Pharmaceutical composition for preventing or treating ovarian failure comprising umbilical cord-derived adherent stem cells

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating ovarian failure, which comprises umbilical cord-derived adhesive stem cells. In one embodiment, the umbilical cord-derived adhesive stem cells have the effect of increasing the number and proportion of follicles or primordial follicles, inducing superovulation of eggs, or promoting the development of fertilized eggs, and are therefore useful for preventing or treating ovarian failure, including early menopause or difficulty in conceiving.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating ovarian failure. [Background technology]

[0002] For women, age and pregnancy are closely related, and pregnancy rates tend to decline with age. The main reasons for this include a decrease in the number and quality of eggs in the ovaries. As the age at marriage increases, even in the absence of a clear cause of infertility, ovarian function declines with age, i.e., a decrease in the number and quality of eggs in the ovaries.

[0003] The ovaries are female reproductive organs that produce eggs and secrete sex hormones such as estrogen, progesterone, and testosterone. They play an important role in maintaining the health of the female reproductive system as well as maintaining the balance of the hormone production system and thus the quality of life of women. Ovarian function can be assessed by measuring changes in follicle-stimulating hormone (FSH), estradiol, or AMH (anti-Mullerian hormone) levels.

[0004] Although assisted reproductive techniques such as in vitro fertilization and intracytoplasmic sperm injection have recently been developed, ovarian dysfunction is virtually impossible to treat even with cutting-edge reproductive medicine technology. Therefore, there is a need to develop methods to suppress ovarian dysfunction. Summary of the Invention [Problem to be solved by the invention]

[0005] One aspect of the present invention is to provide a pharmaceutical composition for preventing or treating ovarian insufficiency, which comprises umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof as an active ingredient.

[0006] Another aspect is to provide a method for preventing or treating ovarian insufficiency, comprising administering to an individual in need thereof an effective amount of umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof.

[0007] Yet another aspect is to provide a method for increasing the number of follicles or primordial follicles in an individual, comprising the step of administering to an individual in need thereof an effective amount of umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof.

[0008] Yet another aspect is to provide a method for promoting ovulation of an egg in an individual, comprising administering to an individual in need thereof an effective amount of umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof.

[0009] Yet another aspect provides a method for promoting the development of a fertilized egg in an individual, comprising administering to the individual in need thereof an effective amount of umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof.

[0010] Yet another aspect is to provide a method for increasing or decreasing, respectively, decreased or increased cell function or activity in ovarian failure, comprising the step of administering an effective amount of umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof to an individual in need thereof.

[0011] The present invention provides use of umbilical cord-derived adherent stem cells, cell populations thereof, or culture media thereof for the prevention or treatment of ovarian insufficiency. [Means for solving the problem]

[0012] One aspect provides a pharmaceutical composition for preventing or treating ovarian insufficiency, which comprises umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof as an active ingredient.

[0013] Another aspect provides a method for preventing or treating ovarian insufficiency, comprising administering to an individual in need thereof an effective amount of umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof.

[0014] Yet another aspect provides a method for increasing the number of follicles or primordial follicles in an individual, comprising administering to the individual in need thereof an effective amount of umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof.

[0015] Yet another aspect provides a method for promoting ovulation of an egg in an individual, comprising administering to an individual in need thereof an effective amount of umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof.

[0016] Yet another aspect provides a method for promoting the development of a fertilized egg in an individual, comprising administering to the individual in need thereof an effective amount of umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof.

[0017] Yet another aspect provides a method for increasing or decreasing, respectively, decreased or increased cell function or activity in ovarian failure, comprising the step of administering an effective amount of umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof to an individual in need thereof.

[0018] As used herein, the term "umbilical cord" refers to the line connecting the mother's body and abdomen so that a mammalian fetus can develop in the placenta, and generally refers to a tissue composed of three blood vessels, namely, two umbilical arteries and one umbilical artery, surrounded by Wharton's jelly. Therefore, as used herein, "umbilical cord-derived adherent stem cells" refer to cells derived from the umbilical cord or Wharton's jelly tissue of the umbilical cord, which have the ability to differentiate into various tissue cells and the ability to adhere to and grow on the surface of a culture vessel.

[0019] The umbilical cord-derived adherent stem cells referred to in this specification are cells having the same cellular characteristics as the cells described in the examples of Korean Patent Application No. 10-2016-0102721, the entirety of which is incorporated herein by reference.

