JP7021828B2 - How to induce meiosis of ovarian-derived stem cells - Google Patents

Description

The present invention relates to a method for inducing meiosis of ovarian-derived stem cells, an additive used in the same method, an oocyte or an egg-like cell obtained by the same method, and the like.

Eggs are formed in the ovary during a limited period of the fetal period, no new eggs are produced after birth, and the number of eggs decreases with age. Due to changes in social structure in recent years, the age of first marriage of women is aging, and the number of patients with ovarian dysfunction due to a decrease in the number of eggs is increasing. As a result, the number of patients presenting infertility has increased rapidly, and even if it is limited to in vitro fertilization, it has exceeded 350,000 in 2013 and is expected to continue to increase in the future. However, at present, the only treatment method for infertility due to egg depletion is in vitro fertilized embryo transfer using the donated egg, and it is impossible to conceive with one's own egg. In order to overcome this limitation, it is necessary to regenerate eggs from their own cells by some method. So far, mice have succeeded in regenerating egg-like cells rather than iPS / ES cells, but in humans, they have succeeded in regenerating eggs. There is not enough evidence yet. Therefore, it has been desired to develop a method for regenerating egg-like cells from human ovarian tissue.

In recent years, a research group such as Tilly of Harvard University has shown that there are cells in the ovary that have the ability to differentiate into egg-like cells having the function of an egg (Patent Document 1). Tilly et al. Disperse ovarian tissue and then use (i) a surface antigen antibody for the DDX4 gene to isolate stem cells (egg-like stem cells) capable of differentiating into egg-like cells. , (Ii) Developed and reported a culture method for differentiating human egg-like stem cells into egg-like cells using four types of growth factors (GDNF, LIF, EGF, bFGF) under a culture medium containing serum (non-patent literature). 1).

However, regarding (i), reproducibility is denied by Non-Patent Documents 2 and 3, and regarding (ii), the ability to differentiate into egg-like cells is denied by Non-Patent Document 4. Many other research groups have also denied the presence of egg-like stem cells, indicating that the work of research groups such as Tilly is irreproducible.

Tilly et al. Isolated egg-like stem cells using DDX4 antibody, but it has been reported that there is a problem with the antibody used for the isolation and that egg-like stem cells are not specifically recovered. No other method for isolating egg-like stem cells has been reported, and no antigen capable of isolating egg-like stem cells has been found.

US 2006/0010508

Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women. White YA, Woods DC, Takai Y, Ishihara O, Seki H, Tilly JL. Nat Med. 2012 Feb 26; 18 (3): 413-21. doi: 10.1038 / nm.2669. Characterization of extracellular DDX4- or Ddx4-positive ovarian cells. Nat Med. 2015 Oct; 21 (10): 1114-6. doi: 10.1038 / nm.3966. Hernandez SF et al. Adult human and mouse ovaries lack DDX4-expressing functional oogonial stem cells. Nat Med. 2015 Oct; 21 (10): 1116-8. Doi: 10.1038 / nm.3775. Zhang H et al FACS-sorted putative oogonial stem cells from the ovary are neither DDX4-positive nor germ cells. Sci Rep. 2016 Jun 15; 6: 27991. Doi: 10.1038 / srep27991. Zarate-Garcia L, Lane SI, Merriman JA, Jones KT.

An object of the present invention is to provide a method for inducing meiosis of ovarian-derived stem cells present in an ovary, with the ultimate goal of producing an egg-like cell having an egg function from human ovarian tissue.

The present inventors have made diligent studies to solve the above problems. As a result, the present inventors do not isolate ovary-derived stem cells from ovarian tissue, and have the function of egg mother cells or eggs from ovary-derived stem cells in co-culture with other somatic cells including ovarian somatic cells. We have developed a method for inducing differentiation of egg-like cells and completed the present invention.
That is, the present invention relates to the following.

[1] (i) A step of culturing ovarian-derived stem cells and ovarian somatic cells in the presence of activin and / or BMP4;
(ii) Culturing in the presence of PKA agonists, RXR agonists, Wnt agonists and Akt agonists; and
(iii) The step of culturing in the presence of RXR agonist, NFκB agonist, Ca agonist and Akt agonist,
A method of inducing meiosis of ovarian-derived stem cells, including.
[2] The method according to [1], wherein the ovarian-derived stem cells and ovarian somatic cells are cryopreserved cells.
[3] The step according to [1], which comprises a step of enzymatically treating the isolated ovarian tissue and dispersing the ovarian cells in a solvent to obtain ovarian-derived stem cells and ovarian somatic cells before the step (i). Method.
[4] The method according to any one of [1] to [3], wherein the activin in the step (i) is activin A.
[5] (ii) The PKA agonist in step (ii) is forskolin, the RXR agonist is 5- {4-[(1-methylcyclohexyl) methoxy] benzyl} -1,3-thiazolidine-2,4-dione, and the Wnt agonist is 2. -Amino-4- [3,4- (methylenedioxy) benzylamino] -6- (3-methoxyphenyl) pyrimidine, and Akt agonist is 2-amino-6-chloro-α-cyano-3- (ethoxycarbonyl) )-The method according to any one of [1] to [4], which is -4H-1-benzopyran-4-ethyl acetate.
[6] The RXR agonist in step (iii) is 5- {4-[(1-methylcyclohexyl) methoxy] benzyl} -1,3-thiazolidine-2,4-dione, and the NFκB agonist is 2-hydroxyglutaric acid, Ca. The agonist is holbol-12-millistate-13-acetate, and the Akt agonist is 2-amino-6-chloro-α-cyano-3- (ethoxycarbonyl) -4H-1-benzopyran-4-ethyl acetate, [ 1] The method according to any one of [5].

