2 Shows one embodiment of the artificial uterus and the kit for producing the artificial uterus of the present invention.

[0089] Figure 3 Figure 3 Shows the artificial uterus (in vitro implantation system) of the present invention and an overview of its preparation.

[0090] Figure 4 Figure 4 ( Figure 4 ​​​​​​​​Panels A and 4B) show the relevant results of the in vitro implantation system (IVIM system) observed under a confocal microscope. Figure 4 Panel A shows that although the blastocyst initially contacts the uterine organoids, it gradually invades the uterine organoids and also shows the interaction with the stromal cells present in the vicinity. Figure 4 Panel B shows that fluorescence of mCherry is observed in the blastocyst, suggesting that epithelial cells are excluded by phagocytosis and invasion occurs.

[0091] Figure 5 Figure 5 Show the relevant results of the in vitro implantation system (IVIM system). Figure 5 Show that in the blastocysts and uterine organoids / stromal cells stained with green and red fluorescence of the Cell Explorer TM Live Cell Tracking Kit, the results observed in Figure 4 Panels A and 4B were also observed.

[0092] Figure 6 Figure 6 ( Figure 6 Panels A - 6D) show the relevant results of the fusing endometrial epithelium and embryo after IVIM. Figure 6 Panel A shows that in the case where no implantation - like reaction occurred in the in vitro implantation experiment, the embryo ( Figure 6 left of Panel A) and the uterine organoids ( Figure 6 right of Panel A) were in a separated state. Figure 6 Panel B shows that if an implantation - like reaction occurred, the removed embryo fused and did not separate. Figure 6 Panel C shows the results of loading blastocysts in the in vitro implantation experimental system, removing all cells after 72 hours, treating with trypsin to form single cells, and performing single - cell RNA expression analysis. According to the gene expression patterns, they were roughly divided into three groups: placental - like cells, endometrial epithelial cells, and endometrial stromal cells. Moreover, cells expressing various marker genes such as Prdm16, Klf4, Rexo1 that play a role in stem cell maintenance, Gata6 that plays a role in endoderm formation, Nodal that plays a role in gastrulation, and Plac8, Klf4, Sdc1 that play a role in placenta formation were dispersed, suggesting that after the embryo develops to the blastocyst stage ( Figure 6 Panel D).

[0093] Figure 7 ​​​​​​Figure 7 ( Figure 7 A to 7F) show the results of confirming the expression of genes known to be involved in implantation through single-cell RNA expression analysis. The expression of WNT signal ( Figure 7 A), Notch signal ( Figure 7 B), matrix metalloproteinase ( Figure 7 C), TGFβ ( Figure 7 D), LIF ( Figure 7 E), integrin ( Figure 7 F), etc. implantation-related genes in cells.

[0094] Figure 8 Figure 8 Show the measurement results of the integrin activation ability of two peptides (Iznm-1 and Iznm-2) obtained from more than 60,000 peptides. At a concentration of 8 μM, an activation effect of about 5-fold was confirmed for Iznm-1, and an activation effect of about 14-fold was confirmed for Iznm-2.

[0095] Figure 9 Figure 9 ( Figure 9 A to 9D) show the results of confirming that the peptide drug Iznm-2 causes integrin activation (Active-Integrinβ1) in embryos. As a result, compared with the control group ( Figure 9 A, 9C), strongly fluorescently stained cells (especially the part indicated by the arrow) were detected in the experimental group ( Figure 9 B, 9D).

[0096] Figure 10 Figure 10 ( Figure 10 A to 10C) show the related results that Iznm-2 promotes the adhesion of embryos (mouse blastocysts) to the glass surface. The embryos in the control group did not adhere, but embryo adhesion to the glass surface was observed in the experimental group with the addition of Iznm-2 ( Figure 10 A, 10B). In addition, the ratio was 0 / 13 in the control, and 13 / 14 when Iznm-2 was added. A significant difference was detected between the two groups (p < 0.01, chi-square test, Figure 10 C).

[0097] Figure 11 Figure 11 Show the related results of the effects of peptide drugs (Iznm-1 and 2) in the in vitro implantation system (the proportion of implantation reactions observed in the in vitro implantation system).

[0098] Figure 12 Figure 12 ​​​​​​​​​​Experimental overview of the effects of Iznm-2 on the implantation and development of transplanted embryos. By staggering the number of days after mating between the transplanted embryos and the recipient mice, the state of misaligned implantation windows was reproduced.

[0099] Figure 13 Figure 13 ( Figure 13 A–13D) Results related to the effects of Iznm-2 on the implantation and development of transplanted embryos. Compared with the Figure 13 control in A, in the experimental group where 8 μM of Iznm-2 was added at the time of embryo transplantation in Figure 13 B, multiple implanted / developed embryos were confirmed at the E15.5 time point ( Figure 13 A–13C). In addition, in the Iznm-2 group, approximately half of the implanted embryos developed, while the others did not develop at all ( Figure 13 C, 13D).

[0100] Figure 14 Figure 14 ( Figure 14 A, 14B) Results related to the effects of Iznm-2 on the implantation and development of transplanted embryos. Figure 14 A shows the embryo morphology at the E15.5 time point, Figure 14 and B is also the HE staining image of the FFPE section of the embryo at the E15.5 time point. Detailed implementation mode

[0101] 1. Artificial uterus

[0102] The present invention provides an artificial uterus, which includes a hydrogel and uterine epithelial organoids configured to be surrounded by the hydrogel. The hydrogel contains endometrial stromal cells and has an extracellular matrix as the main component.

[0103] In this specification, "endometrial stromal cell" refers to the cells that constitute the supporting tissue of endometrial epithelial cells and refers to the cells contained in the endometrial stromal layer. The endometrial stromal cells used in the present invention can be primary endometrial stromal cells isolated from endometrial tissue, or immortalized endometrial stromal cells, or endometrial stromal cells induced from (induced) pluripotent stem cells such as ES cells, nt ES cells, iPS cells, mGS cells, EG cells, and Muse cells.

[0104] In the present invention, the density of endometrial stromal cells present in the hydrogel (cells / cm 3 ​​​​)There is no particular limitation as long as the subsequent fertilized eggs (blastocysts) can implant on the uterine epithelial organoids. For example, it is in the range of 1×10 6 cells / cm 3 ~1×10 7 cells / cm 3 , preferably in the range of 2×10 6 cells / cm 3 ~3×10 6 cells / cm 3 .

[0105] In this specification, "endometrial epithelial cell" is the epithelial cell contained in the uterine mucosa (endometrium), and refers to the cell contained in the endometrial epithelial layer. The endometrial epithelial cell used in the present invention can be a primary endometrial epithelial cell isolated from endometrial tissue, a cultured endometrial epithelial cell, or an endometrial epithelial cell induced from (induced) pluripotent stem cells such as ES cells, nt ES cells, iPS cells, mGS cells, EG cells, Muse cells, etc.

[0106] In this specification, "pluripotent stem cell" refers to a stem cell that can differentiate into various tissues or cells with different morphologies or functions in an organism and has the ability to differentiate into any system of the three germ layers (endoderm, mesoderm, ectoderm). As the pluripotent stem cell used in the present invention, for example, induced pluripotent stem cell (iPS cell), embryonic stem cell (ES cell), embryonic stem cell from a cloned embryo obtained by nuclear transfer (ntES cell), multipotent germline stem cell ("mGS cell"), embryonic germ cell (EG cell) can be mentioned, and preferably iPS cell (more preferably human iPS cell). When the above pluripotent stem cell is an ES cell or any cell from a human embryo, the cell can be a cell prepared by destroying the embryo or a cell prepared without destroying the embryo, and preferably a cell prepared without destroying the embryo.

[0107] ES cells are stem cells established from the inner cell mass of mammalian (early) embryos (e.g., blastocysts) such as those of humans or mice, and have pluripotency and the proliferative ability of self-renewal. ES cells were discovered in mice in 1981 (M.J. Evans and M.H. Kaufman (1981), Nature 292:154-156). Subsequently, ES cell lines were also established in primates such as humans and monkeys (J.A. Thomson et al. (1998), Science 282:1145-1147; J.A. Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; J.A. Thomson et al. (1996), Biol. Reprod., 55:254-259; J.A. Thomson and V.S. Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by removing the inner cell mass from the blastocyst of a fertilized egg of the target animal and culturing the inner cell mass on a feeder layer of fibroblasts. Alternatively, ES cells can also be established using only a single blastomere of an embryo at the cleavage stage before the blastocyst stage (Chung Y et al. (2008), Cell Stem Cell 2:113-117), and can also be established using developmentally arrested embryos (Zhang X et al. (2006), Stem Cells 24:2669-2676.).

[0108] nt ES cells are ES cells derived from cloned embryos produced by nuclear transfer technology and have almost the same characteristics as ES cells derived from fertilized eggs (Wakayama T. et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502). That is, ES cells established from the inner cell mass of blastocysts of cloned embryos obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell are nt ES (nuclear transfer ES) cells. To produce nt ES cells, a combination of nuclear transfer technology (Cibelli J.B. et al. (1998), Nature Biotechnol., 16:642-646) and ES cell production technology (described above) is used (Kaoru Wakayama et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Suppl.), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell can be injected into an enucleated unfertilized egg of a mammal and cultured for several hours to perform reprogramming.

[0109] As the ES cell line used in the present invention, for mouse ES cells, various mouse ES cell lines established by, for example, inGenioustargeting laboratory, RIKEN (The Institute of Physical and Chemical Research), etc. can be used. For human ES cell lines, various human ES cell lines established by, for example, the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, and Cellartis, etc. can be used. Specifically, for example, as human ES cell lines, there can be mentioned: CHB-1 to CHB-12 strains, CHB-1 to CHB-12 strains, RUES1 strain, RUES2 strain, HUES1 to HUES28 strains, etc. sold by ESIBio; H1 strain, H9 strain, etc. sold by WiCell Research; KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, SSES3 strain, etc. sold by RIKEN.

