Ovarian-somatic-cell-like cells and method for producing ovarian-somatic-cell-like cells

Abstract

[Problem] To provide ovarian-somatic-cell-like cells and an efficient method for producing the same. [Solution] A method for producing ovarian-somatic-cell-like cells that includes a step for introducing a factor or a nucleic acid encoding the factor into cells, said factor including NR1H4 and / or WT1.

Description

Ovarian somatic-like cells and method for producing ovarian somatic-like cells

[0001] The present invention relates to ovarian somatic-like cells and a method for producing ovarian somatic-like cells.

[0002] In order to obtain matured eggs in vitro, follicles covering the eggs are required. Therefore, a method for producing cells constituting follicles without collecting them from a living body has been demanded. For example, a method for producing ovarian somatic tissue from pluripotent stem cells, which includes a step of culturing in the presence of a BMP agonist, retinoic acid, an SHH agonist, and an FGF inhibitor (see Patent Document 1), and a method for producing granulosa cells, which includes a step of culturing intermediate mesoderm cells induced from pluripotent stem cells in the presence of follistatin, BMP4, FGF2, and a ROCK inhibitor (see Patent Document 2), etc. are known.

[0003] In addition, a method for inducing cells having the properties of ovarian somatic cells by expressing specific factors in cells such as pluripotent stem cells has been developed. For example, a method for inducing pluripotent stem cells into granulosa cell-like cells by highly expressing transcription factors such as NR5A1 and RUNX family proteins (see Patent Document 3) is known. Furthermore, transcription factors such as NR5A1, RUNX2, TCF21, GATA4, MAFF, etc. have been identified as factors for inducing granulosa cell-like cells of transcription factors (see Non-Patent Document 1), and ovarian support cells (OSC) obtained by overexpressing NR5A1, RUNX2, and GATA4 in induced pluripotent stem cells, and fetal-like ovarian somatic cells (FOSC) obtained by overexpressing NR5A1, RUNX1, GATA4, and FOXL2 have been disclosed (see Non-Patent Document 2).

[0004] International Publication No. 2022 / 163670 International Publication No. 2022 / 094628 International Publication No. 2023 / 192934

[0005] Piechota S. et al. Directed differentiation of human iPSCs to functional ovarian granulosa-like cells via transcription factor overexpression. 2023. https: / / doi.org / 10.7554 / eLife.83291 Piechota S. et al. Human-induced pluripotent stem cell-derived ovarian support cell co-culture improves oocyte maturation in vitro after abbreviated gonadotropin stimulation. 2023. https: / / doi.org / 10.1093 / humrep / dead205

[0006] Methods disclosed in the prior art have the problem that they do not always yield cells with the desired properties. Therefore, there has been a need for a method to obtain cells with the desired properties with high efficiency. The problem that this invention aims to solve is to provide ovarian somatic cell-like cells and an efficient method for producing them.

[0007] The present invention is a method for producing ovarian somatic cell-like cells comprising the step of introducing a factor or a nucleic acid encoding the factor into cells, wherein the factor comprises NR1H4 and / or WT1.

[0008] The aforementioned cells are cells that substantially do not express FOXL2, while the aforementioned ovarian somatic cell-like cells may be FOXL2-expressing cells. Ovarian somatic cell-like cells may include those that express pregranulosa cell marker genes and / or granulosa cell marker genes, but substantially do not express the theca cell marker genes.

[0009] The aforementioned factor may further include one or more selected from the group consisting of TCF21, SIX1, NR2F2, ETV5, and TOX3. Another combination of factors may include at least NR1H4, WT1, and TCF21, and further include NR2F2 and / or SIX1.

[0010] The method may be one in which the above steps are performed two or more times.

[0011] In the method described above, the cells may be pluripotent stem cells or human pluripotent stem cells.

[0012] Another aspect of the present invention is a method for producing ovarian somatic cell-like cells, comprising the step of expressing a factor in cells, wherein the factor comprises NR1H4 and / or WT1. The factor may further comprise one or more selected from the group consisting of TCF21, SIX1, NR2F2, ETV5, and TOX3. In another combination of factors, the factor may comprise at least NR1H4, WT1, and TCF21, and further comprise NR2F2 and / or SIX1.