[7] An additive for meiotic induction culture of ovarian-derived stem cells, which comprises activin and / or BMP4.
[8] An additive for meiotic induction culture of ovarian-derived stem cells, which comprises a PKA agonist, an RXR agonist, a Wnt agonist and an Akt agonist.
[9] An additive for meiotic induction culture of ovarian-derived stem cells, which comprises an RXR agonist, an NFκB agonist, a Ca agonist and an Akt agonist.
[10] (i) A step of culturing ovarian-derived stem cells and ovarian somatic cells in the presence of activin and / or BMP4;
(ii) Culturing in the presence of PKA agonists, RXR agonists, Wnt agonists and Akt agonists; and
(iii) The step of culturing in the presence of RXR agonist, NFκB agonist, Ca agonist and Akt agonist,
A method for producing an oocyte or an egg-like cell having an egg function, which comprises.
[11] (i) A step of culturing ovarian-derived stem cells and ovarian somatic cells in the presence of activin and / or BMP4; and
(ii) Culturing in the presence of PKA agonist, RXR agonist, Wnt agonist and Akt agonist,
A method of inducing mitosis of ovarian-derived stem cells, including.
[12] An additive for mitotic culture of ovarian-derived stem cells, which comprises a PKA agonist, an RXR agonist, a Wnt agonist and an Akt agonist.

As a prior art, a patent application (Patent Document 1) has been filed for a method of producing egg-like cells from human ovary-derived stem cells by in vitro culture, but there is no reproducibility and many doubts have been reported. The present inventors attempted to develop a method for regenerating egg-like cells from ovarian tissue by an in vitro culture method different from the method applied for for a patent, and developed a method for transferring cells in ovarian tissue to meiosis.

INDUSTRIAL APPLICABILITY According to the present invention, a culture method has been developed that enables the induction of meiosis necessary for differentiation into egg-like cells without isolating ovarian-derived stem cells from ovarian tissue as in the prior art. Furthermore, we identified growth factors and agonists that were different from the prior art required at that time, and determined the optimal conditions for in vitro culture that efficiently induces meiosis of cells in ovarian tissue into egg-like cells.

Therefore, according to the present invention, it has become possible to produce cells having stable egg functions under in vitro culture from ovarian tissue cells.

FIG. 1 is a diagram showing a meiosis-inducing medium and a meiosis-inducing protocol used in the examples. FIG. 2 is a diagram showing changes in the expression of germ cell-specific gene markers. A is in vivo (before tissue dispersion), before the start of culture (after dispersion, on the 0th day of culture), after the first stage culture (6th day of culture), and after the second stage culture (12th day of culture). The measurement result of PRDM1 gene expression is shown. B is in vivo (before tissue dispersion), before the start of culture (after dispersion, on the 0th day of culture), after the first stage culture (6th day of culture), and after the second stage culture (12th day of culture). The measurement result of DAZL gene expression is shown. C is in vivo (before tissue dispersion), before the start of culture (after dispersion, on the 0th day of culture), after the first stage culture (6th day of culture), and after the second stage culture (12th day of culture). The measurement results of SYCP3 gene expression are shown. D is an observation result of PRDM1 gene expression in cells before the start of culture (after dispersion, on the 0th day of culture) (photograph substitute for drawings). E is the observation result of DAZL gene expression in the cells after the first stage culture (6th day of culture) (drawing substitute photograph). F is the observation result of SYCP3 gene expression in the cells after the second stage culture (day 12 of the culture) (drawing substitute photograph). FIG. 3 is a diagram showing nuclear translocation of SYCP3 by the third stage culture. A is after the second stage culture (12th day of culture), after the third stage culture using Cocktail A (18th day of culture), and after the third stage culture using Cocktail B (18th day of culture). ) Shows the proportion of SYCP3 nuclear transition cells. B is the observation result of SYCP3 gene expression after the third stage culture using Cocktail A (18th day of culture) and after the third stage culture using Cocktail B (18th day of culture) (drawing substitute). Photo).

The abbreviations used in the present specification and their meanings will be described.
BMP4: Bone Morphogenetic Protein 4
PKA: Protein Kinase A
RXR: Retinoid X Receptor
NFκB: Nuclear Factor kappa B
PMA: Phorbol-12-Myristate-13-Acetate 2-HG: 2-hydroxyglutarate
PBS: Phosphate buffered saline
HBSS: Hanks' Balanced Salt solution
MEMα: Eagle's Minimum Essential Medium Eagle, Alpha Modification
IU: International Units
Hereinafter, the present invention will be described.

(Method of inducing meiosis of ovarian-derived stem cells)
One embodiment of the present invention comprises (i) culturing ovary-derived stem cells and ovarian somatic cells in the presence of Actibin and / or BMP4 (first-stage culture); (ii) PKA agonist, RXR agonist, Including the step of culturing in the presence of Wnt agonist and Akt agonist (second stage culture); and (iii) the step of culturing in the presence of RXR agonist, NFκB agonist, Ca agonist and Akt agonist (third stage culture). The present invention relates to a method for inducing meiosis of ovarian-derived stem cells (hereinafter referred to as "method for inducing meiosis of the present invention"). By the method of inducing meiosis of the present invention, meiosis of ovarian-derived stem cells can be induced stepwise, and differentiation can be induced into oocytes and egg-like cells having an egg function.

Further, another embodiment of the present invention includes the step (i) of subjecting the isolated ovarian tissue to enzymatic treatment and dispersing the ovarian cells in a solvent to obtain ovarian-derived stem cells and ovarian somatic cells. The ovarian-derived stem cells and ovarian somatic cells obtained by the same step are subjected to ovarian-derived stem cell meiosis, which comprises the steps of culturing the ovary-derived stem cells and ovarian somatic cells according to the first to third stages of the culture steps (i) to (iii) of the present invention. Regarding the method of inducing.