[0110] iPS cells are cells reprogrammed by introducing specific factors (nuclear reprogramming factors) into mammalian somatic cells or undifferentiated stem cells. Currently, there are various types of iPS cells. In addition to iPSC established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126:663-676), the following can also be used: iPSC from human cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S. et al., Cell, (2007) 131:861-872.); Nanog-iPSC screened and established using the expression of Nanog as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.); iPSC produced by a method without c-Myc (Nakagawa M, Yamanaka S., et al., Nature Biotechnology, (2008) 26, 101-106); iPSC established by introducing six factors by a virus-free method (Okita K et al., Nat. Methods 2011 May; 8(5):409-12, Okita K et al., Stem Cells. 31(3):458-66.), etc. In addition, the following can also be used: induced pluripotent stem cells established by Thomson et al. by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28 (Yu J., Thomson JA et al., Science (2007) 318:1917-1920.); induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ et al., Nature (2007) 451:141-146); induced pluripotent stem cells produced by Sakurada et al. (Japanese Patent Laid-Open No. 2008-307007), etc.

[0111] In addition, any of the induced pluripotent stem cells known in the art described in all published papers (e.g., Shi Y., Ding S. et al., Cell Stem Cell, (2008) Vol 3, Issue 5, 568-574; Kim JB., Scholer HR. et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA et al., Nature Biotechnology, (2008) 26, No 7, 795-797) or patents (e.g., Japanese Patent Application Laid-Open No. 2008-307007, Japanese Patent Application Laid-Open No. 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852) can be used.

[0112] As the induced pluripotent stem cell line, various iPSC lines established by NIH, RIKEN, Kyoto University, etc. can be used. For example, if it is a human iPSC line, examples include: HiPS-RIKEN-1A line, HiPS-RIKEN-2A line, HiPS-RIKEN-12A line, Nips-B2 line, etc. of RIKEN; 253G1 line, 253G4 line, 1201C1 line, 1205D1 line, 1210B2 line, 1383D2 line, 1383D6 line, 201B7 line, 409B2 line, 454E2 line, 606A1 line, 610B1 line, 648A1 line, 1231A3 line, FfI-01s04 line, etc. of Kyoto University, and preferably the 1231A3 line.

[0113] mGS cells are pluripotent stem cells derived from the testis and are cells that serve as the origin for sperm formation. These cells, like ES cells, can be induced to differentiate into various cell lineages and have properties such as being able to produce chimeric mice when transplanted into mouse blastocysts (Kanatsu-Shinohara M et al. (2003) Biol. Reprod., 69:612-616; Shinohara K et al. (2004), Cell, 119:1001-1012). They can self-renew in a culture medium containing glial cell line-derived neurotrophic factor (GDNF), and furthermore, germ stem cells can be obtained by repeated passage under the same culture conditions as ES cells (Masanori Takebayashi et al. (2008), Experimental Medicine, 26th volume, No. 5 (suppl.), 41-46 pages, Yodosha Co., Ltd. (Tokyo, Japan)).

[0114] EG cells are cells established from primordial germ cells in the fetal period and have the same pluripotency as ES cells. They can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (Matsui Y et al. (1992), Cell, 70:841-847; J.L. Resnick et al. (1992), Nature, 359:550-551).

[0115] In the present invention, pluripotent stem cells obtained by modifying genes on chromosomes using known genetic engineering methods can also be used. The pluripotent stem cells can be cells that can recognize the corresponding differentiation stage based on the expression of a marker gene by frame-integration of a marker gene (e.g., fluorescent proteins such as EGFP, GFP) into a gene encoding a differentiation marker using known methods.

[0116] There are no particular limitations on the source types of the cells (e.g., pluripotent stem cells, endometrial stromal cells, endometrial epithelial cells) used in the present invention. For example, they can be cells of rodents such as rats, mice, hamsters, guinea pigs; Lagomorpha such as rabbits; Artiodactyla such as pigs, cows, goats, sheep; Carnivora such as dogs, cats; Primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, etc. The preferred source type is Primates. In addition, the source type can be cells from the same individual from which fertilized eggs (blastocysts, etc.) suitable for the artificial uterus of the present invention were collected, or can be cells from other individuals different from the individual from which the fertilized eggs (blastocysts, etc.) were collected. Moreover, the source type of the endometrial cells does not have to be the same as the source type of the fertilized eggs (blastocysts, etc.). Also, the source type of the cells constituting the endometrial-like tissue does not have to be the same as the source type of the fertilized eggs (blastocysts, etc.).

[0117] Endometrial stromal cells can be prepared by known methods. Specifically, for example, according to the description in the Journal of Japan Society for Reproductive Medicine, Vol. 57, No. 4, page 401, October 1, 2012, they can be obtained by inducing the differentiation of prepared mesenchymal stem cells.

[0118] The mesenchymal stem cells can be primary cells directly isolated from a biological tissue containing mesenchymal stem cells. Additionally, they can also be mesenchymal stem cells differentiated / induced from an established mesenchymal stem cell line, ES cells, or induced pluripotent stem cells, or cells after cryopreserving them. Here, "directly" means without going through the process of culturing / proliferating in vitro.

[0119] As a method for obtaining mesenchymal stem cells directly isolated from a biological tissue containing mesenchymal stem cells, the desired cells can be obtained by known methods, such as the method described in International Publication No. 2017 / 094879. As the above-mentioned biological tissue containing mesenchymal stem cells, for example, bone marrow, adipose tissue, blood, placenta, umbilical cord, dental pulp, etc. can be listed. Among the above, since adipose tissue can be collected by liposuction or excision of adipose tissue, and the possibility of functional impairment to the organism is small, it is preferably used as the source for collecting the above cells.

[0120] Adipose tissue is a type of biological tissue composed of adipocytes. When used as the source for collecting the above cells, the site of the adipose tissue is not particularly limited. For example, subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat can be listed. Among them, subcutaneous fat can be simply collected under local anesthesia, so the burden on the donor during collection is small, and it can be said to be preferred.

[0121] Endometrial epithelial cells can also be prepared by known methods (Ye L et al. (2011), PLoSone, 6: e21136; Jiang X et al. (2021), Bioactive Materials, 6: 3935 - 3946). Specifically, for example, according to the description of known methods (D'Amour KA et al. (2005), Nature Biotechnology, 23: 1534 - 1541), human / mouse and other pluripotent stem cells can be obtained by inducing the differentiation of prepared embryonic endoderm cells.

[0122] In this specification, an "organoid" is a cell mass, which refers to a structure having a new function that is not present in the individual cells forming the cell mass. Preferably, it is a structure that can differentiate into an organ upon maturation. This differentiation ability can be confirmed, for example, by transplanting the structure into a living organism and studying whether it can differentiate into the target organ (if it differentiates into the target organ, it can be judged as an organoid).

[0123] The "uterine epithelial organoid (endometrial epithelial organoid, endometriumepthelial organoid)" used in the present invention refers to a cell mass that expresses at least Epcam, E-cadherin, cytokeratin, MUC1, estrogen receptor, and progesterone receptor. It exhibits the characteristics of gestational endometrium in response to estrogen (E2) and progesterone (P4) functionally and is further stimulated by human chorionic gonadotropin (hCG), human placental lactogen (hPL), and stromal cell (PRL) signals, and can synthesize PAEP (Glycodelin) or SPP1 (osteopontin). In the artificial uterus of the present invention, uterine epithelial organoids are present in the culture medium. There is no particular limitation on this culture medium, as long as it can maintain the survival of the uterine epithelial organoids, and it can be appropriately selected and used from the culture media described below. In one embodiment, the culture medium can be a WNT medium (with partial modification of Bedzhov, 2014, Nature Protocols) supplemented with 30% KSR (Thermo Fisher), N-acetylcysteine, estrogen, and progesterone.

[0124] The above-mentioned uterine epithelial organoids can be prepared by known methods (e.g., Turco et al., NATURE CELLBIOLOGY 19, 568-577 (2017)). Specifically, for example, first, the uterus is collected from an euthanized mouse and incised, and then finely cut while exposing the epithelium. Alternatively, a part of human uterine tissue is used. They are enzymatically treated with dispase to collect epithelial cells. Next, the epithelial cells are suspended in the basement membrane preparation (e.g., Matrigel) described below and cultured in Advanced DMEM / F12 medium (WNT medium) supplemented with WNT3A, R-spondin1, Noggin, EGF, and Hepes pH7.4 to prepare uterine organoids.

[0125] Regarding the (endometrial) epithelial cells used for producing uterine epithelial organoids, the cells are highly polarized and are divided into an apical side facing the outside of the organism and a lateral basal side facing the basement membrane. Organs (such as the uterus) composed of epithelial cells, etc. face the external environment on the apical side in the living body. On the other hand, the lateral basal side is connected to the extracellular matrix side of the basement membrane via integrin and its receptor.

[0126] The uterine epithelial organoids of the present invention may have the same polarity as the uterus in the living body (i.e., the apical side is located outside facing the external environment, and the lateral basal side is located inside it), or the polarity may be reversed (i.e., the lateral basal side is located outside facing the external environment, and the apical side is located inside it).

[0127] In the case of attempting to perform screening using the fertilized eggs (blastocysts, etc.) described later, the uterine epithelial organoids of the present invention preferably have the same polarity as the uterus in the living body (i.e., the apical side is located outside facing the external environment, and the lateral basal side is located inside it). Therefore, when the polarity of the produced uterine epithelial organoids is opposite to that in the living body (i.e., the lateral basal side is located outside facing the external environment, and the apical side is located inside it), the polarity can be controlled to have the same polarity as the uterus in the living body.

