JP7729579B2 - Culture technology for differentiating ovarian tissue from pluripotent stem cells - Google Patents

Description

The present invention relates to a culture technique for regenerating ovarian tissue from pluripotent stem cells.

The germ cell lineage is the only cell lineage that supports the survival of a species. Among germ cells, the egg plays an extremely important and temporary role, initiating development and driving early development.

Previous studies using mice have reported methods for producing primordial germ cell-like cells from ES cells and iPS cells through in vitro culture (Non-Patent Document 1, Patent Documents 1 and 2), as well as a method for regenerating functional oocytes from primordial germ cell-like cells (Patent Document 3). However, in Non-Patent Document 1, meiosis and follicle formation are carried out and transplantation into mice is used to obtain mature oocytes, and the oocytes are not fully matured through in vitro culture.

Furthermore, while the method of Patent Document 3 matures eggs through in vitro culture, it is necessary to collect ovarian somatic cells (follicles) from the living body to nurture the eggs. This makes it difficult to regenerate eggs from various animals, including humans, from which it is difficult to collect ovarian tissue.

As such, at this stage, there have been no successful examples of creating functional eggs using only pluripotent stem cells, which multiply infinitely, without using living tissue. If functional eggs could be created without using living tissue, it would be possible to elucidate the complex mechanisms of germ cell differentiation and create individuals using gametes produced through in vitro culture. For this reason, there has been a desire for a technology to create functional eggs without using any living cells.

International Publication No. WO2012/020687 International Publication No. WO2014/133194 International Publication No. WO2017/047799

Hayashi K., et al, “Offspring from Oocytes Derived from in Vitro Primordial Germ Cell-like Cells in Mice.” Science 338, 971-975 (2012)

The objective of this invention is to construct an egg production system that is independent of living tissue by inducing differentiation of pluripotent stem cells into ovarian somatic tissue.

In order to solve the above problems, the inventors conducted extensive research and discovered that ovarian somatic cells can be successfully cultured by culturing epiblast-like cells differentiated from pluripotent stem cells in the presence of a BMP agonist and a Wnt agonist, and then culturing them in the presence of a BMP agonist, retinoic acid, an SHH agonist, and an FGF inhibitor.

Furthermore, the inventors discovered that by culturing the obtained ovarian somatic cells with oocytes separately induced from pluripotent stem cells, it is possible to produce eggs without using any living tissue.

The present invention has been completed based on these findings and includes the following broad aspects.
[Section 1]
A method for producing ovarian somatic tissue in vitro from pluripotent stem cells, comprising:
(a) culturing epiblast-like cells differentiated from pluripotent stem cells in the presence of a BMP agonist and a Wnt agonist to induce early mesoderm;
(b) culturing the early mesoderm in the presence of a BMP agonist, retinoic acid, an SHH agonist, and an FGF inhibitor to induce intermediate mesoderm;
A method comprising:
[Section 2]
Item 1. The method according to Item 1, further comprising the step of (c) culturing the intermediate mesoderm in the presence of a BMP agonist and an FGF agonist to induce ovarian somatic tissue.
[Section 3]
Item 3. The method according to Item 1 or 2, wherein the BMP agonist is BMP4 and the Wnt agonist is CHIR99021.
[Section 4]
Item 4. The method according to any one of Items 1 to 3, wherein the BMP agonist is BMP4, the SHH agonist is SHH, and the FGF inhibitor is PD0325901.
[Section 5]
Item 5. The method according to any one of Items 2 to 4, wherein the FGF agonist is FGF9.
[Section 6A]
A method for producing GV stage oocytes, comprising:
(d) culturing the ovarian somatic tissue obtained by the method according to any one of items 1 to 5 and primordial germ cells under conditions that exclude the influence of estrogen or a factor having a function similar to estrogen, to form secondary follicles containing oocytes, a granulosa cell layer, and a theca cell layer;
(e) partially cleaving the bonds between the granulosa cell layer and the theca cell layer in the secondary follicles formed in step (d);
(f) differentiating the oocytes, granulosa cell layer, and theca cell layer constituting the secondary follicles into GV stage oocytes by culturing them in a medium containing a polymer compound;
A method comprising:
[Section 6B]
A method for producing GV stage oocytes, comprising:
(D) Obtaining ovarian somatic tissue by the method according to any one of items 1 to 5;
(E) culturing the ovarian somatic tissue and primordial germ cells under conditions that exclude the influence of estrogen or factors with estrogen-like functions to form secondary follicles containing oocytes, granulosa cell layers, and theca cell layers;
(F) partially cleaving the bonds between the granulosa cell layer and the theca cell layer in the secondary follicles formed in step (E);
(G) differentiating the oocytes, granulosa cell layer, and theca cell layer constituting the secondary follicles into GV stage oocytes by culturing them in a medium containing a polymer compound;
A method comprising:
[Section 7]
Item 7. The method according to Item 6, wherein the polymer compound is at least one compound selected from the group consisting of polyvinylpyrrolidone, ficoll, hydroxypropyl methylcellulose, and serum albumin.
[Section 8A]
A method for producing eggs, comprising:
(g) A method comprising a step of resuming meiosis by in vitro maturation culture of GV stage oocytes obtained by the method according to 6 or 7.
[Section 8B]
A method for producing eggs, comprising:
(H) Obtaining GV stage oocytes by the method described in Item 6 or 7;
(I) a step of resuming meiosis by in vitro maturation culture of the GV stage oocytes.
[Section 9]
Item 6. An ovarian somatic tissue obtained by the method according to any one of Items 1 to 5.
[Section 10]
A GV stage oocyte obtained by the method according to Item 6 or 7.
[Section 11]
Item 9. An ovum obtained by the method according to Item 8.
[Section 12]
A kit for differentiating pluripotent stem cells into ovarian somatic tissue in vitro, comprising:
A kit comprising a BMP agonist, a Wnt agonist, a retinoic acid, an SHH agonist, an FGF inhibitor, or a combination thereof.