The ovary-derived stem cells for inducing meiosis, which are the subjects of the present invention, are stem cells that are present in the ovary and have the ability to differentiate into oocytes and egg-like cells having the function of an egg and mature. .. In the present specification, "ovary-derived stem cells" may be referred to as "egg-like stem cells" or "ovary stem cells", both of which have the same meaning.

Here, the oocyte means an oocyte in the first meiosis phase (primary oocyte) and an oocyte in the second meiosis phase (secondary oocyte), and the oocyte is after fertilization. The state in which the process of is possible is called an egg. In humans and the like, meiosis of oocytes usually progresses to metaphase of the second meiosis, and fertilization becomes possible.

In the method of inducing meiosis of the present invention, co-culture is performed as an ovarian cell group containing ovarian-derived stem cells and ovarian somatic cells. Here, the ovarian somatic cell is not limited as long as it is a somatic cell contained in the ovary and can induce meiosis of the ovary-derived stem cell by co-culturing with the ovary-derived stem cell. Examples of ovarian somatic cells include cells forming follicles such as cumulus cells, granule membrane cells, and pod cells, ovarian epithelial cells, ovarian interstitial cells, and precursor cells of these cells.

(Dispersion of cells)
Hereinafter, a method for preparing an ovarian cell group including ovarian-derived stem cells and ovarian somatic cells will be described.
For ovarian cell groups including ovarian-derived stem cells and ovarian somatic cells, for example, when ovarian tissue is collected, the ovarian tissue is enzymatically treated, the cells are separated from the tissue, and the cells are separated from the tissue (for example, when the enzymatic treatment is performed in a culture medium). , In a culture solution). If necessary, cells may be isolated, recovered, washed, purified, or the like from a cell suspension in which cells are dispersed in a solvent. In the method of the present invention, it is possible to culture the ovary-derived stem cells in the coexistence of the ovary-derived stem cells and the ovary somatic cells without isolating the ovary-derived stem cells from the cell group. The obtained cells can be cryopreserved, thawed at the time of use, and used for culturing. As the method of freezing and thawing, a commonly used method can be applied.

Ovarian tissue can be prepared by collecting the ovaries from animals. The animal from which the ovary is collected is not limited as long as it is an animal having an ovary, and in particular, mammals such as cows, pigs, sheep, goats, dogs, cats, rabbits, hamsters, rats, mice or humans. Can be mentioned.

The isolated ovaries are washed with PBS or the like. Cut the ovaries to a size of 1 mm or less with scissors and transfer to tissue culture medium. As the tissue culture solution, a medium used for normal tissue culture such as HBSS and MEMα can be used.

Subsequently, the tissue in the culture medium is further cut, the ovarian tissue is treated with an enzyme, separated into individual cells, and dispersed in the culture medium. Examples of the enzyme to be used include collagenase and dispase, but the enzyme is not particularly limited as long as it has an enzymatic activity for separating cells adhering or aggregating in a tissue. Such enzymes may be used alone or in combination of two or more. Further, for example, an enzyme having an enzyme activity other than the enzyme activity for separating cells from each other such as DNase may be used together.

The reaction temperature of the enzyme treatment can be appropriately determined according to the type of enzyme, the amount of enzyme used, the volume of tissue, and the like. The reaction temperature is usually close to the temperature inside the living body, for example, 32 to 38 ° C, preferably 36 to 37.5 ° C, more preferably 37 ° C, from the viewpoint of allowing the enzyme to sufficiently act and maintaining the physiological function of the cell. Degree.

The reaction time can be appropriately determined according to the type of enzyme, the amount of enzyme used, the volume of tissue, and the like. The reaction time is usually 5 minutes to 24 hours, preferably 1 to 10 hours, and more preferably about 2 hours.

As the enzyme solution for tissue lysis, an enzyme solution in which the enzyme is dissolved in the above-mentioned tissue culture solution can be used. The tissue-dissolving enzyme solution can be used in an amount that allows the tissue to be sufficiently immersed. Tissue: The tissue-dissolving enzyme solution has a weight ratio of usually 1: 5 to 1:50, preferably 1:10 to 1:40, and more preferably about 1:30. The enzyme concentration in the tissue-dissolving enzyme solution can be appropriately determined according to the volume of the tissue, the type of enzyme, and the like. More specifically, for example, when collagenase is used as an enzyme, the collagenase concentration in the enzyme solution is usually 0.1 unit / mL or more, preferably 1.0 unit / mL or more, and usually 800 units / mL or less, preferably 400. It is unit / mL or less, more preferably about 40 units / mL. Here, 1 unit is a value measured by the method of measuring FALGPA decomposition activity. That is, the amount of enzyme that hydrolyzes 1.0 μmole FALGPA per minute at pH 7.5, 25 ° C, and the presence of calcium ions is defined as 1 FALGPA unit.

After collecting an ovary cell group containing ovary-derived stem cells and ovarian somatic cells, it was determined that the germ cell-specific genes contained the ovary-derived stem cells in the ovarian cell group and induced the formation of germ cells. It is preferable to confirm by expression or the like. The germ cell-specific gene is not particularly limited, and examples thereof include PRDM1. PRDM1 is normally expressed only in fetal primordial germ cells and is an essential gene for germ cell formation. The expression of the gene can be confirmed based on a usual biological method, a molecular biological method, or the like.

In the method for inducing meiosis of the present invention, for example, the ovarian cell group including ovarian-derived stem cells and ovarian somatic cells obtained as described above is cultured by a three-step culture method to reduce the number of ovarian-derived stem cells. It is possible to induce division.
Hereinafter, the culture method at each stage will be described.