[0013] The aforementioned cells are cells that substantially do not express FOXL2, while the aforementioned ovarian somatic cell-like cells may be FOXL2-expressing cells. Ovarian somatic cell-like cells may include those that express pregranulosa cell marker genes and / or granulosa cell marker genes, but substantially do not express the theca cell marker genes.

[0014] In the method described above, the cells may be pluripotent stem cells or human pluripotent stem cells.

[0015] Another aspect of the present invention is ovarian somatic cell-like cells obtained by introducing a factor or nucleic acid encoding the factor into pluripotent stem cells, wherein the factor comprises NR1H4 and / or WT1, the pluripotent stem cells substantially do not express FOXL2, and the ovarian somatic cell-like cells express FOXL2. The factor may further comprise one or more selected from the group consisting of TCF21, SIX1, NR2F2, ETV5, and TOX3. In another combination of factors, the factor may comprise at least NR1H4, WT1, and TCF21, and further comprise NR2F2 and / or SIX1.

[0016] According to the present invention, by introducing previously unknown factors or combinations thereof into cells such as pluripotent stem cells, it is possible to induce cells to become ovarian somatic cell-like cells. The ovarian somatic cell-like cells obtained by the present invention express multiple ovarian somatic cell marker genes, such as FOXL2, and exhibit cell morphology specific to ovarian somatic cells, thus possessing properties similar to ovarian somatic cells.

[0017] This figure shows the percentage of FOXL2-positive cells obtained in the examples. The vertical axis shows the introduced factor, and the horizontal axis shows the percentage of FOXL2-positive cells. This is the flow cytometry analysis result showing the percentage of FOXL2-positive cells obtained in the examples and comparative examples. The horizontal axis shows the fluorescence intensity corresponding to FOXL2 expression, and cells present in Q3 are FOXL2-positive cells. Pluripotent stem cells (iPSCs) are cells before the introduction of the factor and are negative controls. These are phase-contrast / fluorescence microscope images of cells obtained in the examples. "FOXL2" is a fluorescence image showing FOXL2 expression, and "Brightfield + FOXL2" is an image of the fluorescence image and brightfield image superimposed. The scale bar in the figure represents 125 μm. The arrows in the figure indicate FOXL2-positive cells with a spherical morphology. This is the flow cytometry analysis result showing the expression of ovarian somatic cell marker genes in cells obtained in the examples. In each figure, cells present in Q2 are cells that are positive for both FOXL2 and LGR5, AMHR2, or PDGFRa.

[0018] The present invention will be described in detail based on embodiments, but the present invention is not limited to these embodiments.

[0019] In this specification, ovarian somatic cell-like cells refer to cells that have the properties of ovarian somatic cells. Ovarian somatic cells or ovarian somatic cell-like cells refer to cells that have the ability to differentiate into cells that constitute follicles. Specific examples of cells that constitute follicles include pregranulosa cells, granulosa cells, cumulus cells, theca cells, etc. The ovarian somatic cell-like cells obtained in this invention, or cells obtained by differentiating ovarian somatic cell-like cells, constitute follicular structures with oocytes and other cells, and have the function of developing oocytes into eggs.

[0020] One embodiment of ovarian somatic cell-like cells as defined herein expresses at least FOXL2 (Forkhead box protein L2). Furthermore, in addition to FOXL2, ovarian somatic cell marker genes such as LGR5 (Leucine-rich repeat-containing G-protein coupled receptor 5), AMHR2 (anti-Mullerian hormone receptor type 2), or PDGFRa (Platelet-derived growth factor receptor alpha) may also be expressed. The ovarian somatic cell-like cells of the present invention are obtained by introducing a specific factor into cells, and are cells that express genes different from the introduced factor, the genes different from the introduced gene may be ovarian somatic cell marker genes, and more specifically, the expressed genes may be one or more selected from FOXL2, LGR5, AMHR2, and PDGFRa.

[0021] One embodiment of the ovarian somatic cell-like cells of the present invention exhibits diverse morphologies, such as spherical, spindle-shaped, and radially elongated shapes. The diameter of the ovarian somatic cell-like cells can range from approximately 5 micrometers (μm) to 30 μm, and can be approximately 10 μm to approximately 20 μm.