According to the present invention, it is possible to induce differentiation of ovarian somatic tissue from pluripotent stem cells. Furthermore, by culturing the resulting ovarian somatic cells with oocytes separately induced from pluripotent stem cells, it is possible to produce eggs without using any biological tissue.

This shows the results of FACS analysis of T-GFP and PDGFRA in Example (1). Comparison is made between 2 days (upper panel) and 4 days (lower panel) of culture. Since both T-GFP and PDGFRA are positive in early mesoderm, it is clear that 2 days of culture, when both T-GFP and PDGFRA are positive, is preferable. FACS analysis of Osr1-GFP and Foxf1-tdTomato was performed in Example (1). Because Osr1 is a marker for the intermediate mesoderm (IMM) and Foxf1 is a marker for the lateral plate mesoderm (LPM), Osr1-positive and Foxf1-negative conditions are preferred. The expression levels of each marker when the concentrations of BMP4 and CHIR99021 are varied in Example (1) are shown. Conditions that result in high IMM marker levels and low paraxial mesoderm (PM) and LPM marker levels are preferred. This shows the results of Q-PCR analysis of the expression levels of each marker when the concentrations of retinoic acid, SHH, and PD0325901 were varied in Example (2). Because all of these markers are markers for gonadal somatic cell precursors, conditions that result in high values are preferred. The figure shows the number of Gata4-positive and weakly Osr1-positive cells when the concentrations of retinoic acid, SHH, and PD0325901 were varied in Example (2). Conditions that result in a large number of Gata4-positive and weakly Osr1-positive cells are preferred. This is a two-dimensional UMAP plot of E12.5 and FOSCLs in (3) of the Example. This shows the state of culture during secondary follicle formation in Example (5). E12.5 or FOSLCs were cultured together with PGCLCs. FxT: Foxl2-tdTomato, SC: stella-CFP, BV: Blimp1-mVenus 1 shows the results of immunofluorescence analysis showing the state of self-organization during culture during secondary follicle formation in Example (5). 1 shows the results of fluorescence analysis showing the in vitro culture of secondary follicles in Example (6). 1 shows the results of fluorescence analysis showing oocyte maturation in Example (7), showing oocyte-cumulus cell complexes (COCs) before and after culture. These are newborns obtained from FOSCLs in Example (8). In Example (8), the newborn mice are mature living mice. In Example (8), new individuals were born from the mating of live mice whose newborns had matured.

<Production of ovarian somatic tissue from pluripotent stem cells>
As used herein, "pluripotent stem cells" refer to cells that possess both pluripotency, which allows them to differentiate into various cells present in the body, and the ability to self-renew, allowing them to proliferate while maintaining an undifferentiated state. This term encompasses any cell that can be induced to become the epiblast-like cells used in the present invention. Examples of pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ cells (EG cells), spermatogonial stem cells (GS cells), and Muse cells isolated from bone marrow mesenchymal cells. The pluripotent stem cells listed above can be obtained by known methods.

The pluripotent stem cells used in the present invention are derived from mammals. Examples of mammals include mice, rats, humans, monkeys, dogs, pigs, cows, cats, goats, sheep, rabbits, guinea pigs, and hamsters, with mice being preferred.

In the present invention, the ES cells used to induce differentiation into epiblast-like cells can be obtained by known methods. For example, they can be established by collecting the inner cell mass from the blastocyst of a fertilized egg of the target animal and culturing the inner cell mass on Feder cells derived from fibroblasts.

In the present invention, the iPS cells used to induce differentiation into epiblast-like cells may be derived from primary cultures of somatic cells collected from a donor animal, or may be derived from an established cell line. Furthermore, the somatic cells used for iPS cells may, in principle, be derived from either ectodermal or endodermal germ layers. iPS cells can be obtained by known methods. Specifically, this can be done with reference to, for example, Okita K. et al., "Generation of germline-competent induced pluripotent stem cells." Nature 448, 313-317 (2007), Hamanaka S. et al., "Generation of germline-competent rat induced pluripotent stem cells" PLoS One 6(7), e22008 (2011), Ohnuki M. et al., "Generation and characterization of human induced pluripotent stem cells." Curr Protoc Stem Cell Biol. Jun Chapter 4: Unit 4A.2, (2009), etc.

Furthermore, as used herein, "epiblast-like cells (EpiLCs)" are cells derived from pluripotent stem cells and have properties equivalent to those of pre-gastrulation epiblast cells. More specifically, epiblast-like cells (EpiLCs) are defined as cells that have either or both of the following properties:
(1) Increased expression of at least one gene selected from Fgf5, Wnt3, and Dnmt3b compared to pluripotent stem cells before differentiation induction.
(2) A decrease in the expression of at least one gene selected from Gata4, Gata6, Sox17, and Blimp1, compared to pluripotent stem cells before differentiation induction.

Methods for inducing differentiation of epiblast-like cells from ES cells or iPS cells can be performed by referring to known methods, such as Patent Document 1, Cell 146, 519-532 (2011), Science 338, 971-975 (2012), etc.

(a) A step of inducing early mesoderm by culturing epiblast-like cells differentiated from pluripotent stem cells in the presence of a BMP agonist and a Wnt agonist. The method of the present invention comprises "(a) a step of inducing early mesoderm by culturing epiblast-like cells differentiated from pluripotent stem cells in the presence of a BMP agonist and a Wnt agonist."