[0128] The polarity of the uterine epithelial organoids can be confirmed by methods known per se (Forteza R et al., (2016), Molecular Biology of the Cell, 27: 2186 - 2197). Specifically, for example, the uterine epithelial organoids are stained with Hoechst / phalloidin. When the polarity is the same as that in the living body, the actin on the apical side is detected outside by phalloidin facing the external environment, and the DNA present in the lateral basal side is detected inside by the Hoechst dye. On the other hand, when the polarity is opposite to that in the living body, the DNA present in the lateral basal side is detected outside by the Hoechst pigment facing the external environment, and the actin on the apical side is detected inside by phalloidin.

[0129] There is no particular limitation on the method for controlling polarity, as long as the polarity of the uterine epithelial organoids can be changed to the desired polarity. As an example, the method described below can be adopted (Co JY et al., (2019), Cell Reports, 26: 2509-2520). Specifically, for the fabricated uterine epithelial organoids, when attempting to make the outside of the organoids the apical side, the polarity can be controlled by culturing them in a low-adsorption or ultra-low-adsorption culture vessel as described below. As such a culture vessel, for example, an ultra-low-adsorption culture dish (Corning), an ultra-low-adsorption flask (Corning), an ultra-low-adsorption plate (Corning), a PrimeSurface culture dish (Sumitomo Bakelite Co., Ltd.), etc. can be used. The culture medium, culture conditions, or time for controlling polarity can be set appropriately. For example, as the culture medium, the WNT medium described below (WNT3A, R-spondin1, Noggin, EGF, Hepes pH 7.4, and estrogen are added to the Advanced DMEM / F12 medium) can be used. Regarding the culture temperature or culture time, for example, it is 30 to 40 °C, preferably about 37 °C, and the culture is carried out in an atmosphere of air containing CO2. The CO2 concentration is preferably about 2 to 5%, and the culture time is 1 day to several months (for example, 1, 2, 3, 4, 5, 6 months), preferably 2 weeks to 3 months.

[0130] Regarding the uterine tissue after removing the epithelial cells generated when fabricating the uterine epithelial organoids of the present invention, enzymatic treatment can be performed using collagenase, etc., to collect the above-mentioned endometrial stromal cells. In addition, the collected stromal cells can be further cultured (adherent culture) under adsorption conditions using a culture vessel or culture medium as described below. Specifically, for example, they can be cultured on a culture dish treated with cell adhesion in DMEM medium supplemented with 10% serum.

[0131] In this specification, "uterine organoids" and "uterine epithelial organoids" have the same meaning and can be used interchangeably.

[0132] In the artificial uterus of the present invention, the fabricated uterine epithelial organoids are configured to be surrounded by a hydrogel mainly composed of an extracellular matrix. In the present invention, there is no particular limitation on "configured to be surrounded by a hydrogel", as long as the fertilized egg (blastocyst) described below can implant on the uterine epithelial organoids. As a preferred embodiment, for example, a configuration having an opening in the upward direction (upper direction) of the hydrogel configured to surround the uterine epithelial organoids can be cited. As a more preferred embodiment, there is 1 opening. In addition, as a further preferred embodiment, except for one opening, the uterine epithelial organoids are configured such that all parts (surfaces) thereof are surrounded by the hydrogel.

[0133] Examples of the extracellular matrix that can be used in the present invention include laminin (Nat Biotechnol 28, 611-615 (2010)), laminin fragments (Nat Commun 3, 1236 (2012)), basement membrane preparations (Nat Biotechnol 19, 971-974 (2001)), fibronectin, gelatin, collagen, heparan sulfate proteoglycan, entactin, vitronectin, and the like.

[0134] "Laminin" refers to a heterotrimeric molecule composed of α, β, and γ chains, which is an extracellular matrix protein with isotypes having different compositions of subunit chains. Specifically, laminin is a combination of heterotrimers of 5 α chains, 4 β chains, and 3 γ chains, and has approximately 15 isotypes. The numbers of the combined α chains (α1-α5), β chains (β1-β4), and γ chains (γ1-γ3) are used to determine the name of the laminin. For example, the laminin composed of the combination of α5 chain, β1 chain, and γ1 chain is called laminin 511. (Nat Biotechnol 28, 611-615 (2010)).

[0135] The laminin used in the present invention is usually mammalian laminin. The laminin of a mammal that can be used is the same type as the cells constituting the uterine epithelial organoids or the cultured cells. For example, in the culture of human pluripotent stem cells, human laminin (preferably human laminin 511) is used.

[0136] There are no particular limitations on the laminin fragments used in the present invention, as long as the subsequent fertilized eggs (blastocysts) can implant on uterine epithelial organoids. Examples include laminin-111 and fragments containing its E8 region, laminin-211 and fragments containing its E8 region (e.g., iMatrix-211), laminin-121 or fragments containing its E8 region, laminin-221 or fragments containing its E8 region, laminin-332 or fragments containing its E8 region, laminin-3A11 or fragments containing its E8 region, laminin-411 or fragments containing its E8 region (e.g., iMatrix-411), laminin-421 or fragments containing its E8 region, laminin-511 or fragments containing its E8 region (e.g., iMatrix-511, iMatrix-511silk), laminin-521 or fragments containing its E8 region, laminin-213 or fragments containing its E8 region, laminin-423 or fragments containing its E8 region, laminin-523 or fragments containing its E8 region, laminin-212 / 222 or fragments containing its E8 region, laminin-522 or fragments containing its E8 region, etc. Among them, laminin-511 or fragments containing its E8 region are preferred. The E8 fragment of laminin 511 is commercially available and can be purchased, for example, from Nippi Co., Ltd. The laminin or laminin fragment used in the present invention is preferably isolated.

[0137] The "basement membrane preparation" in the present invention refers to a basement membrane preparation containing basement membrane components having the function of controlling epithelial cell-like cell morphology, differentiation, proliferation, motility, functional expression, etc., when desired cells having the ability to form a basement membrane are inoculated thereon and cultured. Here, the "basement membrane component" refers to extracellular matrix molecules in the form of a thin film present between the epithelial cell layer and the interstitial cell layer, etc. in animal tissues. The basement membrane preparation can be produced, for example, by removing cells having the ability to form a basement membrane adhered to a support via the basement membrane with a solution having the ability to dissolve the lipids of the cells or an alkaline solution, etc. from the support. Examples of the basement membrane preparation include commercially available products as basement membrane modulators (e.g., Matrigel TM (manufactured by Corning Inc.: sometimes also referred to as Matrigel hereinafter)) or Geltrex TM (manufactured by Life Technologies), products containing known extracellular matrix molecules (e.g., laminin, type IV collagen, heparan sulfate proteoglycan, entactin, etc.) as basement membrane components.

[0138] Matrigel TMis a basement membrane modulator extracted from Engelbreth Holm Swarm (EHS) mouse sarcoma. Matrigel TM The main components of Matrigel TM are type IV collagen, laminin, heparan sulfate proteoglycan, and entactin. In addition to these, it also includes TGFβ, FGF, tissue plasminogen activator, and growth factors naturally produced by the EHS tumor. The "growth factor-reduced product" of Matrigel TM has a lower growth factor concentration than ordinary Matrigel. Its standard concentrations are as follows: EGF is less than 0.5 ng / ml, NGF is less than 0.2 ng / ml, PDGF is less than 5 pg / ml, IGF1 is 5 ng / ml, and TGFβ is 1.7 ng / ml.

[0139] The cells (e.g., endometrial stromal cells, (endometrial) epithelial cells, etc.) or organoids (uterine epithelial organoids) used in the present invention can be cultured in a medium as described later as needed. The medium can be prepared by adding medium additives to a basal medium as needed.

[0140] Examples of the basal medium include: RPMI-1640 medium, Eagle's MEM (EMEM), Dulbecco's modified MEM (DMEM), Glasgow's MEM (GMEM), α-MEM, 199 medium, IMDM, hybridoma serum-free medium, KnockOut TM DMEM (KO DMEM), Advanced TMCulture media (e.g., Advanced MEM, Advanced RPMI, Advanced DMEM / F-12), chemically defined hybridoma serum-free media, Ham’s Medium F-12, Ham’s Medium F-10, Ham’s Medium F12K, DMEM / F-12, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy’s 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth’s Medium (e.g., Waymouth’s MB752 / 1), CMRL media (e.g., CMRL-1066), Williams’ medium E, Brinster’s BMOC-3 Medium, E8 Medium, StemPro34, MesenPRO RS (the above are from Thermo Fisher Scientific), ReproFF2, Primate ES Cell Medium, ReproStem (the above are from ReproCELL Inc.), ProculAD (Rohto Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (the above are from Lonza), EX-CELL302 medium (SAFC), or EX-CELL-CD-CHO (SAFC), ReproMed TM iPSC Medium (ReproCELL Inc.), Cellartis MSC Xeno-Free Culture Medium (Takara Bio Inc.), TESR-E8 (Veritas Inc.), StemFit (registered trademark) AK02N, AK03N (Ajinomoto Co., Inc.), and their mixtures, etc., but not limited to these.

[0141] In addition, if necessary, physiologically active substances, nutrient factors, etc. required for the survival or proliferation of organoids or cells, etc. can be added to the culture medium. These culture medium additives can be added to the culture medium in advance or during the culture of organoids or cells. The method of addition during the culture can be in any form such as a single solution or a mixed solution of two or more kinds, and can also be added continuously or intermittently.