[0022] One embodiment of the present invention provides a cell population containing at least the ovarian somatic cell-like cells described above. That is, this embodiment may be a method for producing a cell population containing at least ovarian somatic cell-like cells from a cell population containing pluripotent stem cells, and a cell population containing at least ovarian somatic cell-like cells derived from a cell population containing pluripotent stem cells.

[0023] In this embodiment, the cell population includes at least ovarian somatic cell-like cells. Specifically, the cell population may contain 0.1% or more, 0.5% or more, 1% or more, 1.5% or more, 2% or more, or 2.5% or more ovarian somatic cell-like cells. In another embodiment, the cell population may contain ovarian somatic cell-like cells in high proportions such as 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more. The ovarian somatic cell-like cells here may be FOXL2-expressing cells (FOXL2-positive cells).

[0024] The cells used in this invention substantially do not express genes that are characteristically highly expressed in ovarian somatic cells, such as FOXL2. Here, "substantially not expressed" means that in the cells, the amount of their mRNA and / or protein is below the detection limit, or even if detected, these genes are expressed to such an extent that they cannot exert their effects. Gene expression can be confirmed by known methods such as quantitative PCR, Western blotting, and analysis using fluorescent proteins as reporters.

[0025] The cells used in this invention may be pluripotent stem cells. Pluripotent stem cells are cells that have the ability to differentiate into various cells and the ability to self-replicate. Specific examples of pluripotent stem cells include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic germ cells (EG cells), spermatogonial stem cells (GS cells), and bone marrow stem cell-derived Muse cells.

[0026] In the present invention, pluripotent stem cells derived from mammals such as humans, mice, rats, cattle, pigs, horses, sheep, rabbits, dogs, and cats, as well as birds, reptiles, amphibians, and fish, are used. The pluripotent stem cells may be human-derived pluripotent stem cells. One embodiment of the present invention is ovarian somatic cell-like cells derived from human pluripotent stem cells, and a method for producing ovarian somatic cell-like cells using human pluripotent stem cells.

[0027] Each step in the manufacturing method of the present invention may be performed in vitro. Each step in the manufacturing method of the present invention may not be performed in vivo on humans.

[0028] The cell population used in the present invention does not contain, or contains very few, cells that express genes that are characteristically highly expressed in ovarian somatic cells, such as FOXL2. In one embodiment of the present invention, by introducing a specific factor into the cells included in the cell population, a cell population containing cells that express genes that are characteristically highly expressed in ovarian somatic cells, such as FOXL2, can be obtained.

[0029] The present invention provides a method for producing ovarian somatic cell-like cells, comprising the step of introducing a factor or a nucleic acid encoding such factor into cells. The factor may be a protein such as a transcription factor. That is, one embodiment of the present invention may be a method for producing ovarian somatic cell-like cells, comprising the step of introducing a protein such as a transcription factor, or a nucleic acid encoding such protein.

[0030] One embodiment of the present invention is a method for producing ovarian somatic cell-like cells, comprising the steps of 1) providing pluripotent stem cells and 2) introducing a factor or a nucleic acid encoding the factor into the pluripotent stem cells.

[0031] Another embodiment of the present invention is a method for producing ovarian somatic cell-like cells, comprising the steps of: 1) providing pluripotent stem cells; 2) inducing the pluripotent stem cells into mesodermal cells; and 3) introducing a factor or a nucleic acid encoding the factor into the pluripotent stem cells that have been induced into mesodermal cells.

[0032] It has been confirmed that introducing the factors discovered in this invention into pluripotent stem cells yields ovarian somatic cell-like cells without the need to induce the pluripotent stem cells into mesodermal cells. On the other hand, in another embodiment, including the step of inducing pluripotent stem cells into mesodermal cells can increase the efficiency of ovarian somatic cell-like cell production.

[0033] One example of a step in inducing pluripotent stem cells into mesodermal cells is culturing the pluripotent stem cells in a mesodermal differentiation induction medium. A mesodermal differentiation induction medium is a medium capable of inducing the differentiation of pluripotent stem cells, which are capable of mesodermal differentiation, into mesodermal cells. More specifically, a mesodermal differentiation induction medium is a medium containing CHIR99021, BMP4, VEGF, FGF, activin A, etc., but is not limited to these.