(First stage culture)
The first step of culturing in the method of inducing meiosis of the present invention is (i) culturing ovarian-derived stem cells and ovarian somatic cells in the presence of activin and / or BMP4.

For example, a group of ovary cells containing ovary-derived stem cells and ovary somatic cells collected as described above is seeded in a culture dish containing a culture solution. The culture can be performed under either adhesive or non-adhesive conditions, but non-adhesive conditions are preferable.

As the culture medium for culturing the ovarian cell group, those used for normal stem cell culture can be used except that the differentiation-inducing factor of the present invention is added. For example, MEMα, DMEM, GMRM, RPMI-1640, Ham's F-12, IMDM and the like can be mentioned. Additives and the like used in ordinary cell culture can be used as the culture solution without particular limitation as long as the effects of the present invention are not impaired. In particular, it is preferable to use serum or serum substitutes. Further, examples of the additive include 2-mercaptoethanol, sodium pyruvate, amino acids, antibiotics, N-acetylcysteine and the like.

The differentiation-inducing factor of the present invention is added to the culture medium for culturing the ovarian cell group. (i) Activin and / or BMP4 is used as a differentiation-inducing factor in the step. As the differentiation-inducing factor activin, activin A is preferable. The concentration of the differentiation-inducing factor in the culture medium can be appropriately changed depending on the cell source, the number of cells, the amount of the culture medium, etc., and is not limited, but the total amount of the differentiation-inducing factors is usually 1 to 1,000 ng / mL, preferably 10 to 10. It is 200 ng / mL, more preferably about 50 ng / mL. As these factors, commercially available reagents and the like can be used.

The number of cells per culture dish can be changed according to the cell source, the amount of the culture medium, the type of the culture dish, etc. For example, in a culture dish having a dose of 96 wells, the amount of the culture solution is usually 100 to 500. The number of cells per μL, 1 well can usually be about 20,000 to 200,000.

The incubator used for culturing may be any incubator generally used for culturing animal cells. The culture conditions include, for example, 37 ° C. to 39 ° C., a gas phase of 5% carbon dioxide gas 95% air, high humidity conditions, and the like, and an incubator capable of setting such conditions is used.

During the culture period, it is preferable to replace a part of the culture solution with a fresh one once every 2 to 4 days. The culture period can be changed depending on the animal species used and the condition of the collected cells, but is generally about several days to one week.

It is preferable to confirm that germ cells are induced to differentiate and meiosis is induced during or after culturing by expressing a gene specifically expressed before meiosis. The gene specifically expressed before meiosis is not particularly limited, and examples thereof include DAZL. The expression of the gene can be confirmed based on a usual biological method, a molecular biological method, or the like. It is also possible to change the culture period according to the degree of induction of germ cell differentiation and meiosis.

(Second stage culture)
The second step of culturing in the method of inducing meiosis of the present invention is (ii) culturing in the presence of PKA agonist, RXR agonist, Wnt agonist and Akt agonist. Wnt is a secretory glycoprotein that has a β-catenin pathway and planar cell polarity.
: PCP) Involved in the activation of three types of pathways, the calcium pathway and the calcium pathway. The β-catenin pathway regulates signal transduction and regulates cell proliferation and differentiation by regulating protein levels of β-catenin, which functions as a transcriptional promoter. Akt is a serine / threonine kinase that is involved in a widespread signal transduction network and is also known as protein kinase B (PKB).

After the culture in step (i) is completed, the culture broth is removed, washed if necessary, and the culture broth used in step (ii) is added to the culture dish.

As the culture medium for culturing the ovarian cell group, those used for normal stem cell culture can be used except that the differentiation-inducing factor of the present invention is added. As the preferred culture solution and additives, those described above can be used in step (i).

The differentiation-inducing factor of the present invention is added to the culture medium for culturing the ovarian cell group. (ii) At least a PKA agonist is used as a differentiation-inducing factor in the step. Preferred forms are those further using RXR agonists, Wnt agonists and Akt agonists. Although not particularly limited, for example, as PKA agonists, forskolin, CW 008 (4-fluoro N- [5-fluoro-6- (5-methylpyrazole [1,5-A] pyridin-3-yl) -1 H) -Pyrazolo [3,4-B] pyridin-3-yl] benzamide), 8-Bromo-cAMP (8-bromoadenosine-3', 5'-cyclic monophosphate sodium salt) and the like can be used. Although not particularly limited, for example, as the RXR agonist, ciglitazone (5- {4-[(1-methylcyclohexyl) methoxy] benzyl} -1,3-thiazolidine-2,4-dione), retinoic acid and the like can be used. Although not particularly limited, for example, examples of the Wnt agonist include Wnt agonist (2-amino-4- [3,4- (methylenedioxy) benzylamino] -6- (3-methoxyphenyl) pyrimidin) and IM-12 (3). -(4-Fluorophenylethylamino) -1-methyl-4- (2-methyl-1H-indole-3-yl) -1H-pyrrole-2,5-dione) and the like can be used. Although not particularly limited, for example, examples of the Akt agonist include SC79 (2-amino-6-chloro-α-cyano-3- (ethoxycarbonyl) -4H-1-benzopyran-4-ethyl acetate) and HY-N1412 (1, 3 Dicafe oil quinic acid) etc. can be used.