[0142] Examples of physiologically active substances include: insulin, IGF-1, Wnt (such as Wnt1, Wnt2, Wnt3, Wnt3a, Wnt7a, etc.), Noggin, transferrin, albumin, coenzyme Q10, various cytokines (interleukins (such as IL-2, IL-7, IL-15, etc.), stem cell factor (SCF), activin, etc.), various hormones, various growth factors (leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), TGF-β, epidermal growth factor (EGF), etc.), regulators of the Wnt / β-catenin signaling pathway (such as R-spondin1, R-spondin2, R-spondin3, etc.), antagonists of bone morphogenetic protein (BMP) (such as Noggin, etc.). Examples of nutritional factors include: sugars, amino acids, vitamins, hydrolysates, or lipids, etc. Examples of sugars include: glucose, mannose, or fructose, etc., and one kind or a combination of two or more kinds can be used. Examples of amino acids include: L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, or L-valine, etc., and one kind or a combination of two or more kinds can be used. Additionally, amino acids can be acetylated (such as N-acetyl-L-cysteine, etc.). Examples of vitamins include: d-biotin, D-pantothenic acid, choline, folic acid, myo-inositol, nicotinamide, pyridoxal, riboflavin, thiamine, cyanocobalamin, or DL-α-tocopherol, etc., and one kind or a combination of two or more kinds can be used. Examples of hydrolysates include: substances obtained by hydrolyzing soybeans, wheat, rice, peas, corn, cottonseed, yeast extract, etc. Examples of lipids include: cholesterol, linoleic acid, or linolenic acid, etc. Additionally, examples of polysaccharides include: gellan gum, deacylated gellan gum, methylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethyl amylose, xanthan gum, alginic acid, carrageenan, diutan gum, locust bean gum, etc.

[0143] Moreover, in the culture medium, antibiotics such as kanamycin, streptomycin, penicillin, or hygromycin can be added as needed. When adding acidic substances such as sialic acid to the culture medium, it is desirable to adjust the pH of the culture medium to the neutral range suitable for cell growth, i.e., pH 5 to 9, preferably pH 6 to 8.

[0144] The above-mentioned culture medium can be a medium containing serum (e.g., fetal bovine serum (FBS), human serum, horse serum), or a serum-free medium. As the serum, FBS is preferred. From the perspective of preventing the contamination of components from heterologous animals, it is preferred to be serum-free or to use serum from the same animal as the cultured cells. Here, a serum-free medium refers to a medium that does not contain unadjusted or unpurified serum. The serum-free medium may contain components from purified blood or components from animal tissues (e.g., growth factors).

[0145] In the medium for suspension culture, similar to serum, it may or may not contain a serum substitute. As serum substitutes, for example, the following can be listed: albumin substitutes such as albumin, lipid-rich albumin, and recombinant albumin; plant starch, dextran, protein hydrolysate, transferrin or other iron transport proteins, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiol glycerol, or their equivalents. As specific examples of serum substitutes, for example, substances prepared by the method described in WO98 / 30679 or commercially available Knockout Serum Replacement [KSR] (Life Technologies, Thermo Fisher Scientific), Chemically-defined Lipid concentrated (Life Technologies), and L-alanine-L-glutamine dipeptide (e.g., Glutamax (Life Technologies)) can be listed. In addition, as factors from organisms, the following can be listed: platelet-rich plasma (PRP), culture supernatant components of human mesenchymal stem cells, etc.

[0146] The culture medium may contain one or more hormones. As hormones, the following can be listed: estrogen, progesterone, cortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, etc. In addition, hormones can be replaced with artificial or natural compounds having an equivalent steroid structure.

[0147] There is no particular limitation on the culture vessel for culturing the cells (e.g., endometrial stromal cells, (endometrial) epithelial cells, etc.) or organoids (uterine epithelial organoids) used in the present invention. For example, the following can be listed: flasks, tissue culture flasks, culture dishes, Petri dishes, tissue culture dishes, multiwell dishes, microplates, microwells, multiwell plates, porous plates, chamber slides, culture plates, tubes, trays, culture bags, roller bottles.

[0148] When culturing cells under non-adhesive conditions, the incubator is preferably one with low cell adhesiveness, ultra-low cell adhesiveness, or non-adhesiveness. As an incubator with low cell adhesiveness, ultra-low cell adhesiveness, or non-adhesiveness, an incubator whose surface is artificially treated to have low cell adhesiveness, ultra-low cell adhesiveness, or non-adhesiveness, or an incubator that has not been artificially treated (e.g., coated with an extracellular matrix, etc.) to improve cell adhesion can be used. As such an incubator, for example, an ultra-low attachment culture dish (Corning), an ultra-low attachment flask (Corning), an ultra-low attachment plate (Corning), a PrimeSurface culture dish (Sumitomo Bakelite Co., Ltd.), etc. can be used. In addition, when culturing cells under adhesive conditions, it is preferable to coat the incubator with an extracellular matrix, etc. (e.g., laminin, collagen, etc.).

[0149] 2. Manufacturing method of artificial uterus

[0150] The present invention provides a method for manufacturing an artificial uterus, which includes the following steps:

[0151] Step (1), filling an incubator with a solution containing endometrial stromal cells and mainly composed of an extracellular matrix;

[0152] Step (2), pressing a device having a support body with a substance surface and at least one protrusion protruding from the substance surface into the filled solution; and

[0153] Step (3), after the filled solution gels, removing the pressed device.

[0154] In addition, in one aspect of the method for manufacturing an artificial uterus of the present invention, steps (2) and (3) are the following steps (2') and (3'):

[0155] Step (2'), inserting a device having a support body with a substance surface and at least one protrusion protruding from the substance surface into the filled solution; and

[0156] Step (3'), after the filled solution gels, pulling out the pressed device.

[0157] Regarding the "solution containing endometrial stromal cells and mainly composed of an extracellular matrix" used in the present invention, the total amount of the extracellular matrix contained in the solution is not particularly limited as long as the solution can form a hydrogel (can be (hydro)gelated). In addition, the density (number / ml) of the endometrial stromal cells contained in the solution is not particularly limited as long as the fertilized egg (blastocyst) described later can implant on the uterine epithelial organoids. For example, it is 1×10 6pieces / cm 3 ~1×10 7 pieces / cm 3 range, preferably 2×10 6 pieces / cm 3 ~3×10 6 pieces / cm 3 range.

[0158] The device used in the present invention is a device having a support body with an object surface and at least one protrusion protruding from the object surface.

[0159] The above-mentioned support body has an object surface. In the present invention, the "object surface" is a surface that faces and conforms to the object when forming holes in the object. In addition, in the present invention, the "object" refers to a solution containing endometrial stromal cells and mainly composed of extracellular matrix. When forming holes in the object, there may be a gap between the object surface and the object. There is no particular limitation on the overall shape of the support body. For example, it can be a plate shape with a thickness of about 0.5 mm to 1 mm (sometimes also called a film shape or a sheet shape according to the thickness), a column shape (cylindrical shape, prismatic shape, multi-prismatic shape, etc.) that is easy to hold or maintain by hand or tools, etc. The outer peripheral shape of the object surface is not particularly limited as long as it does not interfere with the formation of holes by the protrusions. For example, it can be a circle, an ellipse, a quadrilateral, etc. As an example, the area of the object surface is about 25 to 30 mm 2 or so.

[0160] In the present invention, the "protrusion" has a shape that can function as a mold for forming holes. There is no particular limitation on the length or outer diameter size of the protrusion, the overall shape, the shape of the tip, etc., as long as the fertilized egg (blastocyst) described later can implant on the uterine epithelial organoids in the holes formed by the protrusion. In addition, regarding the length or outer diameter size of the protrusion, the overall shape, the shape of the tip, etc., considering the shrinkage of the formed holes, etc., the size of the target holes to be formed can be appropriately changed. There is no particular limitation on the arrangement pattern of the protrusions, the center-to-center distance, and the gap between adjacent protrusions, as long as the fertilized egg (blastocyst) described later can implant on the uterine epithelial organoids in the holes formed by the protrusion. The device used in the present invention has at least one protrusion. As a specific structure of the protrusion, for example, it can be a cylindrical shape with a length of 1 to 3 mm and an outer dimension of 100 to 200 μm, or a cylindrical shape with the tip of the cylinder rounded.

[0161] In one aspect of the present invention, there is provided a holding structure portion that functions as a handle or a engaging portion when the support body is held by hand or an external device. The holding structure portion may be integrated with the above-mentioned support body, or may be provided as an independent component that can be connected to the above-mentioned support body. In the case where the overall shape of the support body is an easily holdable shape, the support body having such a shape can also be understood as a form in which the support body and the holding structure portion are integrated.

[0162] In the production of the device used in the present invention, for example, the support body, the protrusion, and any holding structure portion can be separately manufactured and then bonded to each other, etc. Alternatively, a part or all of them can be manufactured integrally. In the case of attempting to manufacture integrally, for example, the device can be manufactured using a 3D printer (inkjet method, powder molding method, powder sintering method, etc.).

[0163] There is no particular limitation on the materials of the support body, the protrusion, and the holding structure portion, which are the constituent elements of the device used in the present invention, as long as, after performing steps (ii) and (iii), pores are formed in which the fertilized eggs (blastocysts) described later can implant on the uterine epithelial organoids. Specifically, for example, there can be mentioned: ABS resin (acrylonitrile-butadiene-styrene resin), ASA resin (acrylonitrile-styrene-acrylate resin), PETG resin (glycol-modified polyethylene terephthalate resin), PLA resin (polylactic acid resin), nylon-based resins, or resin materials in which both carbon and other fiber materials are used in these resins, PP (polypropylene)-based powder resins, PS (polystyrene)-based powder resins, metal materials, etc.