Examples of basal media for differentiation induction in (a) above include, but are not limited to, αMEM medium, Neurobasal medium, Neural Progenitor Basal medium, NS-A medium, BME medium, BGJb medium, CMRL 1066 medium, Minimum Essential Medium (MEM), GMEM medium, Eagle MEM medium, Dulbecco's Modified Eagle Medium (DMEM), Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, DMEM/F12 medium, StemPro-34SFM medium, Ham's medium, RPMI 1640 medium, HTF medium, Fischer's medium, and mixtures thereof.

The medium may be serum-containing or serum-free. Serum-free medium is preferred. Serum-free medium (SFM) refers to a medium that does not contain either raw or unpurified serum, including media containing purified blood-derived components or animal tissue-derived components (e.g., growth factors). The serum concentration (e.g., fetal bovine serum (FBS), human serum, etc.) is 0-20%, preferably 0-5%, more preferably 0-2%, and most preferably 0% (i.e., serum-free). SFM may contain any serum substitute. Examples of serum substitutes include albumin (e.g., lipid-rich albumin, albumin substitutes such as recombinant albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. Other examples include KnockOut Serum Replacement (KSR) and Glutamax. These can be used alone or in combination of two or more.

The medium contains BMP agonists and Wnt agonists as essential supplements to the basal medium.

BMP agonists include, for example, BMP family proteins. Examples include BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, BMP11, BMP12, BMP13, BMP14, and BMP15, with BMP4 being particularly preferred. The concentration of the BMP agonist is preferably 0.01 ng/mL or higher, more preferably 0.1 ng/mL or higher. The concentration of the BMP agonist is preferably 10 ng/mL or lower, more preferably 5 ng/mL or lower, and particularly preferably 2 ng/mL or lower.

Examples of Wnt agonists include inhibitors of Wnt family proteins, Frizzled receptor activators, endogenous Wnt antagonists, inhibitors of intracellular β-catenin suppressors, and inhibitors of intracellular β-catenin degradation. GSK-3β inhibitors are preferred, with CHIR99021 being particularly preferred. The concentration of the Wnt agonist is preferably 3 μM or higher, more preferably 5 μM or higher, and particularly preferably 10 μM or higher. Furthermore, the concentration of the Wnt agonist is preferably 50 μM or lower, more preferably 20 μM or lower.

The medium may contain other known additives. The additives are not particularly limited, but examples include growth factors, polyamines, minerals, sugars (e.g., glucose, etc.), organic acids (e.g., pyruvic acid, lactic acid, etc.), amino acids (e.g., non-essential amino acids (NEAA), L-glutamine, etc.), reducing agents (e.g., 2-mercaptoethanol, etc.), vitamins (e.g., ascorbic acid, d-biotin, etc.), steroids, antibiotics (e.g., streptomycin, penicillin, etc.), buffers (e.g., HEPES, etc.), and nutritional additives (e.g., B27 supplement, N2 supplement, StemPro-Nutrient Supplement, etc.). These can be used alone or in combination of two or more. It is preferable that each additive be present within a known concentration range.

In the culture step (a), the culture step of inducing early mesoderm from epiblast-like cells can be carried out, for example, by seeding epiblast-like cells in a known cell-non-adhesive or low-adhesive culture vessel and culturing them. Culture conditions are not limited to the following, but for example, the culture can be carried out in an atmosphere of 1-10% _CO2 /99-90% air. The culture temperature is approximately 30-40°C, preferably approximately 37°C. The culture period is less than 4 days, preferably approximately 2 days (e.g., 48±12 hours, preferably 48±6 hours).

(b) A step of inducing intermediate mesoderm by culturing the early mesoderm obtained by step (a) in the presence of a BMP agonist, retinoic acid, an SHH agonist, and an FGF inhibitor. The present invention further comprises "(b) a step of inducing intermediate mesoderm by culturing the early mesoderm obtained by step (a) in the presence of a BMP agonist, retinoic acid, an SHH agonist, and an FGF inhibitor."

The basal medium for differentiation induction in step (b) above can be the same as the basal medium and serum or serum substitute exemplified for use in step (a).

The essential additives of the basal medium in step (b) above include a BMP agonist, retinoic acid, an SHH agonist, and an FGF inhibitor.

BMP agonists include, for example, BMP family proteins. Examples include BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, BMP11, BMP12, BMP13, BMP14, and BMP15, with BMP4 being particularly preferred. The concentration of the BMP agonist is preferably 0.01 ng/mL or higher, more preferably 0.1 ng/mL or higher. The concentration of the BMP agonist is preferably 10 ng/mL or lower, more preferably 5 ng/mL or lower, and particularly preferably 2 ng/mL or lower.

The concentration of retinoic acid is preferably 0.1 μM or more, more preferably 1 μM or more, and preferably 10 μM or less, more preferably 5 μM or less.

An SHH agonist is a substance that activates the sonic hedgehog pathway and inhibits Wnt signaling, preferably SHH (sonic hedgehog). The concentration of the SHH agonist is preferably 1 ng/mL or higher, more preferably 10 ng/mL or higher. It is also preferably 300 ng/mL or lower, more preferably 50 ng/mL or lower.

The FGF inhibitor is an inhibitor of fibroblast growth factor (FGF), preferably PD0325901. The concentration of the FGF inhibitor is preferably 0.1 μM or higher, more preferably 0.5 μM or higher, and preferably 10 μM or lower, more preferably 5 μM or lower.

Other additives used in the basal medium in step (b) above can be the same as those exemplified for use in step (a).

In addition, step (b) preferably does not contain a Wnt agonist. Addition of a Wnt agonist in step (b) inhibits the formation of intermediate mesoderm.

In the culture of step (b), the culture conditions for inducing early mesoderm to intermediate mesoderm include, but are not limited to, an atmosphere of 1-10% _CO2 /99-90% air. The culture temperature is about 30-40°C, preferably about 37°C. The culture period is less than 3 days, preferably about 2 days (e.g., 48±12 hours, preferably 48±6 hours).