[0020] The umbilical cord-derived adherent stem cells provided herein may express, with respect to cell surface markers, CD200, CD141, CD61, CD87, or SSEA4 positive surface markers at least approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or about 99% of the cells, and express stem cell markers Oct4, Nanog, Tra-1-60, CD3, CD1a, CD11c, CD16, CD86, CD8a, MIC A / B, or CD40 negative markers at least approximately 70% or less, at least 60% or less, at least 50% or less, at least 40% or less, at least 30% or less, at least 20% or less, at least 10% or less, at least 5% or less, or at least 1% or less. In particular, the cells herein may express MIC A / B in at least 10% or less, at least 5% or less, or at least 1% or less of the total cell population. In particular, the cells herein may express SSEA4 in at least 80% or more, at least 85% or more, or at least 90% or more of the total cell population. In particular, the cells of the present invention may express Oct4 and Nanog in at least 10% or less, at least 5% or less, or at least 1% or less of the total cell population. In particular, the cells of the present invention may express CD200 in at least 85% or more, at least 90% or more, or at least 95% or more of the total cell population. Among the surface antigen characteristics, CD61+ may be a surface antigen characteristic that is overexpressed under hypoxic conditions.

[0021] As used herein, the term "positive" can refer to a stem cell marker being present in greater abundance or at a higher concentration than other non-stem cells. That is, a cell is positive for a marker if the marker is present internally or on its surface and can be used to distinguish the cell from one or more other cell types. It can also refer to a cell possessing a sufficient amount of the marker to produce a signal, e.g., a signal from a cell measurement device, that is greater than background. For example, if a cell can be detectably labeled with an antibody specific for CD200, and the signal from this antibody is detectably greater than a control (e.g., background), the cell is "CD200+." As used herein, the term "negative" means that the marker cannot be detected using an antibody specific for a particular cell surface marker compared to background. For example, if a cell cannot be detectably labeled with an antibody specific for CD3, the cell is "CD3-."

[0022] The immunological properties can be determined by conventional methods known in the art, for example, flow cytometry, immunohistochemical staining, or RT-PCR.

[0023] In one embodiment, the umbilical cord-derived adherent stem cells may have one or more properties selected from the following (a) to (e):

[0024] a) higher expression of one or more selected from the group consisting of COL1A1, IGFBP4, TAGLN, STC1, LRRC17 and IL33 compared to bone marrow stem cells;

[0025] b) one or more selected from the group consisting of CCND1, SERPINE1, PRNP, and CYP1B1 are expressed less than in bone marrow stem cells;

[0026] c) maintaining the morphology of adherent fibroblasts during subculture;

[0027] d) the ability to differentiate into adipocytes, osteocytes, or chondrocytes, and

[0028] e) one or more surface antigen characteristics selected from the group consisting of CD200+, Tra-1-60-, CD3-, CD1a-, CD11c-, CD16-, CD86-, CD8a-, CD40-, CD141+, CD61+, CD87+, MIC A / B- and SSEA4+.

[0029] In one embodiment, the umbilical cord-derived adherent stem cells may further have one or more properties selected from the following (f) to (i):

[0030] f) one or more selected from the group consisting of S100A10, BNIP3, IGFBP5, NDUFA4L2, DPYD and SCARA3 are expressed more than in culture under normoxic conditions;

[0031] g) one or more selected from the group consisting of IL8, ALDH1A1, DLC1, CTHRC1, and CPA4 are expressed less than in culture under normoxic conditions;

[0032] h) one or more selected from the group consisting of SNCA, DSG2, NRP2, and PLAT are expressed more than in bone marrow stem cells; and

[0033] i) One or more selected from the group consisting of TPMT, NAGK, and ANXA4 are expressed less than in bone marrow stem cells.

[0034] According to one embodiment, the umbilical cord-derived adherent stem cells may express one or more genes or proteins selected from the group consisting of COL1A1, IGFBP4, TAGLN, STC1, LRRC17, and IL33 at higher levels than bone marrow-derived stem cells. Specifically, the cells may express two or more, three or more, or more specifically all, of the genes or proteins selected from the group consisting of COL1A1, IGFBP4, TAGLN, STC1, LRRC17, and IL33 at higher levels than bone marrow-derived stem cells. Genes that are more highly expressed in the umbilical cord-derived adherent stem cells according to one embodiment compared to the bone marrow-derived stem cells may further include S100A10, SQSTM1, DSTN, DCN, PHGDH, FBLN1, MFGE8, HLA-A, VASN, or KIAA1199. These genes have not been reported to be associated with umbilical cord-derived adherent stem cells. According to one embodiment, the umbilical cord-derived adherent stem cells may exhibit a difference in expression levels of the above genes of at least two-fold compared to bone marrow-derived stem cells. The difference in expression level can be determined, for example, by comparing the expression amount of the gene at the mRNA level, or by microarray analysis.