[0164] In step (ii) of the present invention, there is no particular limitation on the timing of pressing the above-mentioned device, as long as at least a part of the filled solution remains in a solution state. In addition, in step (iii), there is no particular limitation on the timing of removing the device pressed in step (ii), as long as the fertilized eggs (blastocysts) described later can implant on the uterine epithelial organoids in the pores formed after the removal.

[0165] In the method for manufacturing an artificial uterus of the present invention, all the contents described in the above-mentioned "1. Artificial Uterus" are incorporated by reference.

[0166] 3. Kit for manufacturing artificial uterus

[0167] The present invention provides a kit for manufacturing an artificial uterus, which includes: a device having a support body with a substance surface and at least one protrusion protruding from the above-mentioned substance surface; and a solution mainly composed of an extracellular matrix. The kit may optionally contain endometrial stromal cells or uterine epithelial organoids, etc.

[0168] In the kit for manufacturing the artificial uterus of the present invention, all the contents described in the above "1. Artificial uterus" and "2. Manufacturing method of artificial uterus" are incorporated by reference.

[0169] 4, Screening method

[0170] The present invention provides a method for screening an infertility therapeutic agent, which comprises the following steps:

[0171] Step (1), contacting the artificial uterus of the present invention with a fertilized egg in the presence or absence of a test substance;

[0172] Step (2), measuring the implantation rate of uterine epithelial organoids and the fertilized egg; and

[0173] Step (3), when the implantation rate is higher in the presence of a candidate substance than in the absence of the test substance in Step (2), screening the test substance as a candidate substance for a therapeutic or prophylactic drug for infertility.

[0174] In the present invention, the "fertilized egg" refers to a diploid cell formed by the fusion of a sperm and an egg, which is from a mammal. In one embodiment, the fertilized egg is a fertilized egg other than a human. In addition, in the present invention, the fertilized egg contains an early embryo or a blastocyst, and in the case of attempting implantation, a blastocyst is preferably used. When using a blastocyst as the fertilized egg, it is preferable to remove the zona pellucida of the blastocyst. In addition, in this specification, the fertilized egg is sometimes referred to as an "embryo". Moreover, it also includes a fertilized egg produced from pluripotent stem cells (Warmflash A et al., Nature Methods, 11:847-854; Sinunovic M et al., bioRxiv, 330704; Shao Y et al., Nature Communications, 8:208; ten Berge D et al., Cell Stem Cell, 3:508-518; Beccari L et al., Nature, 562:272-276; van den Brink SC et al., Development, 144:3894-3906; Harrison SE et al., Science, 356:eaal1810, Sozen B et al., Nature Cell Biology, 20:978-989; Rivron NC et al., Nature, 557:106-111; Kagawa H et al., Nature, 601:600-605).

[0175] Step (1) of the present invention is a step of bringing the uterine epithelial organoids contained in the artificial uterus of the present invention into contact with a fertilized egg. This contact can be carried out by adding a fertilized egg to a culture medium containing uterine epithelial organoids. There are no particular limitations on this contact. For example, it can be exemplified as 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, and preferably 1 to 3 days. There are no particular limitations on the temperature when bringing the uterine epithelial organoids into contact with the fertilized egg. The temperature is about 30 to 40 °C, preferably about 37 °C, and the culture is carried out in an air atmosphere containing CO2. The CO2 concentration is preferably about 2 to 5%.

[0176] In the screening method of the present invention, the concentration of the test substance can be appropriately adjusted according to the type of the compound (solubility, toxicity, etc.).

[0177] The determination of the implantation rate of the uterine epithelial organoids and the fertilized egg in step (2) of the present invention can be carried out by observing whether the fertilized egg invades the endometrium of the uterine epithelial organoids (in this specification, sometimes referred to as "implantation-like reaction"). Regarding the presence or absence of the implantation-like reaction, for example, it can be observed by the following 1 to 3: 1. Under a fluorescence microscope, the fluorescence emission of the fertilized egg disappears due to this invasion; 2. Under a confocal microscope, it is confirmed that the fluorescence emission of the fertilized egg has invaded; 3. After removing from the artificial uterus, it is confirmed that phenomena such as the fusion of the embryo and the uterine epithelial organoids occur. In addition, when observing under fluorescence, it is desirable to stain the cells on the artificial uterus side and the fertilized egg with their respective fluorescence-emitting dyes. For example, animals that express fluorescent proteins or luminescent proteins through genetic engineering can be used. For example, fertilized eggs obtained by artificial insemination using sperm collected from Rosa26 H2B-EGFP / H2B-EGFP mice express green fluorescent protein (EGFP), while red fluorescent protein (mCherry) is expressed in uterine epithelial organoid / stromal cells cultured from Rosa26 mCherry / mCherry mice. In addition, these can also be fluorescently labeled with fluorescent substances such as Cell tracker (Thermo Fisher) or Cell Explorer (AAT Bioquest). For example, it can be observed by staining uterine epithelial organoid / stromal cells with Cell Explorer TM Immobilized Live Cell Tracking Kit Green Fluorescence and staining fertilized eggs with Cell Explorer TM Live Cell Tracking Kit Red Fluorescence.

[0178] In step (3) of the present invention, the implantation rate in the absence of the test substance to be compared can be measured as a control in each experiment, or the previously measured value can also be used.

[0179] It is considered that the substance obtained by the screening of the present invention also promotes an implantation-like reaction in vivo, and thus is suitable as a therapeutic or prophylactic agent for infertility or a candidate substance thereof. In addition, for the therapeutic or prophylactic agent, the substance can be combined with an agent (for example, a hormonal agent, etc.) used in the treatment of infertility to prepare a combined agent (drug) or a combination agent (drug).

[0180] In the screening method of the present invention, all the contents described in the above "1. Artificial uterus", "2. Method for manufacturing an artificial uterus", and "3. Kit for manufacturing an artificial uterus" are incorporated by reference.

[0181] 4. Integrin activator

[0182] The present invention provides an integrin activator, which comprises:

[0183] (1) A peptide consisting of KFEEERMRCKWMT;

[0184] (2) A peptide consisting of KFEEERSRCKWMT;

[0185] (3) A peptide consisting of an amino acid sequence obtained by deleting, substituting, and / or adding 1 to 3 (i.e., 1, 2, or 3) amino acids in (1) or (2); or

[0186] (4) A peptide obtained by binding any one of (1) to (3) to a membrane-permeable molecule.

[0187] In the present invention, "integrin activation" means increasing cell adhesion via integrin. In addition, the integrin activator of the present invention increases integrin-mediated cell adhesion, and as a result, promotes the implantation (rate) of a fertilized egg (embryo or blastocyst) on the endometrium. Therefore, in one aspect, the integrin activator of the present invention is a promoter for the implantation (rate) of a fertilized egg (embryo or blastocyst) on the endometrium (in the present invention, sometimes simply referred to as "embryo implantation promoter"). In addition, the agent of the present invention or the peptide contained in the agent promotes embryo implantation as a result, and thus infertility treatment, etc. can be carried out by administering the agent to a subject. Therefore, in one aspect, the agent of the present invention is an infertility treatment agent. Moreover, the agent of the present invention can be used in combination with hormonal agents, etc. used in infertility treatment as described later, and can also be formulated as a combined agent or a combination agent.

[0188] As the amino acid sequence of the above (3), for example, the following can be cited: (i) an amino acid sequence in which 1 to several (2, 3, 4, or 5), preferably 1 to 4, more preferably 1 to 3, still more preferably 1 or 2 amino acids in the amino acid sequence of the peptide consisting of KFEEERMRCKWMT (SEQ ID NO: 1) or the peptide consisting of KFEEERSRCKWMT (SEQ ID NO: 2) are replaced by other amino acids, and most preferably an amino acid sequence in which 1 amino acid is replaced by other amino acids; (ii) an amino acid sequence obtained by deleting 1 to several (2, 3, 4, or 5), preferably 1 to 4, more preferably 1 to 3, still more preferably 1 or 2, most preferably 1 amino acid in the amino acid sequence represented by SEQ ID NO: 1 or 2; (iii) an amino acid sequence obtained by inserting 1 to several (2, 3, 4, or 5), preferably 1 to 4, more preferably 1 to 3, still more preferably 1 or 2, most preferably 1 amino acid in the amino acid sequence represented by SEQ ID NO: 1 or 2; (iv) an amino acid sequence obtained by adding 1 to several (2, 3, 4, or 5), preferably 1 to 4, more preferably 1 to 3, still more preferably 1 or 2, most preferably 1 amino acid in the amino acid sequence represented by SEQ ID NO: 1 or 2; or (v) an amino acid sequence formed by combining them.

[0189] In the case of substitution by other amino acids, it is preferably substituted by amino acids with similar physicochemical properties ("similar amino acids"), such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with a hydroxyl group (Ser, Thr), amino acids with a small side chain (Gly, Ala, Ser, Thr, Met), etc., which are classified into the same group of amino acids. It is predicted that substitution with such similar amino acids will not cause a change in the phenotype of the protein (i.e., conservative amino acid substitution). Specific examples of conservative amino acid substitution are well known in the art and are described in various literatures (for example, see Bowie et al., Science, 247: 1306-1310 (1990)). In addition, in the case of substitution, deletion or insertion of the amino acid sequence as described above, there is no particular limitation on the position of the substitution, deletion or insertion, as long as it can increase cell adhesion mediated by integrin. In one embodiment regarding insertion, as the amino acid sequence of (3) above, an amino acid sequence obtained by inserting 1 or 2 amino acids at the N-terminus and / or C-terminus of SEQ ID NO: 1 or 2 can be cited. In addition, the substituted amino acid can be substituted with a non-natural artificial amino acid. In particular, amino acids modified with fluoride (F), chloride (Cl), bromide (Br), or iodide (I) have a greater impact on cell permeability or stability in the blood, so they are preferably used as substitutions.