(c) A step of inducing ovarian somatic tissue by culturing the intermediate mesoderm obtained by step (b) in the presence of a BMP agonist and an FGF agonist. The present invention further comprises "(c) a step of inducing ovarian somatic tissue by culturing the intermediate mesoderm obtained by step (b) in the presence of a BMP agonist and an FGF agonist."

The basal medium for differentiation induction in step (c) above can be the same as the basal medium and serum or serum substitute exemplified for use in step (a).

The additives to the basal medium in step (c) above preferably contain a BMP agonist and an FGF agonist.

BMP agonists include substances that activate BMP or the BMP signaling pathway, such as BMP family proteins. Examples include BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP9, BMP10, BMP11, BMP12, BMP13, BMP14, and BMP15, with BMP4 being particularly preferred. The concentration of the BMP agonist is preferably 0.01 ng/mL or higher, more preferably 1 ng/mL or higher. The concentration of the BMP agonist is preferably 100 ng/mL or lower, more preferably 30 ng/mL or lower.

FGF agonists include substances that activate FGF or the FGF signaling pathway, such as FGF family proteins. Examples include FGF9, bFGF, FGF2, and FGF4, with FGF9 being preferred. The concentration of the FGF agonist is preferably 0.01 ng/mL or higher, more preferably 0.1 ng/mL or higher. The concentration of the FGF agonist is preferably 100 ng/mL or lower, more preferably 10 ng/mL or lower.

Other additives used in the basal medium in step (c) above can be the same as those exemplified for use in step (a).

In the culture of step (c), the culture conditions for inducing intermediate mesoderm into ovarian somatic tissue include, but are not limited to, an atmosphere of 1-10% _CO2 /99-90% air. The culture temperature is about 30-40°C, preferably about 37°C. The culture period is less than 4 days, preferably about 2 days (e.g., 48±12 hours, preferably 48±6 hours).

As used herein, "ovarian somatic tissue" refers to cells derived from pluripotent stem cells that have properties equivalent to those of fetal ovarian somatic cells, and that have the ability to differentiate into granulosa cells, theca cells, etc. that will form follicles in the future. More specifically, ovarian somatic tissue is defined as cells that have the following properties:
(1) Increased gene expression of Nr5a1 compared to epiblast-like cells.
Therefore, induction of differentiation into ovarian somatic tissue can be confirmed, for example, by analyzing the expression levels of ovarian somatic tissue marker genes.

The present invention also provides a reagent kit for inducing differentiation of pluripotent stem cells into ovarian somatic tissue. The kit of the present invention may contain a BMP agonist, a Wnt agonist, a retinoic acid, an SHH agonist, an FGF inhibitor, or a combination thereof. Preferably, the kit contains all of a BMP agonist, a Wnt agonist, a retinoic acid, an SHH agonist, and an FGF inhibitor. The kit may also contain other reagents necessary for culture, equipment such as wells, etc.

(d) A step of culturing the ovarian somatic tissue obtained in step (c) and primordial germ cells under conditions that eliminate the influence of estrogen or factors with estrogen-like functions to form secondary follicles. The present invention further comprises "(d) a step of culturing the ovarian somatic tissue and primordial germ cells under conditions that eliminate the influence of estrogen or factors with estrogen-like functions to form secondary follicles" using the ovarian somatic tissue obtained in step (c).

As used herein, "primordial germ cells" refer to cells that are destined to differentiate into germ cells, and that differentiate into eggs or sperm via oogonia or spermatogonia. Primordial germ cells may be derived from a living organism, or may be primordial germ cell-like cells (PGCLCs) differentiated from pluripotent stem cells. The "primordial germ cells" used in the present invention also include primary oocytes, which have progressed in development from primordial germ cells and have begun meiosis. Primordial germ cells derived from a living organism or primordial germ cell-like cells derived from pluripotent stem cells, also include cells whose genes have been modified using genetic engineering techniques.

Methods for inducing differentiation of primordial germ cells from pluripotent stem cells, particularly iPS cells and ES cells, can be performed with reference to, for example, Non-Patent Document 1, Hayashi K. et al., "Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells," Cell, Aug. 19, 146(4), 519-32 (2011), etc.

In addition, it is preferable to carry out a culture step prior to step (d) to prepare an aggregate consisting of primordial germ cells and the ovarian somatic tissue obtained in step (c). The method for preparing an aggregate consisting of primordial germ cells and the ovarian somatic tissue obtained in step (c) can be carried out with reference to, for example, K. Hayashi, O. Hikabe, Y. Obata, Y. Hirao, "Reconstitution of mouse oogenesis in a dish from pluripotent stem cells." Nat Protoc 12, 1733-1744 (2017)," Patent Document 3, Non-Patent Document 5, etc. For example, this can be achieved by mixing, aggregating, and culturing the primordial germ cells and the ovarian somatic tissue obtained in step (c) in GK15 medium containing retinoic acid and Y27632 (medium prepared by adding 15% KSR, 1x GlutaMax, 1x penicillin/streptomycin (100 U/ml penicillin and 0.1 mg/ml streptomycin), 100 μM 2-mercaptoethanol, 1 μM retinoic acid, and 10 μM Y27632 to GMEM). It is preferable to use a low-adhesion culture dish for culturing. For example, the culture period for producing aggregates can be 1 to 4 days, preferably 2 days.

Furthermore, the ratio of the primordial germ cells to the ovarian somatic cell tissue obtained in step (c) when mixed is not limited as long as the produced aggregates form secondary follicles and functional GV-stage oocytes. However, in the case of mice, for example, the cell number ratio of pluripotent stem cell-derived PGCLCs to gonad-derived somatic cells is preferably 1:5 or more and 1:40 or less, and more preferably 1:15 or more and 1:25 or less.