The concentration of the differentiation-inducing factor in the culture solution is usually 1 to 100 μg / mL, preferably 1 to 10 μg / mL for PKA agonists, which can be appropriately changed depending on the cell source, the number of cells, the amount of the culture solution, etc. It is mL, or usually 1 to 100 μM, preferably 1 to 10 μM, and more preferably about 5 μM. If it is an RXR agonist, it is usually 1 to 100 μg / mL, preferably 1 to 10 μg / mL, or usually 1 to 100 μM, preferably 1 to 10 μM, and more preferably about 5 μM. If it is a Wnt agonist, it is usually 1 to 100 μg / mL, preferably 1 to 10 μg / mL, or usually 1 to 100 μM, preferably 1 to 20 μM, and more preferably about 10 μM. If it is an Akt agonist, it is usually 1 to 100 μg / mL, preferably 1 to 10 μg / mL, more preferably about 4 μg / mL, or usually 1 to 100 μM, preferably 1 to 10 μM. As these factors, commercially available reagents and the like can be used.

As the number of cells per culture dish and the culture conditions, those described above can be used in step (i).

During the culture period, it is preferable to replace a part of the culture solution with a fresh one once every 2 to 4 days. The culture period can be changed depending on the animal species used and the condition of the collected cells, but is generally about several days to one week.

It is preferable to confirm that germ cells are induced to differentiate and meiosis is induced during or after culturing by expressing a gene specifically expressed during meiosis. The gene specifically expressed during meiosis is not particularly limited, and examples thereof include SYCP3.
The expression of the gene can be confirmed based on a usual biological method, a molecular biological method, or the like. It is also possible to change the culture period according to the degree of induction of germ cell differentiation and meiosis.

(Culturing in the third stage)
The third step of culturing in the method for inducing meiosis of the present invention is (iii) culturing in the presence of RXR agonist, NFκB agonist, Ca agonist and Akt agonist.

After the culture in step (ii) is completed, the culture broth is removed, washed if necessary, and the culture broth used in step (iii) is added to the culture dish.

As the culture medium for culturing the ovarian cell group, those used for normal stem cell culture can be used except that the differentiation-inducing factor of the present invention is added. As the preferred culture solution and additives, those described above can be used in step (i).

The differentiation-inducing factor of the present invention is added to the culture medium for culturing the ovarian cell group. (iii) As the differentiation-inducing factor in the step, an RXR agonist, an NFκB agonist, a Ca agonist and an Akt agonist are used. As the RXR agonist and Akt agonist, those used in step (ii) can be used. Although not particularly limited, for example, 2-HG, bethulinic acid and the like can be used as the NFκB agonist. Although not particularly limited, for example, PMA, bryostatin and the like can be used as the Ca agonist.

The concentration of the differentiation-inducing factor in the culture medium can be appropriately changed depending on the cell source, the number of cells, the amount of the culture medium, and the like, and is not limited. The concentration of the RXR agonist and the Akt agonist can be the same as the amount used in step (ii). If it is an NFκB agonist, it is usually 1 to 100 mg / mL, preferably 1 to 10 mg / mL, or usually 1 to 100 mM, preferably 1 to 50 mM, and more preferably about 20 mM. If it is a Ca agonist, it is usually 0.1 to 100 μg / mL, preferably 0.5 to 10 μg / mL, or usually 1 to 100 μM, preferably 1 to 10 μM, and more preferably about 1 μM. As these factors, commercially available reagents and the like can be used.

As the number of cells per culture dish and the culture conditions, those described above can be used in step (i).

During the culture period, it is preferable to replace a part of the culture solution with a fresh one once every 2 to 4 days. The culture period can be changed depending on the animal species used and the condition of the collected cells, but is generally about several days to one week.

It is preferable to confirm that germ cells are induced to differentiate and meiosis is induced during or after culturing by expressing a gene specifically expressed during meiosis. The gene specifically expressed during meiosis is not particularly limited, and examples thereof include SYCP3. In the third stage culture, the transition to the meiotic phase can be confirmed, for example, by confirming the translocation of SYCP3 into the nucleus. The expression of the gene can be confirmed based on a usual biological method, a molecular biological method, or the like. It is also possible to change the culture period according to the degree of induction of germ cell differentiation and meiosis.

(Additive)
A further embodiment of the invention relates to an additive for meiotic induction culture of ovarian-derived stem cells, including activin and / or BMP4. Further, another embodiment of the present invention relates to an additive for meiotic induction culture of ovarian-derived stem cells, which comprises a PKA agonist, an RXR agonist, a Wnt agonist and an Akt agonist. Further, another embodiment of the present invention relates to an additive for meiotic induction culture of ovarian-derived stem cells, which comprises an RXR agonist, an NFκB agonist, a Ca agonist and an Akt agonist. Hereinafter, these are referred to as "additives of the present invention".

The additives of the present invention are used in the culture medium of each stage in the method of inducing meiosis of ovary-derived stem cells of the present invention.

Examples of the additive of the present invention include a reagent used for research and development purposes (for example, in vitro or in vivo research and development), an additive as a material for producing a pharmaceutical product, and the like.

Since the additive of the present invention has an action of inducing meiosis of ovary-derived stem cells, a reagent for inducing meiosis in culturing ovary-derived stem cells, an egg having an egg mother cell or egg function from ovary-derived stem cells. It can be suitably used as a reagent for producing like cells, a material for producing a pharmaceutical drug for producing an egg mother cell or an egg-like cell having an egg function from an ovary-derived stem cell, and the like. When the additive of the present invention is used as a manufacturing material for a reagent or a pharmaceutical product, it may contain additional components such as a stabilizer and a solvent in addition to the compound which is a differentiation-inducing factor of the active ingredient. good. Further, the additive of the present invention may be the compound itself which is a differentiation inducing factor. Further, the additive of the present invention may be used in combination with an agent containing one or more compounds which are differentiation-inducing factors, or may contain all the compounds.