[0190] As a membrane-permeable molecule, there is no particular limitation as long as it is a molecule that increases hydrophobicity by binding to a peptide and improves its affinity for the cell membrane. Specifically, for example, saturated fatty acids can be cited. More specifically, octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, and stearic acid can be cited. In addition, polyethylene glycol can be reversibly bound by a method known per se (Utatsu et al., Materials Today Bio, Volume 12, September 2021, 100160).

[0191] For example, (N-terminal) myristoylation with myristic acid or palmitoylation with palmitic acid results in an increase in the hydrophobicity of the peptide and an improvement in its affinity for the cell membrane, so they are preferred.

[0192] The integrin activator of the present invention may contain, in addition to the peptide of the present invention, a pharmaceutically acceptable carrier as described later, etc., and can be administered orally or parenterally to mammals, etc.

[0193] Dosage forms of the integrin activator of the present invention include, for example: oral preparations such as tablets (e.g., sugar-coated tablets, film-coated tablets, sublingual tablets, buccal tablets, orally disintegrating tablets), pills, granules, powders, capsules (e.g., soft capsules, microcapsules), syrups, emulsions, suspensions, films (e.g., orally disintegrating films, oral mucosal patches), etc. In addition, dosage forms of the integrin activator of the present invention include, for example: parenteral preparations such as injections, infusions, transdermal agents (e.g., iontophoresis transdermal agents), suppositories, ointments, nasal agents, pulmonary agents, eye drops, vaginal agents, vaginal capsules, etc. In addition, the integrin activator of the present invention can be a controlled release preparation such as an immediate release preparation, a sustained release preparation (e.g., sustained release microcapsules).

[0194] The integrin activator of the present invention can be manufactured by known manufacturing methods commonly used in the pharmaceutical formulation art (e.g., methods described in the Japanese Pharmacopoeia). In addition, in the integrin activator of the present invention, excipients, binders, disintegrants, lubricants, sweeteners, surfactants, suspending agents, emulsifiers, colorants, preservatives, fragrances, flavoring agents, stabilizers, thickeners, and other additives commonly used in the pharmaceutical field can be appropriately and quantitatively contained as needed. Examples of pharmacologically acceptable carriers include these additives.

[0195] For example, tablets can be manufactured using excipients, binders, disintegrants, lubricants, etc., and pills and granules can be manufactured using excipients, binders, disintegrants. In addition, powders and capsules can be manufactured using excipients, etc., syrups can be manufactured using sweeteners, etc., and emulsions or suspensions can be manufactured using suspending agents, surfactants, emulsifiers, etc.

[0196] Examples of excipients include: lactose, sucrose, glucose, starch, sucrose, microcrystalline cellulose, licorice powder, mannitol, sodium bicarbonate, calcium phosphate, calcium sulfate.

[0197] Examples of binders include: 5 - 10 wt% starch slurry, 10 - 20 wt% gum arabic solution or gelatin solution, 1 - 5 wt% tragacanth solution, carboxymethyl cellulose solution, sodium alginate solution, glycerol.

[0198] Examples of disintegrants include: starch, calcium carbonate.

[0199] Examples of lubricants include: magnesium stearate, stearic acid, calcium stearate, purified talc powder.

[0200] Examples of sweeteners include: glucose, fructose, invert sugar, sorbitol, xylitol, glycerol, simple syrup.

[0201] Examples of surfactants include: sodium dodecyl sulfate, polysorbate 80, sorbitan monofatty acid ester, polyethylene glycol 40 stearate.

[0202] Examples of suspending agents include: gum arabic, sodium alginate, sodium carboxymethyl cellulose, methyl cellulose, bentonite.

[0203] Examples of emulsifiers include: gum arabic, tragacanth, gelatin, polysorbate 80.

[0204] For example, when the medicament of the present invention is a tablet, the tablet can be compression-molded by adding, according to a method known per se, excipients (e.g., lactose, sucrose, starch), disintegrants (e.g., starch, calcium carbonate), binders (e.g., starch, gum arabic, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose) or lubricants (e.g., talc, magnesium stearate, polyethylene glycol 6000) to the peptide of the present invention, and then, if necessary, coated by a method known per se for the purpose of masking taste, enteric solubility or sustained release. As the coating agent for coating, for example, hydroxypropyl methylcellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, polyethylene glycol, Tween 80, Pluronic F68, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxymethyl cellulose acetate succinate, Eudragit (manufactured by ROHM GmbH, Germany, methacrylic acid / acrylic acid copolymer) and dyes (e.g., iron oxide red, titanium dioxide) can be used.

[0205] As the above-mentioned injection, in addition to intravenous injection, it also includes subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, drip injection, etc.

[0206] Such an injection is prepared by a method known per se, that is, by dissolving, suspending or emulsifying the peptide of the present invention in a sterile aqueous solution or an oily solution. Examples of the aqueous solution include: physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride), etc. The aqueous solution may contain a suitable cosolvent, such as an alcohol (e.g., ethanol), a polyol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant (e.g., polysorbate 80, HCO-50). Examples of the oily solution include: sesame oil, soybean oil, etc. The oily solution may contain a suitable cosolvent. Examples of the cosolvent include: benzyl benzoate, benzyl alcohol, etc. In addition, in the injection, a buffer (e.g., phosphate buffer, sodium acetate buffer), an analgesic (e.g., benzalkonium chloride, procaine hydrochloride), a stabilizer (e.g., human serum albumin, polyethylene glycol), a preservative (e.g., benzyl alcohol, phenol), etc. can be incorporated. The prepared injection is usually filled in ampoules.

[0207] The content of the peptide of the present invention in the agent of the present invention varies depending on the form of the preparation, and is generally about 0.01 to about 100% by weight, preferably about 2 to about 85% by weight, and more preferably about 5 to about 70% by weight relative to the whole preparation.

[0208] The content of the additive in the agent of the present invention varies depending on the form of the preparation, and is generally about 1 to about 99.9% by weight, preferably about 10 to about 90% by weight relative to the whole preparation.

[0209] The peptide of the present invention is stable and has low toxicity, and can be used safely. The daily dosage of the peptide of the present invention varies depending on the state or body weight of the patient, the type of compound (amino acid), the administration route, etc. For example, in the case of oral administration to a patient for the treatment of infertility, the daily dosage for an adult (body weight about 60 kg) is about 5 to 500 mg in terms of the peptide of the present invention. They can be administered once or divided into several times.

[0210] In the case of parenteral administration of the peptide of the present invention, it is usually administered in the form of a liquid preparation (for example, an injection) or a vaginal capsule. The single dosage of the peptide of the present invention also varies depending on the administration object, the target organ, the symptom, the administration method, etc. For example, usually per 1 kg of body weight, about 0.08 to about 8 mg is administered vaginally, by percutaneous injection or intravenously.

[0211] The peptide of the present invention can be used in combination with other drugs. Specifically, for example, the peptide of the present invention can be used in combination with a hormonal agent, etc. In this specification, other drugs that can be used in combination with the peptide of the present invention are sometimes referred to as "combination drugs".

[0212] As the hormonal agent, for example, ovulation inducers, hCG preparations, follicle-stimulating hormone (estrogen) agents, luteinizing hormone (progesterone) agents, GnRH preparations (GnRH agonists / antagonists) can be cited. In addition, as other agents, hyperprolactinemia treatment drugs (for example, CABASER, etc.), endometriosis / breast disease treatment drugs (for example, danazol, etc.) can be cited.

[0213] In particular, as the follicle-stimulating hormone (estrogen) agent, for example, estradiol, estriol, ethinylestradiol, estradiol cypionate, estradiol valerate, etc. can be cited. In addition, as the luteinizing hormone (progesterone) agent, progesterone, medroxyprogesterone acetate, dienogest, norethisterone, dienogest, etc. can be cited.

[0214] By combining the peptide of the present invention with a co-administered drug, the following excellent effects (1) to (5) can be obtained: (1) Compared with the case of administering the peptide of the present invention or the co-administered drug alone, the dosage thereof can be reduced; (2) The drug to be co-administered with the peptide of the present invention can be selected according to the symptoms of the patient (mild, severe, etc.); (3) A long treatment period can be set; (4) The persistence of the treatment effect can be pursued; and (5) By co-administering the peptide of the present invention and the co-administered drug, a synergistic effect can be obtained.

[0215] Hereinafter, the case of co-administering the peptide of the present invention and the co-administered drug is referred to as "the co-administered agent of the present invention". When using the co-administered agent of the present invention, there is no limitation on the administration timing of the peptide of the present invention and the co-administered drug. The peptide of the present invention and the co-administered drug can be simultaneously administered to the administration subject, or can be administered with a time difference. In the case of administering with a time difference, the time difference varies depending on the active ingredient, dosage form, and administration method to be administered. For example, in the case of first administering a co-administered drug for improving the endometrial state, after administering the co-administered drug, in the case of natural pregnancy, the peptide of the present invention can be administered between 1 week and 10 days after sexual intercourse, and in the case of artificial insemination embryo transfer, the peptide of the present invention can be administered at the time of transplantation. In the case of first administering the peptide of the present invention, a co-administered drug such as a progesterone preparation for maintaining the pregnancy state can be administered after administering the peptide of the present invention. The dosage of the co-administered drug can follow the dosage used clinically and can be appropriately selected according to the administration subject, administration route, disease, combination, etc.

[0216] In addition, the peptide of the present invention is not limited to being used for humans. For example, it can also be used to improve the implantation efficiency in livestock animals such as racehorses or beef cattle.