[0034] One embodiment of the present invention is a method for producing ovarian somatic cell-like cells, comprising the step of introducing one or more factors selected from NR1H4, WT1, TCF21, SIX1, NR2F2, ETV5, and TOX3, or nucleic acids encoding these factors, into cells.

[0035] Another embodiment of the present invention is a method for producing ovarian somatic cell-like cells, comprising the step of introducing a factor or a nucleic acid encoding a factor into cells, wherein the factor comprises at least NR1H4, WT1, and TCF21. In yet another embodiment, the factor may selectively comprise SIX1, NR2F2, ETV5, and TOX3.

[0036] NR1H4 (Nuclear Receptor subfamily 1, group H, member 4) is a nuclear receptor involved in bile acid metabolism and is also called the Farnesoid X receptor (FXR). Human NR1H4 is disclosed as Q96RI1 in UniprotKB, and mouse NR1H4 is disclosed as Q60641 in UniprotKB.

[0037] WT1 (Wilms tumor 1), also known as Wilms tumor protein or WT33, is a transcription factor with a zinc finger motif and is known to be involved in cell proliferation and differentiation. Human WT1 is disclosed as P19544 in UniprotKB, and mouse WT1 is disclosed as P22561 in UniprotKB. Isoforms such as the +KTS isoform and -KTS isoform may also be included in WT1.

[0038] TCF21 is an abbreviation for Transcription factor 21, and is a transcription factor that has a basic helix-loop-helix (bHLH) motif. Human TCF21 is disclosed as O43680 in UniprotKB, and mouse TCF21 is disclosed as O35437 in UniprotKB.

[0039] SIX1 is an abbreviation for Sine oculis homeobox homolog 1 and is a transcription factor belonging to the homeobox gene. Human SIX1 is disclosed as Q15475 in UniprotKB, and mouse SIX1 is disclosed as Q62231 in UniprotKB.

[0040] NR2F2 is an abbreviation for Nuclear receptor subfamily 2 group F member 6, and is a nuclear receptor belonging to the NR2F subfamily. Human NR2F2 is disclosed as P10588 in UniprotKB, and mouse NR2F2 is disclosed as P43136 in UniprotKB.

[0041] ETV5 is an abbreviation for ETS translocation variant 5 and is a transcription factor also known as Ets-Related Molecule (ERM). Human ETV5 is disclosed as P41161 in UniprotKB, and mouse ETV5 is disclosed as Q9CXC9 in UniprotKB.

[0042] TOX3 is an abbreviation for TOX high mobility group box family member 3, and is a transcription factor belonging to the high mobility group box (HMG) protein superfamily. Human TOX3 is disclosed as O15405 in UniprotKB, and mouse TOX3 is disclosed as Q80W03 in UniprotKB.

[0043] In one embodiment, a nucleic acid encoding a factor is introduced into a cell. The nucleic acid introduced into the cell contains an open reading frame encoding the above-described protein. Further, the nucleic acid encoding the factor is introduced alone or in combination with the nucleic acid and a promoter. The promoter is not limited as long as it promotes transcription of the nucleic acid encoding the factor in the cell into which it is introduced. The cell into which the nucleic acid has been introduced expresses or overexpresses the factor encoded by the nucleic acid.

[0044] In one embodiment, the nucleic acid is introduced into the cell using a vector designed to express or overexpress a protein in the cell. A viral vector and / or a non-viral vector is used as the vector. When introducing a plurality of factors, the plurality of factors may be incorporated into one vector, or one or more genes may be incorporated into a plurality of vectors.

[0045] Specific examples of the viral vector include a retroviral vector, a lentiviral vector, a Sendai virus vector, an adenoviral vector, an adeno-associated virus vector, a herpes virus vector, an Epstein-Barr virus vector, a vaccinia virus vector, a poxvirus vector, a poliovirus vector, a silbis virus vector, a rhabdovirus vector, a paramyxovirus vector, an orthomyxovirus vector, and the like.

[0046] The non-viral vector is a plasmid vector, an artificial chromosome, or the like, and specific examples include a plasmid vector using a transposon system and an episomal vector.