[0035] Furthermore, umbilical cord-derived adherent stem cells according to one embodiment may express less of one or more genes or proteins selected from the group consisting of CCND1, SERPINE1, PRNP, and CYP1B1 than bone marrow-derived stem cells. Specifically, the cells may express less of two or more, or more than three, or more specifically all, genes or proteins selected from the group consisting of CCND1, SERPINE1, PRNP, and CYP1B1 than bone marrow-derived stem cells. Genes or proteins that are less expressed in umbilical cord-derived adherent stem cells according to one embodiment compared to bone marrow-derived stem cells may include MTA2A, TM4SF1, HIST1H4C, and NME1. These genes or proteins have not been reported to be associated with umbilical cord-derived adherent stem cells. Umbilical cord-derived adherent stem cells according to one embodiment may exhibit a two-fold or greater difference in expression of the genes or proteins compared to bone marrow-derived stem cells. The difference in expression level may be determined, for example, by comparing gene expression levels at the mRNA level. The difference in expression level may also be determined, for example, by microarray analysis.

[0036] The umbilical cord-derived adherent stem cells may also have the morphology of subcultured fibroblasts. In one embodiment, the cells may have the properties of cells that require adhesion to a surface for growth in vitro and may exhibit the specific morphology of spindle-shaped fibroblasts.

[0037] In another embodiment, the umbilical cord-derived adherent stem cells may have colony-forming ability, and the cells may have increased colony-forming ability compared to cells cultured under normoxic conditions.

[0038] The umbilical cord-derived adherent stem cells can also differentiate into adipocytes, osteocytes, chondrocytes, etc. The cells may be induced to differentiate along specific cell lineages, including, for example, adipocyte differentiation, chondrocyte differentiation, osteoblast differentiation, hematopoietic cell differentiation, myocyte differentiation, vascular cell differentiation, neuronal differentiation, and hepatocyte differentiation.

[0039] The umbilical cord-derived adherent stem cells can also differentiate into adipocytes, osteocytes, chondrocytes, etc. The cells may be induced to differentiate along specific cell lineages, including, for example, adipocyte differentiation, chondrocyte differentiation, osteoblast differentiation, hematopoietic cell differentiation, myocyte differentiation, vascular cell differentiation, neuronal differentiation, and hepatocyte differentiation.

[0040] The term "differentiation" refers to the phenomenon in which cells undergo structural and functional specialization during cell division and growth, i.e., the change in morphology and function of cells, tissues, etc. of an organism in order to perform their intended tasks. Differentiation into specific cell types can be measured by methods well known in the art, and differentiation into specific cells can be induced by known methods. Furthermore, differentiation can be confirmed by examining cell morphology using a light microscope or confocal microscope, while measuring changes in cell surface markers (e.g., staining cells with tissue-specific or cell marker-specific antibodies) and morphology using techniques such as flow cytometry or immunocytochemistry, or by measuring changes in gene expression using techniques well known in the art, such as PCR and gene expression profiling.

[0041] As used herein, the term "isolation of umbilical cord-derived adherent stem cells" can refer to the removal of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells that are normally associated with the stem cells in an intact mammalian umbilical cord. A population of cells containing stem cells from an organ is said to be "isolated" when less than 50% of the total cells in that organ are normally associated with the stem cells in the intact state.

[0042] The ovarian failure may be any selected from primary ovarian failure, secondary ovarian failure, hyperandrogenemia, polycystic ovary syndrome, amenorrhea, oligomenorrhea, early menopause, and infertility.

[0043] In one embodiment, the stem cells may increase the number of follicles or primordial follicles.

[0044] In one embodiment, the stem cells may promote ovulation of an egg.

[0045] In one embodiment, the stem cells may promote the development of a fertilized egg.

[0046] In one embodiment, the stem cells may restore the function or activity of any cell selected from the group consisting of B cells, dendritic cells, and fibroblasts, which are decreased due to ovarian failure.

[0047] In one embodiment, the stem cells may reduce the number or activity of mural granulosa lutein cells or atretic follicles that are increased in ovarian failure.

[0048] In one embodiment, the stem cells may have increased expression or activity of Cd 19 or Cd 209a.

[0049] In one example, the stem cells may increase the expression or activity of Itm2a or decrease the expression or activity of Parm1 or Chf.

[0050] In one embodiment, the cells may be any one selected from the group consisting of B cells, dendritic cells, and fibroblasts, which are decreased in the ovarian failure.

[0051] In one embodiment, the increased number of mural granulosa lutein cells or atretic follicles may be due to ovarian failure.

[0052] Furthermore, the above-mentioned aspect includes a pharmaceutical composition containing the culture medium of the umbilical cord-derived adherent stem cells. Also, for example, the present specification provides a pharmaceutical composition containing the umbilical cord-derived adherent stem cells, a cell population thereof, or a culture medium thereof as an active ingredient.