As used herein, culturing "under conditions that eliminate the effects of estrogen or factors with estrogen-like functions" includes culturing in the presence of an "estrogen inhibitor." The "estrogen inhibitor" used in the present invention is an agent that has the effect of inhibiting the activation of estrogen receptors, and examples thereof include estrogen receptor antagonists, ICI 182,780 ((7R,9S,13S,14S,17S)-7-(9-(4,4,5,5,5-Pentafluoropentylsulfinyl)nonyl)-7,8,9,11,12,13,14,15,16,17-decahydro-13-methyl-6H-cyclopenta[a]phenanthrene-3,17-diol), tamoxifen citrate, 4-hydroxytamoxifen, MPP (4-[1-(4-hydroxyphenyl)-4-methyl-5-[4-[2-(1-piperidinyl)ethoxy]phenyl]-1H-pyrazol-3-yl]-phenol), and PHTPP. (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol), G15 ((3aS,4R,9bR)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline), etc. can be used. These can be used alone or in combination of two or more. Among these, ICI 182,780 is particularly preferred. Furthermore, the estrogen inhibitor is not limited to the above, and any inhibitor that can inhibit estrogen receptor activation and can differentiate primordial germ cells into functional oocytes can be used. It is preferable to add the estrogen inhibitor before oocyte cyst breakdown and/or primordial follicle formation. Furthermore, it is not necessary to culture the aggregates "under conditions that eliminate the influence of estrogen or factors with estrogen-like functions" from the start of production, but it is preferable to culture the aggregates "under conditions that eliminate the influence of estrogen or factors with estrogen-like functions" at least during the period of oocyte cyst breakdown and/or primordial follicle formation.

The concentration of estrogen inhibitors added to the culture medium is preferably in the range of 0.01 to 50 μM, and more preferably in the range of 0.1 to 10 μM.

The basal medium for differentiation induction in step (d) is the same as the basal medium exemplified for use in step (a), to which serum or a serum substitute is added. The concentration of serum (e.g., fetal bovine serum (FBS), human serum, etc.) is 0.1-20%, preferably 2-10%.

Culturing "under conditions that eliminate the effects of estrogen or factors with estrogen-like functions" also includes culturing in serum-free medium. Serum-free medium (SFM) refers to a medium that does not contain any untreated or unpurified serum, and examples include media containing purified blood-derived components or animal tissue-derived components (e.g., growth factors). SFM can be similar to the basal medium exemplified for use in step (a), and may contain any alternative serum. Examples of alternative serum include SPS (Serum Protein Substitute), KSR (KnockOut Serum Replacement), and SSS (serum substitute supplement), which can be used alone or in combination. SPS or KSR is preferred. The alternative serum is preferably added to the medium at a concentration of 5% to 20%, with 10% being more preferred.

The period of culture using serum-free medium is preferably the period during which the breakdown of oocyte cysts and the formation of primordial follicles are completed. During the culture period of primordial germ cells in step (d), culture can be performed using serum-free medium throughout. Culture can also be performed by switching from serum medium to serum-free medium as appropriate. During the period when serum-free medium is not used, it is preferable to culture using a medium in which serum such as FBS has been added to the basal medium exemplified for use in step (a).

Furthermore, other additives used in the basal medium in step (d) can be the same as those exemplified for use in step (a).

In the culture of step (d), the culture conditions for inducing secondary follicles are not limited to the following, but can be, for example, 1-10% _CO2 /99-90% air atmosphere. The culture temperature is approximately 30-40°C, preferably approximately 37°C. The culture period is preferably 11-25 days counting from the start of culture of the previously prepared aggregates. The culture period in step (d) varies depending on the animal species from which the cultured primordial germ cells are derived, but is preferably set to the period required for the primordial germ cells to form secondary follicles in vivo.

(e) A step of partially severing the bonds between the granulosa cell layer and the theca cell layer among the oocytes, granulosa cell layers, and theca cell layers that constitute the formed secondary follicles. The present invention further comprises, for the secondary follicles obtained by step (d), "(e) a step of partially severing the bonds between the granulosa cell layer and theca cell layer among the oocytes, granulosa cell layers, and theca cell layers that constitute the formed secondary follicles." The method of step (e) can be performed with reference to, for example, Patent Document 3.

The secondary follicles obtained by culturing in step (d) have a structure in which a layer of granulosa cells surrounds the oocyte, and a layer of theca cells surrounds the granulosa cell layer. In step (e), the bond between the granulosa cell layer and theca cell layer that form the follicle is partially severed.

Partial severing of the bond between the granulosa cell layer and theca cell layer can be achieved by enzymatic treatment and/or physical treatment. A preferred method is to sever the bond by combining enzymatic treatment and physical treatment.

Enzyme treatment can be performed by dissolving and diluting commercially available collagenase in a known medium. The basal medium for differentiation induction in step (e) is the same as the basal medium exemplified for use in step (a). Examples of collagenase include type I collagenase, type II collagenase, type III collagenase, type IV collagenase, type V collagenase, type VI collagenase, and type VII collagenase, with type I collagenase being preferred. The collagenase concentration is preferably 0.05-0.5%, and more preferably 0.1%. The temperature is preferably in the range of 30-40°C, more preferably 37°C. The treatment time is preferably 2-15 minutes.

Physical treatment can be performed, for example, by pipetting using a glass capillary or a pipetman. Partial severing of the bond between the granulosa cell layer and the thecal cell layer can be performed when all or part of the thecal cell layer has been detached from the follicle.