Further, the additive of the present invention may contain a component of the culture medium of each of the above-mentioned stages in the method for inducing meiosis of the ovary-derived stem cells of the present invention. In this case, the medium used in the method of the present invention can be given only by dissolving in water or the like. In this case, the blending amount of the compounds which are various differentiation-inducing factors contained in the additive is the method in which the final concentration of each compound in the medium induces the meiotic division of the ovary-derived stem cells of the present invention described above when diluted. , Prepare to be the same as the concentration of the differentiation-inducing factor in the culture medium of each stage of the above. The additive of the present invention can be produced according to a usual pharmaceutical method, pharmaceutical method and the like.

(Method of producing oocyte or egg-like cell having egg function)
A further embodiment of the invention is (i) culturing ovary-derived stem cells and ovarian somatic cells in the presence of activin and / or BMP4; (ii) culturing in the presence of PKA agonists, RXR agonists, Wnt agonists and Akt agonists. And (iii) a method for producing an egg mother cell or an egg-like cell having an egg function, which comprises a step of culturing in the presence of an RXR agonist, an NFκB agonist, a Ca agonist and an Akt agonist (hereinafter, "the present invention". It is referred to as "method for producing an egg mother cell or an egg-like cell").

Regarding the ovary-derived stem cells and ovarian somatic cells used in the method for producing oocytes or oocytes of the present invention, the culture conditions in the culture of each stage of (i) to (iii), the above-mentioned meiosis of the present invention. You can refer to the cells, culture conditions, etc. in the method of inducing.

By the method for producing an oocyte or an egg-like cell of the present invention, the oocyte and the egg-like cell having the function of an egg are produced by promoting the induction of differentiation of ovarian-derived stem cells and inducing meiosis in stages. Can induce differentiation. The production of egg-like cells can be confirmed by the expression of the gene specifically expressed during the above-mentioned meiosis phase. It can also be confirmed by observing the morphology of cells using a microscope. Further, the oocyte obtained by inducing the differentiation of the ovary-derived stem cell by the method for producing an oocyte or an oocyte of the present invention is further matured by a normal maturation culture method of the oocyte, if necessary. You can also let them.

(Method of inducing mitosis of ovarian-derived stem cells, additives)
A further embodiment of the invention is (i) culturing ovarian-derived stem cells and ovarian somatic cells in the presence of activin and / or BMP4; and (ii) in the presence of PKA agonists, RXR agonists, Wnt agonists and Akt agonists. The present invention relates to a method for inducing mitosis of ovary-derived stem cells, which comprises a step of culturing (hereinafter referred to as "method for inducing mitosis of ovary-derived stem cells of the present invention").
Further, another embodiment of the present invention relates to an additive for mitotic culture of ovarian-derived stem cells, which comprises a PKA agonist, an RXR agonist, a Wnt agonist and an Akt agonist.

Regarding the ovary-derived stem cells and ovarian somatic cells used in the method for inducing somatic cell division of ovary-derived stem cells of the present invention, the culture conditions in the culture of each stage of (i) to (ii), the culture in step (ii). Except for the period, the cells, culture conditions, etc. in the above-mentioned method for inducing meiosis of the present invention can be referred to.
The culture period of step (ii) in the method for inducing mitosis of ovarian-derived stem cells of the present invention is set longer than that in the above-mentioned method for inducing meiosis of the present invention. That is, in the state where the protein essential for the meiosis phase was induced in the ovary-derived stem cells by the culture of the step (ii), the meiosis of the ovary-derived stem cells was not induced by continuing the culture of the step (ii). Somatic cell division is induced.
(ii) The culture period of the step can be changed depending on the animal species used and the condition of the collected cells, but is generally about 1 to 2 weeks.

The details of the present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

<Materials and methods>
Human-derived ovarian tissue was prepared by recovering the ovarian medulla portion excluding the cortex containing the follicle of the excised ovary used for treatment.
The expression of PRDM1, DAZL, and SYCP3 genes in cultured cells was measured by measuring the expression level of mRNA by real-time PCR. To observe the expression distribution of PRDM1, DAZL, and SYCP3 proteins in cultured cells, the cultured cells were used with anti-PRDM1 antibody, anti-DAZL antibody, anti-SYCP3 antibody, anti-DAPI antibody (for contrast staining), and secondary antibody for immunofluorescence staining. The antibody was stained with a fluorescent microscope and observed with a fluorescent microscope.

<Example 1> Dispersion of human ovarian tissue
A tissue lysate in which Collagenase (Worthington Biochemical Corporation, (40 U / mL) and DNase (0.1 mg / mL) was dissolved in HBSS was used. The ovarian tissue was cut with scissors to a size of 1 mm or less, and the tissue: tissue by weight ratio. The solution was added in an amount of about 1:30 and reacted in an incubator at 37 ° C. for 2 hours.

About half of the tissue was dissolved in 2 hours. The semi-dissolved solution was pipetted 2-3 times, the HBSS supernatant was pipetted and collected in a 50 mL tube via a 40 μm cell strainer filter. To the remaining undissolved tissue, the same amount of HBSS as the initial amount was added, pipetting was performed about 10 times, and the supernatant was collected again. This operation was repeated 3 to 5 times, and the isolated cells were collected.

The suspension containing the collected cells was centrifuged at 300 G at 4 ° C for 15 minutes, and the supernatant was discarded after centrifugation. Further, the same amount of HBSS was added, and the cells were washed repeatedly by centrifugation and discarding the supernatant three times. After washing the cells was completed, a certain amount of cell suspension was collected, the number of cells was counted, and the expression of PRDM1, DAZL, and SYCP3 genes was measured and observed. The rest is used for cell culture described later or cryopreserved. 2A and 2D are data using cells cryopreserved using Cerbunker 1 (Nippon zenyaku kogyo CO). For cryopreservation, use 1 mL of cell bunker 1 for 2 x 10 ⁶ cells, -80.
It was frozen and stored in a freezer at ° C.