[0217] As an administration regimen in the case of co-administering the peptide of the present invention and the co-administered drug, for example, the following can be cited: (1) Administration of a single preparation obtained by simultaneously formulating the peptide of the present invention and the co-administered drug; (2) Simultaneous administration of two preparations obtained by separately formulating the peptide of the present invention and the co-administered drug through the same administration route; (3) Administration of two preparations obtained by separately formulating the peptide of the present invention and the co-administered drug through the same administration route with a time difference; (4) Simultaneous administration of two preparations obtained by separately formulating the peptide of the present invention and the co-administered drug through different administration routes; (5) Administration of two preparations obtained by separately formulating the peptide of the present invention and the co-administered drug through different administration routes with a time difference (for example, administering in the order of the peptide of the present invention → co-administered drug, or in the reverse order).

[0218] The dosage of the co-administered drug can be appropriately selected according to the dosage used clinically. In addition, the mixing ratio of the peptide of the present invention and the co-administered drug can be appropriately selected according to the administration subject, administration route, target disease (especially infertility), symptoms, combination, etc.

[0219] Hereinafter, examples are given to more specifically illustrate the present invention, but the present invention is not limited by any of these examples.

[0220] Example

[0221] Experimental animals

[0222] All animal experiments were carried out using the animal experiment facilities of Kansai Medical University after obtaining approval from the Animal Experiment Committee. The mouse strains used were C56BL / 6, B6D2F1, Rosa26 mCherry / mCherry , Rosa26 H2B-EGFP / H2B-EGFP and ICR. The animals were 8 to 20 weeks old.

[0223] Cell culture

[0224] Uteri were collected from euthanized C56BL / 6 mice or Rosa26 mCherry / mCherry mice, incised, finely cut while exposing the epithelium, enzymatically treated with dispase, and epithelial cells were collected. The epithelial cells were suspended in Matrigel and cultured in Advanced DMEM / F12 medium (WNT medium) supplemented with WNT3A, R-spondin1, Noggin, EGF, and Hepes pH 7.4 to produce uterine organoids (Turco, 2017, Nature Cell Biology). The uterine tissue from which epithelial cells had been removed was enzymatically treated with collagenase, and stromal cells were collected. The stromal cells were cultured on a cell adhesion-treated culture dish in DMEM medium supplemented with 10% serum. Uterine organoids and stromal cells from C56BL / 6 mice were fluorescently labeled with a Cell Explorer TM Live Cell Tracking Kit Green Fluorescence (AAT Bioquest) immediately before the artificial implantation experiment.

[0225] Embryo culture

[0226] Female B6D2F1 mice were given CARD HyperOva (Kudou), and 48 hours later, human chorionic gonadotropin (Aska Pharmaceutical) was administered to mate them with male B6D2F1 mice. The female mice were euthanized the next day, and 2-cell stage embryos were collected from the oviducts and cultured in KSOM medium (Ark Resources) for 3 days to develop into blastocysts, which were fluorescently labeled with a Cell Explorer TM Live Cell Tracking Kit Red Fluorescence (AAT Bioquest) immediately before the artificial implantation experiment. Additionally, in the case of artificial insemination, Rosa26 H2B-EGFP / H2B-EGFPMale mice were euthanized, and sperm was collected from the cauda epididymis and cryopreserved using CARD FERTIUP mouse sperm cryopreservation solution (Jiudao). Next, female B6D2F1 mice were given CARD HyperOva (Jiudao), and 48 hours later, human chorionic gonadotropin (Aska Pharmaceutical) was administered. The female mice were euthanized the next day, and unfertilized eggs were collected from the oviducts. The unfertilized eggs were fertilized with sperm that had been thawed and recovered in CARD FERTIUP mouse sperm pre-culture medium, resulting in fertilized eggs fluorescently labeled with EGFP, which developed into blastocysts.

[0227] Example 1: Miniature uterus and in vitro implantation experiment

[0228] Uterine organoids are spherical cell masses composed of a single layer of columnar epithelial cells in Matrigel ( Figure 1 A). The uterine organoids together with Matrigel were suspended in PBS supplemented with 5 mM EDTA and rotated at 4°C for 60 minutes to dissolve Matrigel. The uterine organoids were sorted one by one into 96-well ultra-low attachment culture dishes (Costar) and cultured in WNT medium supplemented with estrogen for 4 days to reverse polarity. The polarity of epithelial cells can be judged according to the positional relationship between the nucleus stained with Hoechst and the actin fibers stained with phalloidin. In uterine organoids cultured in Matrigel, the nucleus and actin fibers are oriented from the outside to the inside of the sphere ( Figure 1 F right), and in uterine organoids cultured in ultra-low attachment culture dishes, the nucleus and actin fibers are oriented from the inside to the outside of the sphere ( Figure 1 F left). The actin fiber side is the lumen side, which is the side in contact with the embryo. Thereafter, they were cultured for another 1 day in WNT medium supplemented with estrogen and progesterone. The stromal cells were suspended in Matrigel, placed on a glass-bottom culture dish, and a mold made with a 3D printer was placed and fixed ( Figure 1 C–E), and a structure with a hole having a diameter of 200 μm was fabricated. The uterine organoids and blastocysts stripped of the zona pellucida were placed in the hole ( Figure 1 B, Figure 2 , Figure 3 ), and cultured in WNT medium (Bedzhov, 2014, Nature Protocols, partially modified, implantation medium) supplemented with 30% KSR (Thermo Fisher), N-acetylcysteine, estrogen, and progesterone, and the implantation-like reaction was observed using a confocal fluorescence microscope FV3000 (Olympus).

[0229] By setting up a simple CO2 incubator (Tokai Hit) on a confocal fluorescence microscope FV3000, the implantation-like reaction was observed under the same in vivo environment of 37 °C and 5% CO2. To observe the green fluorescence of the EGFP or Cell Explorer TM live cell tracing kit, a 488 nm laser was used. To observe the red fluorescence of the mCherry or Cell Explorer TM live cell tracing kit, a 561 nm laser was used, and three-dimensional fluorescence observation was performed for 3 days. The blastocyst initially contacted the uterine organoids, but slowly invaded into the uterine organoids ( Figure 4 A). In addition, the interaction with the surrounding stromal cells was also observed ( Figure 4 A). Moreover, the fluorescence of mCherry was also observed in the blastocyst, suggesting that the epithelial cells were excluded by phagocytosis and invasion occurred ( Figure 4 B). In addition, this observation was also performed in blastocysts and uterine organoids / stromal cells stained with green and red fluorescence of the Cell Explorer TM live cell tracing kit ( Figure 5 ).

[0230] Example 2: Verification of in vitro implantation experiment

[0231] In the in vitro implantation experiment, in the case where no implantation-like reaction occurred, the embryos ([[]]END]] Figure 6 A left) and uterine organoids ( Figure 6 A right) were in a separated state. In contrast, if an implantation-like reaction occurred, the removed embryos fused and did not separate ( Figure 6 B). In the in vitro implantation experiment system, blastocysts were added. After 72 hours, all the cells were taken out, treated with trypsin to form single cells, and single-cell RNA expression analysis was performed. For the single-cellized cells, dead cells were fluorescently stained with 7-AAD, and live cells were separated and collected using a cell sorter (SH800S, Sony). The collected cells were tagged with RNA for each cell using Chromium (10×Genomics) at the Genetic Information Experiment Center affiliated with the Institute of Microbiology, Osaka University, and next-generation sequencing was performed. The gene expression data was analyzed using SEURAT (https: / / satijalab.org / seurat / ). According to the gene expression pattern, the analyzed cell populations could be divided into three types: placental-like cells, endometrial stromal cells, and endometrial epithelial cells ( Figure 6C). It is considered that these are cells derived from blastocysts, stromal cells, and uterine organoids added to the in vitro implantation experimental system, respectively. Moreover, cells expressing Prdm16, Klf4, Rexo1 that play a role in stem cell maintenance, or Gata6 that plays a role in endoderm formation, Nodal that plays a role in gastrulation, and Plac8, Klf4, Sdc1, etc. that play a role in placenta formation are dispersed, suggesting that after the embryo develops to the blastocyst stage ( Figure 4 D).

[0232] Example 3: Expression of Implantation-Related Genes

[0233] The expression of genes known to play a role in implantation was confirmed by single-cell RNA expression analysis in Example 2. The expression of WNT signal ( Figure 7 A), Notch signal ( Figure 7 B), matrix metalloproteinase ( Figure 7 C), TGFβ ( Figure 7 D), LIF ( Figure 7 E), integrin ( Figure 7 F), etc. was observed in both cells from the embryo and cells from the endometrium, suggesting that the phenomena observed in Examples 1 and 2 are reactions similar to implantation occurring in vivo.

[0234] Example 4: Peptide Activity Assay (Outline)

[0235] The activity of the peptide was detected by measuring the ability to form an integrin activation complex using AlphaScreen. Among more than 60,000 peptides, Iznm-1 (Myr-KFEEERMRCKWMT (MW: 1984.45)) and Iznm-2 (Myr-KFEEERSRCKWMT (MW: 1940.33)) with the highest integrin activation ability were found. At a concentration of 8 μM, an activation effect of approximately 5-fold was confirmed for Iznm-1, and an activation effect of approximately 14-fold was confirmed for Iznm-2 ( Figure 8 ).