The treatment to disrupt the bond between the granulosa cell layer and the thecal cell layer is preferably performed between days 0 and 7 after the start of follicle culture, and more preferably between days 2 and 4 of culture. It is preferable to pre-culture the secondary follicles for about 1 to 3 days without performing the treatment to disrupt the bond between the granulosa cell layer and thecal cell layer immediately after isolation, as this allows the follicles to adhere to the insert membrane and become stable. The medium used for such pre-culture can be the same as the medium used in step (f) below. In a preferred embodiment, GDF9 and/or BMP15 can be added to the medium used for pre-culture. Adding these compounds to the medium used for pre-culture of secondary follicles can further promote the proliferation of granulosa cells. For example, GDF9 and BMP15 can be added to αMEM at a ratio of 10 to 20 ng/ml, preferably 15 ng/ml.

(f) A step of differentiating the oocytes into GV stage oocytes by culturing the oocytes, granulosa cell layer, and theca cell layer constituting the secondary follicles in a medium containing a polymer compound. The present invention further comprises "(f) a step of differentiating the oocytes into GV stage oocytes by culturing the oocytes , granulosa cell layer, and theca cell layer constituting the secondary follicles in a medium containing a polymer compound" using the secondary follicles obtained in step (e). The method of step (f) can be performed with reference to, for example, Patent Document 3.

The medium used for the in vitro culture of secondary follicles of the present invention is a basal medium to which a polymer compound has been added. The basal medium for differentiation induction in step (f) is the same as the basal medium exemplified for use in step (a). In addition to the polymer compound, the basal medium may also contain, as appropriate, fetal bovine serum (FBS), follicle-stimulating hormone (FSH), etc.

The polymer compounds used in the present invention can be any of those commonly used in secondary follicle culture. In particular, polymer compounds that satisfy the following criteria are preferred: high water solubility, extremely low cytotoxicity, lack of properties that destabilize the pH of the culture medium during culture, and stable long-term maintenance of initial characteristics. Furthermore, compounds that do not impair oocyte viability when used in culture, prevent the shedding of somatic cells such as granulosa cells and theca cells surrounding the oocyte, and maintain the oocyte-centered structure (no irregular or extensive cell proliferation), and do not affect differentiation into functional oocytes, are preferred. Examples of suitable polymers include synthetic polymers, polysaccharide polymers, proteins, and proteoglycans. Synthetic polymers include polyvinylpyrrolidone (PVP; molecular weight approximately 360,000) and polyvinyl alcohol (PVA; molecular weight approximately 70,000-100,000). Examples of polysaccharide polymers include dextran, hydroxyethylated starch, cellulose derivatives (e.g., hydroxypropylmethylcellulose), a synthetic polymer of sucrose called ficoll (molecular weight 400,000) (Ficoll (registered trademark)), and glycosaminoglycans such as hyaluronic acid and chondroitin sulfate. Examples of proteins include serum albumin (molecular weight approximately 69,000). Examples of proteoglycans include chondroitin sulfate proteoglycan. These can be used alone or in combination of two or more.

Particularly preferred polymer compounds in the present invention include, but are not limited to, PVP, Ficoll (registered trademark), hydroxypropyl methylcellulose, and serum albumin.

The concentration of the polymer compound added can be, for example, within the range of approximately 1-12% (w/v) of the basal medium, preferably 1-8% (w/v), more preferably 1-4% (w/v), and most preferably approximately 2% (w/v).

The culture period for secondary follicles in step (f) is preferably set to the period required for the oocytes in the secondary follicles to form functional GV-stage oocytes, and is preferably 12 to 16 days, for example.

(g) A step of resuming meiosis by in vitro maturation culture of the GV stage oocytes obtained by step (f ) The present invention further comprises "(g) a step of resuming meiosis by in vitro maturation culture of the GV stage oocytes obtained by step (f)" using the GV stage oocytes obtained by step (f). In step (g), the GV stage oocytes can be matured into eggs by carrying out a known culture method that is commonly used for in vitro maturation of immature oocytes.

As used herein, "egg" refers to an egg that has reached metaphase II of meiosis (MII) and is arrested at the MII stage. Furthermore, the eggs obtained in the present invention may be functional eggs. Here, a functional egg refers to an egg that has the ability to develop into a normal individual through fertilization with sperm, etc., and that has the ability for that individual to produce normal offspring.

The process of maturing GV stage oocytes into eggs is usually carried out by collecting oocyte-cumulus cell complexes (COCs) from a culture dish after oocyte development, washing them with a maturation medium, and then transferring them to a final maturation medium. The same basal medium as exemplified for use in step (a) can be used as the maturation medium. Sodium pyruvate, antibiotics, gonadotropins, growth factors, serum, follicular fluid, etc. can be added to the basal medium as appropriate. These can be used alone or in combination of two or more.

The eggs obtained in this way can be used for conventional in vitro fertilization, as well as for producing parthenogenetic embryos and as recipient eggs in the production of cloned animals.

The present invention will be further explained below using examples, but the present invention is not limited to these examples.

<Materials>
ES cells
The ES cells used to induce ovarian somatic tissues (FOSLCs) were established from blastocysts of mice with the following genetic backgrounds:
・T-GFP mESCs (B6D2F1 female and T _{nEGFP-CreERT2/-} male) ・Osr1-GFP mESCs for knock-in of Foxf1-tdTomato (B6D2F1 female and Osr1-GFP male)
・Osr1-GFP mESCs for knock-in of Gata4-CFP (C57Bl/6J female and Osr1-GFP male)
・Nr5a1-hCD271 mESCs for knock-in of Foxl2-tdTomato (129X1/Svj female and Nr5a1-hCD271 male)
(ICR, C57Bl/6J, 129X1/Svj, and B6D2F1 mice were all purchased from Japan SLC, and _{TnEGFP-CreERT2/-} , Osr1-GFP, and Nr5a1-hCD271 mice were derived from C57Bl/6J mice.)