It was found that PRDM1-positive cells, which are germ cell-specific genes, are induced to differentiate from ovarian-derived stem cells in the ovarian constituent cells 3 hours after tissue dispersion by collagenase (Fig. 2A: real-time). PRDM1 mRNA expression level by PCR was measured as the expression level expressed relative to Log using the expression level of the housekeeping gene B2M, D: PRDM1 staining by immunofluorescence staining, the photo on the right is a merged image of PRDM1 and contrast staining (DAPI)) ..

<Example 2> Induction of meiosis First-stage culture (DAZL-induced culture)
I) Preparation of MEM-A Based on MEMα of the basal culture solution, 4% KSR (KockOut Serum replacement (registered trademark)), L-Alanyl-L-Glutamine (2000 μM), 2-Mercaptoethanol Solution (100 μM) ), Sodium Pyruvate (1000 μM), MEM non-essential Amino Acids Solution, N-acetyle-L-Cysteine (100 μg / mL) with the addition of the antibiotic Antibiotic-Antimycotic Mixed Solution (Penicillin, Streptomycin, Amphotericin B). Was used. To induce differentiation, the cytokines BMP4 and Activin A were added at 50 ng / mL each.

II) Culturing the collected cells The collected cells were collected in Example 1 and used for culturing. A 96-Well non-adhesive type was used for the cultured dish. As for the collected cells, 50,000 cells (or cells cryopreserved after collection can be used, 70,000 in the case of frozen cells) are uniformly sprinkled in 1 well, and MEM-A is added (100 μl /). Well) and cultured for 6 days. In addition, MEM-A was replaced on the 3rd day of culture. After 6 days of culture, PRDM1, DAZL, and SYCP3 gene expressions were measured and observed.

The ovary-derived cell group containing the PRDM1-positive cell group obtained by ovarian tissue dispersal was mixed with a culture medium (MEM-A) containing BMP4 [50 ng / mL] (WAKO) and Activin A [50 ng / mL] (WAKO) 6 After day-to-day culture, DAZL gene-positive cells expressed before meiosis appeared (Fig. 2B: DAZL mRNA expression level by real-time PCR as Log relative expression level using the expression level of housekeeping gene B2M. Measurement, E: DAZL staining by immunofluorescence staining, the photo on the right is a merged image of DAZL and contrast staining (DAPI)).

Second-stage culture (SYCP3-induced culture)
I) Preparation of MEM-B Based on MEMα of basal culture solution, 4% KSR, L-Alanyl-L-Glutamine (2000 μM), 2-Mercaptoethanol Solution (100 μM), Sodium Pyruvate (1000 μM), MEM A non-essential Amino Acids Solution, N-acetyle-L-Cysteine (100 μg / mL) plus the antibiotic Antibiotic-Antimycotic Mixed Solution was used. Forskolin, an agonist, is used to induce differentiation.
(WAKO) (PKA agonist) [5 μM], 5- {4-[(1-methylcyclohexyl) methoxy] benzyl} -1,3-thiazolidine-2,4-dione (Ciglitazone, WAKO) (RXR agonist) [5 μM], 2-Amino-6-chloro-α-cyano-3- (ethoxycarbonyl) -4H-1-benzopyran-4-acetic acid ethyl ester (SC79, Merck) (Akt agonist) [4 μg / mL], 2 -Amino-4- [3,4- (methylenedioxy) benzylamino] -6- (3-methoxyphenyl) pyrimidine (Wnt agonist, Santa Cruz Biotechnology) (Wnt agonist)
[10 μM] was added.

II) Culture the collected cells
A group of cells cultured with MEM-A for 6 days was used. MEM-B (100 μl / well) was added to the new Dish, and the cell population was cultured for 6 days. In addition, MEM-B was replaced on the 3rd day of culture. After 6 days of culture in the second stage (12 days after culture from the first stage), PRDM1, DAZL, and SYCP3 gene expressions were measured and observed.

A group of cells containing DAZL-positive cells obtained by the first-stage culture was used as Cocktail A: Forskolin (PKA agonist) [5 μM], Ciglitazone (RXR agonist) [5 μM], SC79 (Akt agonist) [4 μg / mL. ], By culturing in a culture medium (4% KSR) supplemented with Wnt agonist [10 μM] for 6 days, the expression of SYCP3, which is essential for the meiosis phase, could be induced. However, protein localization was in the cytoplasm, and no nuclear translocation was observed for SYCP3 functioning during meiosis (Fig. 2C: real-time).
The expression level of SYCP3 mRNA by PCR was measured as the expression level relative to Log using the expression level of the housekeeping gene B2M, F: SYCP3 staining by immunofluorescence staining, the photo on the right is a merged image of SYCP3 and counterstain (DAPI)). ..

Third stage culture (SYCP3 nuclear transfer culture)
I) Preparation of MEM-C Based on MEMα of the basal culture solution, 4% KSR, L-Alanyl-L-Glutamine (2000 μM), 2-Mercaptoethanol Solution (100 μM), Sodium Pyruvate (1000 μM), A MEM non-essential Amino Acids Solution with the antibiotic Antibiotic-Antimycotic Mixed Solution added was used. Agonists PMA (WAKO) (Ca agonist) [1 μM], Ciglitazone (RXR agonist) [10 μM], SC79 (Akt agonist) [4 μg / mL], 2-HG (Cayman Chemical) (NFκB) Agonist) [20 mM] was added.

II) Culture the collected cells
A group of cells cultured with MEM-B for 6 days was used. MEM-C (100 μl / well) was added to the new Dish, and the cell population was cultured for 6 days. In addition, MEM-C was replaced on the 3rd day of culture. After 6 days of culture in the third stage (18 days after culture from the first stage), PRDM1, DAZL, and SYCP3 gene expressions were measured and observed.