[0053] The dosage of the cell therapy or pharmaceutical composition according to one embodiment is 1.0 x 10 based on umbilical cord-derived adherent stem cells. 3 ~1.0×10 10 cells / kg (body weight) or individual, or 1.0 x 10 7 ~1.0×10 8 The dosage may be determined by cell / kg (body weight) or individual. However, the dosage may vary depending on factors such as the formulation method, administration method, the patient's age, body weight, sex, condition, diet, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will be able to appropriately adjust the dosage taking these factors into consideration. The number of administrations may be one or more than two times within the range of clinically acceptable side effects, and the administration site may be one or more than two sites. For non-human animals, the dosage may be the same as that for humans per kg or per individual, or may be calculated by converting the dosage based on, for example, the volume ratio (e.g., average value) of organs (e.g., heart) between the target animal and humans. Examples of target animals for treatment in one embodiment include humans and other mammals, specifically humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, pigs, etc.

[0054] A cell therapy or pharmaceutical composition according to one embodiment may contain the umbilical cord-derived adherent stem cells as an active ingredient and a pharmaceutically acceptable carrier and / or additives. For example, sterile water, physiological saline, conventional buffers (e.g., phosphate, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (e.g., ascorbic acid), surfactants, suspending agents, isotonicity agents, or preservatives may be included. For local administration, it is also preferable to combine the cell therapy or pharmaceutical composition with organic materials such as biopolymers, or inorganic materials such as hydroxyapatite, specifically collagen matrices, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. When the cell therapy or pharmaceutical composition according to one embodiment is prepared into a dosage form suitable for injection, the cell aggregates may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution.

[0055] In one embodiment, umbilical cord-derived adherent stem cells can be used in a variety of therapeutic protocols in which bodily tissues or organs are augmented, repaired, or replaced by engraftment, transplantation, or infusion of a desired cell population, e.g., a stem cell or derived cell population. The umbilical cord-derived adherent stem cells can replace or augment existing tissue, develop new or altered tissue, or integrate with biological tissues or structures. Furthermore, in therapeutic protocols in which stem cells from tissues other than the umbilical cord are typically used, the stem cells can be replaced with the umbilical cord-derived adherent stem cells described herein.

[0056] The cell therapy drug or pharmaceutical composition according to one embodiment may contain, as needed depending on the administration method or formulation, a suspending agent, a solubilizing agent, a stabilizer, an isotonicity agent, a preservative, an anti-adsorption agent, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc. Pharmaceutically acceptable carriers and formulations compatible with the present invention, including those exemplified above, are described in detail in the literature "Remington's Pharmaceutical Sciences, 19th ed., 1995."

[0057] According to one embodiment, the cell therapy drug or pharmaceutical composition may be prepared in unit dose form or in a multi-dose container by formulating it with pharmaceutically acceptable carriers and / or excipients according to a method that can be easily carried out by a person skilled in the art to which the invention pertains. In this case, the dosage form may be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or in the form of a powder, granules, tablet, or capsule. Furthermore, the cell therapy drug may be formulated into an injectable preparation. In this case, known conventional ingredients for formulation may be used, and the preparation may be prepared by a conventional method. [Effects of the Invention]

[0058] According to one embodiment, umbilical cord-derived adhesive stem cells have the effect of increasing the number and proportion of follicles or primordial follicles in an individual, inducing superovulation of eggs, or promoting the development of fertilized eggs, and are useful for preventing or treating ovarian failure, including early menopause or infertility. [Brief explanation of the drawings]

[0059] [Figure 1] FIG. 1 is a graph confirming the estrous cycle in mice in which POI was induced by CP treatment. [Figure 2] FIG. 2 is a graph confirming the estrous cycle in mice in which POI was induced by CP treatment. [Figure 3] FIG. 3 is a graph showing ovarian weight in mice in which POI was induced by CP treatment. [Figure 4] FIG. 4 shows histological photographs and a graph showing the number and percentage of follicles in mice in which POI was induced by CP treatment. [Figure 5] FIG. 5 is a tissue photograph showing the number and proportion of follicles and primordial follicles after administration of stem cells in one example. [Figure 6] FIG. 6 is a graph showing the number and percentage of follicles and primordial follicles measured after administration of stem cells according to one embodiment. [Figure 7] FIG. 7 is a tissue photograph showing ovarian apoptosis caused by administration of stem cells in one example. [Figure 8] FIG. 8 is a graph showing the measurement of ovarian apoptosis following administration of stem cells according to one embodiment. [Figure 9] FIG. 9 is a tissue photograph showing an area stained with AMH (Anti-Mullerian Hormone) following administration of stem cells according to one example. [Figure 10] FIG. 10 is a graph showing the measurement of the AMH (Anti-Mullerian Hormone) stained area after administration of stem cells according to one example. [Figure 11] FIG. 11 is a micrograph showing the number of superovulated oocytes following administration of stem cells in one example. [Figure 12]FIG. 12 is a graph showing the number of superovulated eggs measured by administration of stem cells according to one embodiment. [Figure 13] FIG. 13 is a photomicrograph showing the proportion of two-cells and blastocysts in in vitro fertilization using stem cells according to one embodiment. [Figure 14] FIG. 14 shows the results of measuring the ratio of two-cells to blastocysts in in vitro fertilization using stem cells in one example. [Figure 15] FIG. 15 is a graph showing the results of an analysis of immune cell populations present in the ovaries. [Figure 16] FIG. 16 is a graph showing changes in immune cell populations due to administration of stem cells according to one example. [Figure 17] FIG. 17 is a graph showing the results of an analysis of stromal cell populations present in the ovary. [Figure 18] FIG. 18 is a graph showing changes in stromal cell populations due to administration of stem cells according to one example. [Figure 19] FIG. 19 is a graph showing the results of an analysis of the granulosa cell population present in the ovary. [Figure 20] FIG. 20 is a graph showing changes in granulosa cell populations due to administration of stem cells according to one example. DETAILED DESCRIPTION OF THE INVENTION