<Induction of pluripotent stem cells into primordial germ cell-like cells (PGCLCs)>
Pluripotent stem cells were induced to primordial germ cell-like cells (PGCLCs) according to the method described in Patent Document 3, Hayashi K. et al., "Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells," Cell, Aug. 19, 146(4), 519-32 (2011).

The ES cells used to induce PGCLCs were an ES cell line established from blastocysts of mice (the Jackson Laboratory) transfected with Blimp1-mVenus and Stella-ECFP (BVSC). Blimp1 is a marker gene for PGC-like cells (PGCLCs), and Stella is a marker gene for PGCLCs and oocytes. The karyotype of the ES cells used in this example was female, with 40 chromosomes (38XX).

ES cells used for PGCLC induction were cultured for 2 days under feeder-free conditions (5% CO2, 95% air, 37°C) in N2B27 medium containing 2i ( _PD0325901 , 0.4 μM: Stemgent, San Diego, CA; CHIR99021, 3 μM: Stemgent) and LIF (1000 μg/ml). ES/iPS cells were then differentiated into epiblast-like cells (EpiLCs) by culturing them for 2 days on culture dishes coated with human plasma-derived fibronectin in N2B27 medium containing activin (20 ng/ml), bFGF (12 ng/ml), and KSR (1%). The resulting EpiLCs were cultured in suspension in serum-free medium (GK15; GMEM (Invitrogen) containing 15% KSR, 0.1 mM NEAA, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine) containing BMP4 (500 ng/ml; R&D Systems), LIF (1000 U/ml; Invitrogen), SCF (100 ng/ml; R&D Systems), and EGF (50 ng/ml; R&D Systems) in low-adhesion 96-well culture dishes (NUNC) for 6 days to induce differentiation into PGCLCs.

(1) Induction of early mesoderm from ES cells
Epiblast-like cells were induced from each type of ES cell line using the same method as described in "Induction of Primordial Germ Cell-Like Cells (PGCLCs) from Pluripotent Stem Cells." The resulting EpiLCs were cultured in serum-free medium (GK7.5; GMEM (Invitrogen) containing 7.5% KSR, 0.1 mM NEAA, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine) containing BMP4 (R&D Systems), CHIR99021 (R&D Systems), and EGF (50 ng/ml; R&D Systems) in low-adhesion 96-well culture dishes (NUNC) and induced to differentiate into early mesoderm. Each EpiLC was cultured at 3 × ¹⁰ cells/well. The BMP4 concentrations tested were 1 ng/ml, 3 ng/ml, and 10 ng/ml, and the CHIR99021 concentrations tested were 3 μM, 8 μM, and 14 μM. The results are shown in Figures 1 to 3. Based on these results, the optimal concentrations of BMP4 and CHIR99021 were determined to be 1 ng/ml and 14 μM, respectively.

(2) Induction of early mesoderm into intermediate mesoderm
After 43-45 hours of culture under the optimal conditions found in (1) (day 2), the medium was changed to serum-free GK7.5 medium containing BMP4 (1 ng/ml; R&D Systems), retinoic acid (3 μM; Sigma), SHH (30 ng/ml; R&D Systems), PD0325901 (1 μM; Stemgent), and EGF (50 ng/ml; R&D Systems) for differentiation into intermediate mesoderm. Retinoic acid concentrations were tested at 0 μM, 0.3 μM, and 3 μM. SHH concentrations were tested at 0 ng/ml and 30 ng/ml. PD0325901 concentrations were tested at 0 μM and 1 μM. The results are shown in Figures 4 and 5. From these results, the optimal concentrations of retinoic acid, SHH, and PD0325901 were determined to be 3 μM, 30 ng/ml, and 1 μM, respectively.

(3) Induction of ovarian somatic cells (FOSLCs) from intermediate mesoderm
After 47-49 hours under the optimal conditions in (1) and (2) (4 days after the start of culture), the medium was changed to serum-free GK7.5 medium containing BMP4 (20 ng/ml; R&D Systems) and FGF9 (2 ng/ml; Peprotech), and the cells were cultured to induce differentiation into FOSLCs.

FACS analysis was then performed using anti-hCD271 antibodies to select Nr5A1-hCD271-positive FOSLCs. As shown in Figure 6, these cells resemble E12.5 (gonad somatic cells of 12.5-day-old ICR mice).

(4) Aggregation culture of ovarian somatic cells (FOSLCs) and primordial germ cell-like cells (PGCLCs)
Following the method described in Hayashi K. et al., "Reconstitution of mouse oogenesis in a dish from pluripotent stem cells," Nat Protoc 12, 1733-1744 (2017), FOSLCs were cultured with PGCLCs on day 5 of culture. PGCLCs and FOSLCs were cultured for 2 days in GK15 medium (GMEM supplemented with 15% KSR, 0.1 mM NEAA, 1 nM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine) containing retinoic acid (1 μM) and Y27632 (10 μM). The PGCLCs and FOSLCs were cultured at 5 × 10 ³ cells/well and 7.5 × 10 ⁴ cells/well and 10 × 10 ⁴ cells/well, respectively. As a control, 7.5×10 ⁴ gonadal somatic cells/well from 12.5-day-old ICR mice were used and cultured with PGCLCs under the same conditions.