[0047] In one embodiment, the factor is introduced into the cell by a method such as electroporation, microinjection, lipofection, or the like. When the factor is a protein, it can be introduced into the cell by a method of fusing with a protein transduction domain or a cell membrane-permeable peptide.

[0048] In this embodiment, the step of introducing the factor only needs to be performed at least once. In another embodiment, the step of introducing the factor may be performed multiple times. Introducing the factor multiple times may result in obtaining a larger number of target cells. In this embodiment, the number of times the factor is introduced may be two, three, four, or five or more times.

[0049] Another embodiment of the present invention includes the steps of introducing a nucleic acid encoding a factor into a cell and expressing the factor in the cell. This step may be the step of expressing the factor in the cell by introducing the nucleic acid, etc., into the cell by the introduction step described above. The factor may be one or more selected from NR1H4, WT1, TCF21, SIX1, NR2F2, ETV5, and TOX3. The factor may include at least NR1H4, WT1, and TCF21, and selectively include SIX1, NR2F2, ETV5, and TOX3.

[0050] In this embodiment, the step of expressing the factor in cells only needs to be performed at least once. In another embodiment, the step of expressing the factor in cells may be performed multiple times. Expressing the factor multiple times may result in obtaining a larger number of target cells. In this embodiment, the number of times the step of expressing the factor can be two, three, four, or five or more times.

[0051] In the above embodiment, nucleic acids can be incorporated into an expression vector containing the nucleic acid and a promoter and introduced into cells. The promoter used here is not limited, but examples include those that are active in the introduced cells and those whose activity is induced by the presence of a specific substance. Examples of promoters that are active in the introduced cells include the CAG promoter, CMV promoter, EF1-α promoter, UbC promoter, and PGK promoter. Examples of promoters whose activity is induced by the presence of a specific substance include the tetracycline-inducible promoter and the estrogen-inducible promoter.

[0052] The step of expressing the factor within cells may be the step of incubating cells into which the expression vector described above has been introduced. Furthermore, if an expression vector incorporating a promoter whose activity is induced by the presence of a specific substance is introduced, the step of expressing the factor within cells may be the step of incubating cells into which the expression vector has been introduced in the presence of that specific substance.

[0053] The factors of the present invention may include not only the proteins described above, but also derivatives and / or modified forms of the factors, factors having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology to the factors and performing substantially the same function as the factors, and one or more factors selected from factors in which one or more amino acids are substituted, deleted, inserted, and / or added and which perform substantially the same function as the factors. Furthermore, the nucleic acids that can be introduced into cells in the present invention may include one or more nucleic acids, DNA, and RNA that encode the factors described above.

[0054] Furthermore, another embodiment of the present invention may include a step of selecting ovarian somatic cell-like cells. In the cell selection step, cells into which the factor has been introduced are selected from the other cells in the cell population. The cell population obtained by selecting cells into which the factor has been introduced has a higher proportion of the target cells compared to the cell population before selection.

[0055] In one embodiment, even if the proportion of ovarian somatic cell-like cells is low in the cell population into which the factor has been introduced, the selection step can yield a cell population with a higher proportion of the desired cells. Therefore, it is not essential that the cell population obtained by introducing the factor has a high proportion of ovarian somatic cell-like cells before selection; a proportion of, for example, 10% or less, 1% or less, or 0.1% or less may be acceptable.

[0056] The cell selection step may involve separating the target cells from other cells. The cell selection step may also involve selecting cells that express ovarian somatic cell marker genes. For example, by introducing nucleic acids encoding a fusion protein that combines an ovarian somatic cell marker gene with a fluorescent protein reporter, cells induced to resemble ovarian somatic cells are separated from other cells using fluorescence-activated cell sorting (FACS).

[0057] In another embodiment, the cell selection step may involve selecting target cells based on drug resistance genes to drugs to which the cells are sensitive. For example, by introducing an expression vector containing a factor and drug resistance genes into cells and incubating the resulting cell population in a medium containing the drug, a cell population with a high proportion of the target cells can be obtained.

[0058] Furthermore, the present invention provides pregranulosa cell-like cells, granulosa cell-like cells, cumulus cell-like cells, and / or follicles composed of these cells, obtained by inducing the ovarian somatic cell-like cells described above. This embodiment includes the step of culturing the ovarian somatic cell-like cells. The culturing step may be any step of culturing the ovarian somatic cell-like cells until they acquire properties similar to pregranulosa cells, granulosa cells, and / or cumulus cells.