[0236] Example 5(1): Increase in Activation of Integrin β1 Caused by Peptide Drugs

[0237] To confirm that the peptide drug Iznm-2 actually induces integrin activation in embryos, blastocysts without zona pellucida were placed in a glass-bottom dish (Matsunami) and cultured in implantation medium for 3 days. An antibody that recognizes activated integrin β1 (MAB2259Z, Merck) and an anti-mouse secondary antibody conjugated with Alexa594 were added to the culture medium. The experiment was conducted with an experimental group supplemented with Iznm-2 and a control group without supplementation. After culturing, the embryos were fixed with 4% formaldehyde, the nuclei were stained with Hoechst, and observations were made using an FV3000 or a fluorescence microscope CKX-53 (Olympus). As a result, compared with the control group ( Figure 8 A, Figure 8 C), strongly fluorescently stained cells were detected in the experimental group ( Figure 8 B, Figure 8 D).

[0238] Example 5(2): Promotion of embryo adhesion to the glass surface by the peptide drug

[0239] In the experiment of Example 5(1), the embryos in the control group did not adhere, but in the experimental group supplemented with Iznm-2, embryo adhesion to the glass surface was observed ( Figure 10 A, Figure 10 B). The proportion was 0 / 13 in the control group and 13 / 14 in the Iznm-2 supplemented group, and a significant difference was detected between the two groups (p<0.01, chi-square test, Figure 10 C).

[0240] Example 6: Effect of the peptide drug in the in vitro implantation system

[0241] In the in vitro implantation system of the present invention, the presence or absence of an implantation-like reaction was determined based on whether the embryos fused with the uterine organoids as shown in Figure 6 A and 6B ( Figure 11 ). In the control group, an implantation-like reaction was observed at a rate of 5 / 22 (22.7%). In contrast, in an experimental system in which stromal cells were excluded from the in vitro implantation system, an implantation-like reaction was observed at a rate of 1 / 22 (4.5%) (p<0.01, chi-square test and residual analysis). In addition, in the experiment supplemented with Iznm-1, it was 6 / 14 (42.9%), and in the experiment supplemented with Iznm-2, it was 12 / 19 (63.2%), and a significant difference was detected in the latter (p<0.01, chi-square test and residual analysis).

[0242] Example 7: Effect of the peptide drug on embryo development

[0243] To confirm the effect of the peptide drug on embryonic development, the following experiment was conducted. ICR male mice at 8 weeks of age were subjected to vasectomy. ICR female mice in proestrus were mated with vasectomized male mice, and mice with a vaginal plug on the next day were used as pseudopregnant female mice. Two days after confirming the vaginal plug, blastocyst-stage B6D2F1 mouse embryos were transplanted. The blastocysts were transferred to KSOM medium supplemented with 32 μM Iznm-2 immediately before transplantation, and the embryos were transplanted together with the medium. The volume of the medium injected into the uterus during transplantation was 5 μl or less. Embryo transfer was performed as follows. The unilateral skin and abdominal wall on the dorsal side of an anesthetized pseudopregnant female mouse were incised to expose the uterus outside the body, perforated with a 26G needle, and the embryos together with the medium were injected into it using a glass tube. After transplantation, the uterus was returned to the body and sutured. The transplanted embryos developed to E14 in the transplanted mice, and then the surrogate mice were euthanized, removed, and studied for development. The removed embryos developed normally, indicating that Iznm-2 had no significant effect on embryonic development( Figure 14 ).

[0244] Example 8: Effect of Peptide Drug on Embryo Transfer

[0245] To confirm the effect of the peptide drug on embryo transfer, the following experiment was conducted. ICR male mice at 8 weeks of age were subjected to vasectomy. ICR female mice in proestrus were mated with vasectomized male mice, and mice with a vaginal plug on the next day were used as pseudopregnant female mice. Two days after confirming the vaginal plug, blastocyst-stage B6D2F1 mouse embryos were transplanted. The blastocysts were transferred to KSOM medium supplemented with 8 μM Iznm-2 or not supplemented (control) immediately before transplantation, and the embryos were transplanted together with the medium. The volume of the medium injected into the uterus during transplantation was 5 μl or less. The blastocysts to be transplanted were transplanted 4.5 days after mating, while the pseudopregnant mice were transplanted 2.5 days after mating. The different transplantation dates after mating reproduced the difference between the time when the endometrium accepts the fertilized egg and the time when the fertilized egg reaches the endometrium (the so-called "implantation window difference"), which is one of the causes of infertility( Figure 12 ). Embryo transfer was performed as follows. The unilateral skin and abdominal wall on the dorsal side of an anesthetized pseudopregnant female mouse were incised to expose the uterus outside the body, perforated with a 26G needle, and the embryos together with the medium were injected into it using a glass tube. After transplantation, the uterus was returned to the body and sutured. The transplanted embryos developed to E15.5 in the transplanted mice, and then the surrogate mice were euthanized, removed, and studied for implantation rate and development( Figure 13A, 13B). In the control group, no implantation was confirmed in 24 transplanted embryos. In the Iznm-2 addition group, 22 implantations were confirmed in 41 transplanted embryos. Additionally, 12 of them were confirmed to develop normally ( Figure 13 C). The implanted embryos were divided into two groups: those with no development at all and those with normal development ( Figure 13 D). It can be speculated that the effect of the drug in the early stage of human pregnancy resulted in two outcomes, similar to the all-or-none rule, that is, either it has an effect or it has no effect.

[0246] Example 9: Effects of peptide drug on embryonic development

[0247] The development of E15.5 embryos obtained in Example 8 was confirmed. The shape of the embryos was normal ( Figure 14 A). Additionally, paraffin blocks of the embryos were prepared, and their sections were stained with hematoxylin-eosin to confirm the normal development of organs such as the brain / heart / digestive organs ( Figure 14 B).

[0248] Example 10: Effects of peptide drug on the mother

[0249] By intraperitoneally administering an excessive amount of Iznm-2 to female mice, the side effects of the peptide drug were studied. In Example 8, 8 μM of Iznm-2 and embryos were simultaneously administered into the uterus in an amount of 5 μl or less. In this experiment, administration was carried out at a concentration of 8 μM relative to the total body weight of the mice. Iznm-2 was dissolved in corn oil, and the control was given corn oil without the peptide drug. Blood was collected 24 hours after administration, and the blood components as indicators of the status of the liver and kidneys were analyzed.

[0250] Compared with the control (mice given corn oil), the blood test results of mice (mothers) intraperitoneally administered with the peptide drug (Iznm-2) of the present invention are shown in Table 1.

[0251] [Table 1]

[0252]

[0253] As shown in Table 1, it was confirmed that the peptide drug of the present invention has no major adverse effects on the mother.

[0254] From the results of the above examples, it can be understood that the peptide of the present invention is extremely safe for the development of the mother or embryo. Additionally, it can also be understood that even if the state of the uterus is incomplete, the peptide of the present invention can improve the adhesion between the embryo and the uterus, thereby directly increasing the implantation rate.

[0255] Industrial Applicability

[0256] According to the present invention, by activating integrin, the implantation rate of embryos after natural pregnancy or artificial insemination can be increased, and thus it is useful. In addition, the agent of the present invention can be used in combination with agents conventionally used in infertility treatment, and thus can be one of the countermeasures for solving the problem of declining birthrate. In addition, it also leads to a reduction in infertility treatment costs or increased subsidies related to such treatment. Moreover, according to the present invention, the evaluation of substances that can increase the embryo implantation rate can be carried out quickly and reliably, and thus the present invention is also very useful for obtaining such substances.

[0257] This application is based on Japanese Patent Application No. 2022-183639 (filing date: November 16, 2022), the content of which is incorporated herein by reference in its entirety.

Claims

1. An embryo implantation promoter or infertility treatment agent, comprising: (1) A peptide consisting of KFEEERMRCKWMT; (2) A peptide consisting of KFEEERSRCKWMT; (3) A peptide consisting of an amino acid sequence obtained by deleting, substituting, and / or adding 1 to 3 amino acids in (1) or (2); or (4) A peptide obtained by binding a membrane-permeable molecule to any one of (1) to (3).

2. The agent according to claim 1, wherein The peptide obtained by binding a membrane-permeable molecule is a myristoylated peptide.

3. The agent according to claim 1 or 2, comprising a hormonal agent.

4. An integrin activator, comprising: (1) A peptide consisting of KFEEERMRCKWMT; (2) A peptide consisting of KFEEERSRCKWMT; (3) A peptide consisting of an amino acid sequence obtained by deleting, substituting, and / or adding 1 to 3 amino acids in (1) or (2); or (4) A peptide obtained by binding a membrane-permeable molecule to any one of (1) to (3).

5. An artificial uterus, comprising a hydrogel and uterine epithelial organoids configured to be surrounded by the hydrogel, the hydrogel containing endometrial stromal cells and having an extracellular matrix as a main component, and having an opening in an upward direction of the hydrogel configured to surround the uterine epithelial organoids.

6. The artificial uterus according to claim 5, wherein, The extracellular matrix contains laminin and / or a fragment thereof.

7. The artificial uterus according to claim 5, wherein, For the uterine epithelial organoids, the outside of the organoids is the apical side.

8. A kit for manufacturing an artificial uterus, comprising: A device having a support body with an object surface and at least one protrusion protruding from the object surface; And a solution having an extracellular matrix as a main component.

9. A method for manufacturing an artificial uterus, comprising the following steps: Step (1), filling a culture vessel with a solution containing endometrial stromal cells and having an extracellular matrix as a main component; Step (2), pressing a device having a support body with an object surface and at least one protrusion protruding from the object surface into the filled solution; and Step (3), after the filled solution is gelled, removing the pressed device.

10. A method for screening an infertility treatment agent, comprising the following steps: Step (1), contacting the artificial uterus according to claim 5 with a fertilized egg in the presence or absence of a test substance; Step (2), measuring the implantation rate of the uterine epithelial organoids and the fertilized egg; and Step (3), when the implantation rate is higher in the presence of a candidate substance than in the absence of the test substance in step (2), screening the test substance as a candidate substance for a treatment or preventive drug for infertility.

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