[0060] Hereinafter, preferred embodiments will be presented to facilitate understanding of the present invention. However, the following embodiments are provided to facilitate understanding of the present invention, and the content of the present invention is not limited to the following embodiments. Although various modifications can be made to the embodiments, the embodiments are not limited to the embodiments disclosed below and may be realized in various forms.

[0061] Embodiment. Preparation and characterization of umbilical cord-derived adherent stem cells 1. Preparation of umbilical cord-derived adherent stem cells Umbilical cord-derived adherent stem cells were prepared in the same manner as in Korean Patent Application No. 10-2016-0102721, the entirety of which is incorporated herein by reference.

[0062] Umbilical cords were isolated from placental tissue collected from healthy mothers who had given normal deliveries and provided informed consent. The isolated umbilical cords were washed 2-5 times with Ca / Mg-free DPBS to remove blood. Without removing the outer amniotic membrane, the two arteries and one vein were removed, and the umbilical cords were then cut into pieces approximately 1-5 mm in size. The umbilical cords were then attached to a culture vessel and cultured for 10-15 days. After confirming cell growth from the cultured tissue, umbilical cord-derived adherent stem cells were isolated by treatment with 200 U / ml collagenase I for 5-6 hours. To confirm cell outgrowth from the umbilical cord-attached tissue before and after collagenase I treatment, cell types were identified under a light microscope at 40x and 100x magnification. The isolated cells were then cultured as PO in MEMα GlutaMAX (CS-CM medium) containing 25 ng / ml FGF4, 1 μg / ml heparin, and 10% FBS at 37°C under hypoxic conditions (3% O2). The CS-CM medium was then replaced every 3–4 days to remove cells that had not adhered to the flask bottom. For the first passage, the cells were subcultured using Invitrogen's TrypLE, an animal component-free (ACF) recombinant enzyme, for a short period (3 min) in a 37°C incubator. Hereinafter, the isolated stem cells will be referred to as "CordSTEM."

[0063] 2. Characterization of Umbilical Cord-Derived Adherent Stem Cells Flow cytometry analysis and immunofluorescence staining analysis were performed to analyze the surface proteins of the umbilical cord-derived adherent stem cells prepared in Example 1. This was performed using the same method as described in Korean Patent Application No. 10-2016-0102721, the entire contents of which are incorporated herein by reference.

[0064] Specifically, for flow cytometry analysis, cells were washed with DPBS and then placed in DPBS containing 2% FBS. They were incubated for 20 minutes on ice with Tra-1-60, CD3, CD1a, CD11c, CD16, CD14, CD86, CD8a, CD19, CD40, CD80, CD200, CD141, CD61, CD87, MIC A / B, and SSEA4 markers. Surface antigens were then analyzed using a flow cytometer (FACS Calibur, Becton Bickinson). Furthermore, immunofluorescence staining was performed to confirm the expression of embryonic stem cell markers Oct4 and Nanog. First, cells were washed three times with DPBS and then fixed in 4% paraformaldehyde in a culture vessel at room temperature for 10 minutes. After fixation, cells were washed three times with DPBS. Next, cells were permeabilized with 0.2% Triton X-100 solution at room temperature for 10 minutes, followed by three washes with DPBS. After blocking with 10% normal goat serum at room temperature for 30 minutes, primary antibodies (Oct4, Nanog) were added and incubated overnight at 4°C in the dark. After washing three times with DPBS, secondary antibodies were added and incubated at room temperature for 1 hour. Finally, after washing three times with DPBS, the sections were observed under a fluorescence microscope.