Coktail B: PMA (Ca agonist) [1 μM], Ciglitazone (RXR agonist) [10 μM], SC79 (Akt agonist) [4 μg / mL] ], 2-HG (NFkB agonist) [20 mM] was added and cultured for 6 days, and SYCP3 was transferred to the nucleus and the initiation of meiosis was observed. In addition, when continuously cultured in Coktail A, SYCP3 did not translocate to the nucleus, and SYCP3-positive cells divided into somatic cells. Therefore, it was clarified that Cocktail A has the effect of promoting the proliferation of germ cells, and Coktail B has the effect of promoting the nuclear translocation of SYCP3 and inducing the translocation to the meiotic phase (Fig. 3: Immunofluorescent staining). SYCP3 staining by SYCP3 staining, A: Percentage of cells showing SYCP3 nuclear translocation in SYCP3-positive cells, B: SYCP3 staining, SYCP3 signal is observed in the nucleus by cacktail B).

The present invention can be applied to research reagents and the like.

Claims (9)

(i) A step of culturing ovarian-derived stem cells and ovarian somatic cells in the presence of activin A and B MP4;
(ii) Forskolin, 5- {4-[(1-methylcyclohexyl) methoxy] benzyl} -1,3-thiazo
Lysine-2,4-dione, 2-amino-4- [3,4- (methylenedioxy) benzylamino] -6- (3-methoxyphenyl) pyrimidine, and 2-amino-6-chloro-α-cyano -3- (ethoxycarbonyl) -4H-1-benzopyran-4- Culturing in the presence of ethyl acetate; and
(iii) 5- {4-[(1-Methylcyclohexyl) methoxy] benzyl} -1,3-thiazolidine-2,4-dio
, 2-Hydroxyglutarate, phorbol-12-millistate-13-acetate, and 2-amino-6-chloro-α-cyano-3-(ethoxycarbonyl) -4H-1-benzopyran-4-ethyl acetate present The process of culturing under,
A method of inducing meiosis of ovarian-derived stem cells, including. The method according to claim 1, wherein the ovarian-derived stem cells and ovarian somatic cells are cells cryopreserved. (I) The method according to claim 1, comprising the step of enzymatically treating the isolated ovarian tissue and dispersing the ovarian cells in a solvent to obtain ovarian-derived stem cells and ovarian somatic cells prior to the step (i). To be used in the method according to any one of claims 1 to 3 , which comprises activin A and B MP4.
Additive for meiosis-inducing culture of ovarian-derived stem cells. Forskolin, 5- {4-[(1-methylcyclohexyl) methoxy] benzyl} -1,3-thiazoli
Din-2,4-dione, 2-amino-4- [3,4- (methylenedioxy) benzylamino] -6- (3-methoxyphenyl) pyrimidine, and 2-amino-6-chloro-α-cyano Addition for meiosis-inducing culture of ovary-derived stem cells for use in the method according to any one of claims 1 to 3, comprising -3- (ethoxycarbonyl) -4H-1-benzopyran-4-ethyl acetate . Agent. 5- {4-[(1-Methylcyclohexyl) methoxy] benzyl} -1,3-thiazolidine-2,4-dione, 2-hydroxyglutaric acid, phorbol-12-millistate-13-acetate, and 2-amino Includes -6-chloro-α-cyano-3- (ethoxycarbonyl) -4H-1-benzopyran-4-ethyl acetate ,
An additive for meiotic induction culture of ovary-derived stem cells for use in the method according to any one of claims 1 to 3 . (i) A step of culturing ovarian-derived stem cells and ovarian somatic cells in the presence of activin A and B MP4;
(ii) Forskolin, 5- {4-[(1-methylcyclohexyl) methoxy] benzyl} -1,3-thiazo
Lysine-2,4-dione, 2-amino-4- [3,4- (methylenedioxy) benzylamino] -6- (3-methoxyphenyl) pyrimidine, and 2-amino-6-chloro-α-cyano -3- (ethoxycarbonyl) -4H-1-benzopyran-4- Culturing in the presence of ethyl acetate; and
(iii) 5- {4-[(1-Methylcyclohexyl) methoxy] benzyl} -1,3-thiazolidine-2,4-dio
, 2-Hydroxyglutarate, phorbol-12-millistate-13-acetate, and 2-amino-6-chloro-α-cyano-3-(ethoxycarbonyl) -4H-1-benzopyran-4-ethyl acetate present The process of culturing under,
A method for producing an oocyte or an egg-like cell having an egg function, which comprises. (i) Culturing ovarian-derived stem cells and ovarian somatic cells in the presence of activin A and B MP4; and (ii) forskolin, 5- {4-[(1-methylcyclohexyl) methoxy] benzyl} -1, 3-Thiazo
Lysine-2,4-dione, 2-amino-4- [3,4- (methylenedioxy) benzylamino] -6- (3-methoxyphenyl) pyrimidine, and 2-amino-6-chloro-α-cyano -3- (ethoxycarbonyl) -4H-1-benzopyran-4- step of culturing in the presence of ethyl acetate,
A method of inducing mitosis of ovarian-derived stem cells, including. Forskolin, 5- {4-[(1-methylcyclohexyl) methoxy] benzyl} -1,3-thiazoli
Din-2,4-dione, 2-amino-4- [3,4- (methylenedioxy) benzylamino] -6- (3-methoxyphenyl) pyrimidin, and 2-amino-6-chloro-α-cyano An additive for inducing somatic cell division-induced culture of ovarian-derived stem cells for use in the method according to claim 8, which comprises -3- (ethoxycarbonyl) -4H-1-benzopyran-4-ethyl acetate .

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