[0059] One embodiment of ovarian somatic cell-like cells in the present invention is an ovarian somatic cell-like cell that has acquired properties similar to granulosa cell-like cells and / or granulosa cell-like cells. These cells express at least one of the pre-granulosa cell marker genes and / or granulosa cell marker genes, and substantially do not express the theca cell marker genes.

[0060] Here, pregranulosa cell marker genes specifically include FOXL2, WNT6, IRX3, LHX2, POU6F2, CYP19A1, ZFPM2, and EMX2. Granulosa cell marker genes specifically include MAGED2, AMH, LIMS2, LAMA1, ST6GAL2, and MFAP2. Theca cell marker genes specifically include CYP11A1, LHCGR, STAR, HSD3B2, HSD17B1, and ANPEP.

[0061] The expression level of marker genes can be expressed as a relative expression level. Ovarian somatic cell-like cells of one embodiment of the present invention exhibit relatively high expression levels of pregranulosa cell marker genes and / or granulosa cell marker genes, and relatively low expression levels of theca cell marker genes, compared to cells obtained by introducing other factors.

[0062] Furthermore, the present invention includes a method for co-culturing the aforementioned ovarian somatic cell-like cells, or pregranulosa cell-like cells or granulosa cell-like cells induced from said cells, with oocytes, and oocytes obtained by co-culturing.

[0063] The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0064] 1. We attempted to produce ovarian somatic cell-like cells by introducing factors into human iPS cells (802-3G strain, ReproCELL, Inc.) and using FOXL2 gene expression as an indicator. We lipofection human iPS cells with a CRISPR-Cas9 vector that cleaves near the stop codon of the gene encoding FOXL2, and a donor vector containing a homology arm with a nucleotide sequence adjacent to the gene region, and containing the genes for FOXL2, the red fluorescent protein tdTomato, and drug resistance between the 5' and 3' arms. This resulted in the creation of iPS cells in which the gene encoding tdTomato was inserted downstream of the gene encoding FOXL2.

[0065] Furthermore, a vector was constructed to overexpress one transcription factor selected from TCF21, WT1-KTS splicing variant (hereinafter referred to as WT1-KTS), NR1H4, and other transcription factors in iPS cells. Specifically, a CAG promoter and destabilization domain (DD) were introduced into the PiggyBAC vector to obtain the PB-CAG-DD vector. Next, the nucleic acids encoding the aforementioned transcription factors were introduced into the PB-CAG-DD vector using the In-Fusion HD cloning kit (Takara Bio Inc.).

[0066] iPS cells were placed in a 24-well plate containing 500 μL of culture medium (Dulbeccoo's Modified Eagle Medium containing 10 wt% fetal bovine serum (FBS) and 10 μM CHIR99021), with a total volume of 4.0 × 10⁶ cells. 4 Cells were seeded and cultured for 2 days. Lipofection solutions containing 500 ng of vector, i.e., 50 μL of Opti-MEM (Thermo Fisher Scientific) and 2 μL of Lipofectamine Stem Transfection Reagent (Thermo Fisher Scientific), were added to each well and cultured for 2 days. On the second day of culture, the medium was changed to Stemfit® AK02N containing penicillin / streptomycin and Shield1 (Takara Bio Inc.) to induce expression, and the cells were cultured on a larger scale. On the eighth day of culture, the number of cells expressing FOXL2 (hereinafter referred to as "FOXL2-positive cells") was measured using an Attune NxT flow cytometer (Thermo Fisher Scientific). The expression level of cells without the factor introduced (negative control) was set as the threshold.

[0067] The results are shown in Figure 1. When one factor selected from TCF21, WT1-KTS, NR1H4, NR5A1, TOX3, ETV5, RUNX1, RUNX2, SIX1, TGIF, SALL1, IFI27, and SC22D3 was introduced into iPS cells, FOXL2-positive cells were obtained. In particular, TCF21, WT1-KTS, and NR1H4 were confirmed to produce FOXL2-positive cells at extremely high levels compared to other transcription factors.