[0065] As a result, the umbilical cord-derived adherent stem cells according to one embodiment were cells that selectively showed positivity for CD200, CD141, CD61, CD87, and SSEA4, and were cells that selectively showed negativity for TRA-1, CD3, CD1a, CD11c, CD16, CD86, CD8a, CD40, and MIC A / B, and additionally, CD61 was selectively shown to be positivity under hypoxic conditions.Furthermore, it was found that the umbilical cord-derived adherent stem cells according to one embodiment did not express Oct4 and Nanog proteins, which are specific markers for embryonic stem cells.

[0066] Experimental example: Verification of non-clinical efficacy for ovarian failure 1. Establishment of a POI (Primary Ovarian Insufficiency) Mouse Model To establish the POI mouse model, 6-week-old C57BL / 6N female mice were administered cyclophosphamide (CP) at concentrations of 100, 200, 300, and 400 mg / kg, and the estrous cycle, ovary weight, and follicle counting were measured. The results are shown in Figures 1 to 4.

[0067] As shown in Figures 1 to 4, it was confirmed that CP administration resulted in ovarian dysfunction, and in subsequent experiments, the group administered 200 mg / kg of CP was used to verify the effects.

[0068] 2. Non-clinical efficacy verification To confirm the effect of the stem cells prepared in Example 1 on ovarian dysfunction, on Day 7 after CP administration, CordSTEM according to one embodiment was administered intravenously at a dose of 100 μL / head. On Day 17, the mice were euthanized and samples were taken.

[0069] The test groups were divided as follows:

[0070] Group 1: Normal group (N=10)

[0071] Group 2: Sham (CP 200 mg / kg administration group, N=10)

[0072] Group 3: Low-concentration cell administration group (N = 10, CordSTEM 5.33 x 10 4 cells / mouse)

[0073] Group 4: Medium concentration cell administration group (N=10, CordSTEM 8x10 4 cells / mouse)

[0074] Group 5: High-concentration cell administration group (N = 10, CordSTEM 1.07 x 10 5 cells / mouse)

[0075] The test evaluation items are as follows: (1) Histological examination On the day of necropsy, mice were euthanized and both ovaries were removed. Both ovaries were weighed and fixed in 4% paraformaldehyde solution. After fixation, paraffin blocks were prepared from the two ovarian tissues. Sectioning was performed by cutting the ovarian tissue into 10 consecutive slices at 5 μm thickness, designated as one section. This process was repeated until the entire ovarian tissue was cut, with 55 μm intervals between each section. (1-1) H&E (follicle counting), (1-2) TUNEL analysis (apoptosis), and (1-3) IHC (anti-mouse hemoglobin-myotrophic lateral sclerosis) were then performed.

[0076] (2) Confirmation of the number of superovulated eggs After a single intraperitoneal administration of 200 mg / kg of CP, CordSTEM was administered on day 7. On day 21 after test substance administration, 7.5 IU PMSG (Pregnant Mare Serum Gonadotropin) was administered intraperitoneally. Forty-eight hours later, 7.5 IU hCG (Human Choriogonical Gonadotropin) was administered intraperitoneally. Fifteen hours later, the oviducts of superovulated female mice were removed. Cumulus-oocyte complexes (COCs) were extracted from the oviducts and treated with 10 μl of hyaluronidase to remove cumulus cells, after which the number of oocytes was counted.

[0077] (3) In vitro fertilization: Verification of egg fertilization rate and embryo development rate Seven days after test substance administration, 7.5 IU PMSG was administered intraperitoneally. 48 hours later, 7.5 IU hCG was administered intraperitoneally. 15 hours later, the oviducts of superovulated female mice were removed. Sperm was expressed from the epididymis of male mice and cultured for 1 hour (37°C, 5% CO2). COCs were removed from the oviduct and treated with 10 μl of hyaluronidase to remove cumulus cells. Oocytes were collected in the center of a dish, and 10 μl of sperm was spread around them and cultured for 4 hours (37°C, 5% CO2). The oocytes were washed to remove all sperm, and then cultured to observe embryonic development.

[0078] The results of (1-1) are shown in Figures 5 and 6, the results of (1-2) are shown in Figures 7 and 8, the results of (1-3) are shown in Figures 9 and 10, the results of (2) are shown in Figures 11 and 12, and the results of (3) are shown in Figures 13 and 14.

[0079] As shown in Figures 5 and 6, administration of stem cells according to one embodiment was found to increase the number of follicles, the percentage of follicles in the ovaries, and the number of primordial follicles in a dose-dependent manner compared to untreated controls.

[0080] Furthermore, as shown in Figures 7 and 8, it was found that administration of stem cells according to one embodiment significantly reduced ovarian apoptosis compared to the control group.