<(5) Creation of secondary follicles>
After (4), the cells were cultured for 3 days in IVD-αMEM medium (αMEM supplemented with 2% fetal calf serum (FCS), 150 μM ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 55 μM 2-mercaptoethanol) containing BMP2 (150 ng/ml; Peprotech) and retinoic acid (100 nM). On day 4, the medium was changed to IVD-SP medium (StemPro-34 SFM (Life Technologies) supplemented with 10% FCS, 150 μM ascorbic acid, 2 mM GlutaMax, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 55 μM 2-mercaptoethanol) containing BMP2 (150 ng/ml; Peprotech) and retinoic acid (100 nM). On the fifth day of culture, the cells were cultured in IVD-SP medium without BMP2 or retinoic acid. From days 7 to 10, IVD-SP medium supplemented with ICI182780 (500 nM) was used. Culture to form secondary follicles was performed on Transwell-Col for a total of 23 days. The progress is shown in Figures 7 and 8. Figure 7 shows that self-organization progresses with the passage of culture days. On day 21 of culture, strong expression of the oocyte marker Stella was confirmed.

(6) Secondary follicle culture (IVG)
We attempted to produce oocyte-cumulus cell complexes (COCs) by in vitro culture of the secondary follicles obtained in (5) as described below.

Secondary follicles obtained during culture were physically isolated using a tungsten plate on a Transwell-Col. Isolated secondary follicles were cultured for two days on a Transwell-Col in IVG-αMEM medium (αMEM containing 5% FCS, 2% polyvinylpyrrolidone (Sigma), 150 μM ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 100 μM 2-mercaptoethanol, 55 μg/ml sodium pyruvate, and 0.1 IU/ml FSH) supplemented with GDF9 and BMP15 (both at 15 ng/ml). On the second day of culture, the IVG-αMEM medium was changed to one without GDF9 and BMP15, and culture was continued. On the third day of culture, 1.5 mL of IVG-αMEM medium was added to cover the follicles. On day 4 of culture, the secondary follicles were treated with 0.1% type I collagenase at room temperature for 5 minutes to partially separate the bonds between the granulosa cell layer and theca cell layer. They were then cultured on a Transwell-Col using the above-mentioned IVG-αMEM medium (without GDF9 or BMP15). As a result, COCs could be isolated from developed secondary follicles on day 12 of culture using a glass capillary. The culture progress is shown in Figure 9.

(7) In vitro oocyte maturation (IVM)
The COCs obtained in (6) were transferred to IVM medium (αMEM supplemented with 5% FCS, 25 μg/ml sodium pyruvate, 1x penicillin/streptomycin, 0.1 IU/ml FSH, 4 ng/ml EGF, and 1.2 IU/ml hCG) and cultured for 16 hours (Figure 10). After that, cumulus cells were dissociated from the oocytes with hyaluronidase, and those that had released the first polar body were used as MII oocytes for in vitro fertilization.

(8) In vitro fertilization of externally matured eggs and embryo transfer
MII oocytes obtained in (7) were co-cultured with sperm in HTF medium (ARK Resources). After approximately 6 hours, the fertilized oocytes were transferred to fresh HTF medium and cultured for 16 hours. After culture, embryos that reached the 2-cell stage were transferred to the oviducts of ICR mice on day 0.5 of pseudopregnancy.

Nineteen days after transplantation, newborn pups were obtained by Caesarean section (Figure 11). The resulting newborns were nursed by their foster mothers and allowed to grow to adulthood. As a result, the resulting newborn pups developed into apparently normal adults (Figure 12). Furthermore, after maturation, the adult mice successfully gave birth to the next generation, both male and female (Figure 13). Thus, it was confirmed that functional GV-stage oocytes and eggs can be obtained by the ex vivo culture method of the present invention, even when using FOSLCs derived from pluripotent stem cells.

Claims (12)

A method for producing ovarian somatic tissue in vitro from pluripotent stem cells, comprising:
(a) culturing epiblast-like cells differentiated from pluripotent stem cells in the presence of a BMP agonist and a Wnt agonist to induce early mesoderm;
(b) culturing the early mesoderm in the presence of a BMP agonist, retinoic acid, an SHH agonist, and an FGF inhibitor to induce intermediate mesoderm;
A method comprising: The method of claim 1, further comprising the step of (c) culturing the intermediate mesoderm in the presence of a BMP agonist and an FGF agonist to induce ovarian somatic tissue. The method of claim 1 or 2, wherein the BMP agonist is BMP4 and the Wnt agonist is CHIR99021. The method according to any one of claims 1 to 3, wherein the BMP agonist is BMP4, the SHH agonist is SHH, and the FGF inhibitor is PD0325901. The method of any one of claims 2 to 4, wherein the FGF agonist is FGF9. A method for producing GV stage oocytes, comprising:
(d) culturing the ovarian somatic tissue obtained by the method according to any one of claims 1 to 5 and primordial germ cells under conditions that exclude the influence of estrogen or a factor having a function similar to estrogen, to form secondary follicles containing oocytes, a granulosa cell layer, and a theca cell layer;
(e) partially cleaving the bonds between the granulosa cell layer and the theca cell layer in the secondary follicles formed in step (d);
(f) differentiating the oocytes into GV stage oocytes by culturing the oocytes, granulosa cell layer, and theca cell layer that constitute the secondary follicles in a medium containing a polymer compound. The method according to claim 6, wherein the polymer compound is at least one compound selected from the group consisting of polyvinylpyrrolidone, ficoll, hydroxypropyl methylcellulose, and serum albumin. A method for producing eggs, comprising:
(g) A method comprising a step of resuming meiosis by in vitro maturation culture of GV stage oocytes obtained by the method of claim 6 or 7. Ovarian somatic tissue obtained by the method of any one of claims 1 to 5. GV stage oocytes obtained by the method of claim 6 or 7. Eggs obtained by the method of claim 8. A kit for differentiating pluripotent stem cells into ovarian somatic tissue in vitro, the kit comprising a BMP agonist, a Wnt agonist, retinoic acid, an SHH agonist, and an FGF inhibitor .