[0081] Furthermore, as shown in Figures 9 and 10, administration of stem cells according to one embodiment was found to significantly increase the amount of AMH, which is associated with ovarian function such as menopause or polycystic ovary syndrome, compared to the control group.

[0082] Furthermore, as shown in Figures 11 and 12, administration of stem cells according to one embodiment was found to significantly increase the number of superovulated eggs in an animal model in which ovarian function was impaired and egg ovulation was reduced.

[0083] Furthermore, as shown in Figures 13 and 14, administration of stem cells according to one embodiment was found to significantly increase the development of fertilized eggs during in vitro fertilization (proportion of two-cell and blastocyst embryos) compared to the control group.

[0084] The above results indicate that administration of stem cells according to one embodiment may be useful in preventing or treating ovarian failure, including early menopause, infertility, or polycystic ovarian syndrome.

[0085] 3. Biomarker validation at the single-cell level To confirm the therapeutic mechanism of ovarian dysfunction using the stem cells prepared in Example 1, biomarkers were identified at the single cell level.

[0086] Ovarian samples from the normal, negative control, low-dose CordSTEM, and high-dose CordSTEM groups were minced with a surgical knife, immersed in 0.2% collagenase, and incubated at 37°C for 20 minutes. The collagenase was inactivated by adding FACS buffer (2% FBS / PBS), and then washed twice by centrifugation. Single cells isolated from the ovarian tissue were incubated with the following FACS antibodies: PE-Cy7-conjugated CD140a antibody and Alexa488-conjugated CD45 antibody. After filtering dead cells using DAPI, CD140a+ cells, CD45+ cells, and other cells (CD140a- / CD45-) were separated into 96-well plates containing lysis buffer (0.1% Triton X-100, 1U / μl RNase inhibitor, 0.25μM oligo dT, and 2.5mM dNTPs) at one cell per well, frozen in liquid nitrogen, and stored at -80°C.

[0087] After thawing a frozen 96-well plate on ice, reverse transcription reagents (1X Maxima H-buffer, 1M Betaine, 5uM MgCl2, 1uM TSO, 40U / μl RNase inhibitor, 200U / μl Maxima H-RTase) were added and reverse transcription reactions were initiated. Whole genome amplification reagents (1X KAPA HiFi buffer, 300uM dNTPs, 500uM MgCl2, 0.167uM, 0.1uM ISPCR primers, 1u / μl KAPA HiFi DNA polymerase) were added to the 96-well plate, and whole genome amplification reactions were initiated. The amplified cDNA was purified using sera-mag speed beads and QC was performed via GAPDH qPCR. Samples with negative Ct values ​​were excluded from sequencing library construction. To prepare a sequencing library, a single-cell cDNA library was added with 1X homemade Tn5 buffer and Tn5, incubated at 55°C for 5 minutes, and then placed on ice. Neutralize tagment buffer was then added and incubated at room temperature for 5 minutes. NEXTERA index and sequencing library amplification reagents (1X KAPA bf, 300 μM dNTPs, 500 μM MgCl2, 1 μ / μl KAPA enzyme) were added and incubated. After the complete reaction, all samples were pooled and 5X the volume of PB used for sequencing was added. The PCR product was then purified using a PCR product purification kit. The purified DNA was quantitatively analyzed using a TapeStation. 0.3X beads were added to the library and magnetically separated. The magnetically separated supernatant was collected, and 0.6X beads were added and magnetically separated. The supernatant was removed, washed with 80% ethanol, and the ethanol was completely removed. EB buffer was added, and the library was eluted by magnetic separation. Libraries used for sequencing were stored at 4°C. Sequencing libraries were subjected to paired-end sequencing using a NextSeq550 instrument.

[0088] Next, the sequencing reads were aligned to the mm10 mouse transcriptome using STAR aligner, and TPM and counts were calculated for each sample using RSEM. TPM values ​​were transformed into log2(TPM+1). Cells with 500 or more genes detected and genes with at least five genes detected were selected for analysis. Analysis was performed using the Numpy, SciPy, Matplotlib, sckit-learn, and pandas packages in a Python environment. Variable genes were selected, and principle component analysis and tSNE visualization were performed based on them. Unsupervised hierarchical clustering was performed to analyze cell groups, and differentially expressed and marker genes were analyzed using the DESeq2 package in the R software environment.

[0089] Statistical analysis of the quantitative results was performed using Prism Version 5.03. Student's t-test was performed to compare the normal group with the negative control group, and one-way ANOVA was used to compare the negative control group with the test substance-treated group. Statistical significance was determined when p<0.05.

[0090] The results of the immune cell population analysis are shown in FIGS. 15 and 16, the results of the stromal cell population analysis are shown in FIGS. 17 and 18, and the results of the granulosa (GC) cell population analysis are shown in FIGS. 19 and 20.

[0091] 15 and 16, it was confirmed that the B cell population and dendritic cell population, which were reduced in mice in which POI was induced by CP treatment, were restored by administration of stem cells according to one embodiment, and in particular, it was found that the dendritic cell population in the high-concentration cell administration group was restored to a level equivalent to that of the normal group. These results indicate that the restoration of ovarian function by stem cells according to one embodiment may occur through the restoration of dendritic cells present in the ovaries.

[0092] As shown in Figures 17 and 18, it was found that the fibroblast 3 cell group (Gsn), which was reduced in mice in which POI was induced by CP treatment, did not recover even after administration of stem cells according to one embodiment, but the fibroblast 2 cell group (Itm2a) recovered after administration of stem cells according to one embodiment.

[0093] 19 and 20, it was confirmed that the mural granulosa lutein cell group (Parm1) and atretic follicle group (Cfh), which were increased in mice in which POI was induced by CP treatment, were reduced after administration of stem cells according to one embodiment. In particular, it was found that the mural granulosa lutein cell group and atretic follicle group were restored to the same level as the normal control group. Considering that atretic follicles are cells that cannot develop into follicles and die, it was confirmed that the cells according to one embodiment reduce atretic follicles and are therefore significantly effective in treating ovarian dysfunction.

[0094] From the above results, it was found that stem cells according to one embodiment restore the functions of B cells, dendritic cells, fibroblasts, mural granulosa lutein cells, and atretic follicles in the POI mouse model. These results indicate that stem cells according to one embodiment induce the recovery of surrounding constituent cells that are important for oocyte development and maturation, thereby restoring ovarian function.

Claims

1. A pharmaceutical composition for preventing or treating ovarian insufficiency, comprising umbilical cord-derived adherent stem cells as an active ingredient.

2. The pharmaceutical composition of claim 1, wherein the umbilical cord-derived adherent stem cells have one or more surface antigen characteristics selected from the group consisting of D200+, Tra-1-60-, CD3-, CD1a-, CD11c-, CD16-, CD86-, CD8a-, CD40-, CD141+, CD61+, CD87+, MIC A / B-, and SSEA+.

3. 2. The pharmaceutical composition according to claim 1, wherein the ovarian failure is any one selected from primary ovarian failure, secondary ovarian failure, hyperandrogenemia, polycystic ovary syndrome, amenorrhea, oligomenorrhea, early menopause, and infertility.

4. 2. The pharmaceutical composition of claim 1, wherein the stem cells increase the number of ovarian follicles or primordial follicles.

5. The pharmaceutical composition of claim 1 , wherein the stem cells promote ovulation of an egg.

6. The pharmaceutical composition of claim 1 , wherein the stem cells promote the development of a fertilized egg.

7. The pharmaceutical composition of claim 1, wherein the stem cells restore the function or activity of any cell selected from the group consisting of B cells, dendritic cells, and fibroblasts, which is decreased in ovarian failure.

8. The pharmaceutical composition of claim 1, wherein the stem cells reduce the number or activity of mural granulosa lutein cells or atretic follicles that are increased in ovarian failure.

9. 2. The pharmaceutical composition of claim 1, wherein the stem cells increase the expression or activity of Cd 19 or Cd 209a.

10. The pharmaceutical composition of claim 1, wherein the stem cells increase the expression or activity of Itm2a or decrease the expression or activity of Parm1 or Chf.

11. The stem cells were 1.0 x 10 ３ ~1.0 x 10 １０ The pharmaceutical composition of claim 1, wherein the composition is administered in an amount of 1000 mg / kg of cells / individual.

12. A method for preventing or treating ovarian insufficiency, comprising administering to an individual in need thereof an effective amount of umbilical cord-derived adherent stem cells.

13. A method for increasing the number of ovarian follicles or primordial follicles in an individual in need thereof, comprising administering to the individual an effective amount of umbilical cord-derived adherent stem cells.

14. A method for promoting ovulation of an egg in an individual, comprising administering to the individual in need thereof an effective amount of umbilical cord-derived adherent stem cells.

15. A method for promoting the development of a fertilized egg in an individual in need thereof, comprising administering to the individual an effective amount of umbilical cord-derived adherent stem cells.

16. A method for increasing or decreasing, respectively, decreased or increased cellular function or activity in ovarian failure, comprising administering to an individual in need thereof an effective amount of umbilical cord-derived adherent stem cells.

17. The method according to claim 16, wherein the cells are any one selected from the group consisting of B cells, dendritic cells, and fibroblasts, which are decreased in the ovarian failure.

18. The method according to claim 16, wherein the cells increased in the ovarian failure are mural granulosa luteal cells or atretic follicles.

19. Use of umbilical cord-derived adherent stem cells for the manufacture of a pharmaceutical preparation for the prevention or treatment of ovarian failure.

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