[0068] 2. Multiple transcription factors were introduced into human iPS cells, and the production of ovarian somatic cell-like cells was attempted using FOXL2 gene expression as an indicator. In Comparative Example A and Examples A to C, the following combinations of transcription factors were used: Comparative Example A: NR5A1, GATA4, and RUNX2 (combination disclosed in Non-Patent Literature 2), Example A: NR1H4, WT1-KTS, and TCF21, Example B: NR1H4, WT1-KTS, TCF21, and NR2F2, Example C: NR1H4, WT1-KTS, TCF21, NR2F2, and SIX1. A vector was constructed using the method described in "1", and the above combinations of transcription factors were introduced into human iPS cells, and the number of cells expressing FOXL2 was measured.

[0069] The results are shown in Figure 2. In Comparative Example A, 22.4% of FOXL2-positive cells were obtained, while in Examples A to C, 76.7% to 83.2% of FOXL2-positive cells were obtained, indicating that these examples showed significantly higher induction efficiency compared to the comparative example. Furthermore, when the morphology of the cells obtained in Examples A and B was observed using a phase-contrast / fluorescence microscope, spherical cells expressing FOXL2 were found, as shown by the arrows in Figure 3.

[0070] Using the method described above, NR1H4, WT1-KTS, TCF21, NR2F2, and SIX1 were introduced into iPS cells in Example C, NR5A1, GATA4, and RUNX2 (combination disclosed in Non-Patent Literature 2) in Comparative Example A, and NR5A1, GATA4, RUNX1, and FOXL2 (combination disclosed in Non-Patent Literature 2) in Comparative Example B. The iPS cells before introduction, and the cells obtained in Comparative Example A, Comparative Example B, and Example C were cultured for a further 10 days, and RNA-seq analysis was performed. The relative expression levels of marker genes in pregranulosa cells, granulosa cells, and theca cells are shown below. The cells obtained in this example showed higher expression levels of marker genes in pregranulosa cells and granulosa cells, and lower expression levels (substantially absent) of marker genes in theca cells compared to the cells obtained in the comparative examples.

[0071]

[0072]

[0073]

[0074] 3. We attempted to produce ovarian somatic cell-like cells by introducing multiple transcription factors into human iPS cells and using FOXL2 gene expression as an indicator. In Examples D to E, the following combinations of transcription factors were used: Example D: NR1H4, WT1-KTS, TCF21, SIX1, and ETV5; Example E: NR1H4, WT1-KTS, TCF21, and SIX1. A vector was constructed using the method described in "1", introduced into iPS cells, and the number of cells expressing FOXL2, as well as LGR5, AMHR2, and PDGFRa, which are gene markers for ovarian somatic cells other than FOXL2, was measured.

[0075] The results are shown in Figure 4. In the figure, the number labeled Q2 indicates the percentage of cells expressing both FOXL2 and other ovarian somatic cell markers. In this example, it was confirmed that the cells obtained expressed not only FOXL2 but also other ovarian somatic cell gene markers as proteins.

[0076] 4. Place human iPS cells into a 24-well plate containing 500 μL of culture medium (Stemfit® AK02N, Ajinomoto Healthy Supply Co., Ltd.) at a rate of 4.0 × 10⁶ ４ Cells were seeded and cultured for 2 days in Dulbecco's modified Eagle medium containing 10% by weight fetal bovine serum (FBS) and free of CHIR99021. Otherwise, the following combinations of transcription factors were introduced under the conditions described in "1". Example F: NR1H4, TCF21, and SIX1. Example G: WT1-KTS, TCF21, and SIX1. Example H: WT1-KTS, NR1H4, and SIX1. Example I: WT1-KTS, NR1H4, and TCF21. Example J: WT1-KTS, NR1H4, TCF21, and SIX1. In all of Examples F to J, FOXL2-positive cells were confirmed, although the efficiency was lower than in Examples D to E.

[0077] The method for producing ovarian somatic cell-like cells of the present invention may further encompass the following embodiments.

[0078] Embodiment 1 1) A step of providing pluripotent stem cells 2) A step of inducing the pluripotent stem cells into mesodermal cells 3) A step of introducing a factor or nucleic acid encoding the factor into the pluripotent stem cells induced into mesodermal cells 4) A step of selecting cells that express a marker gene for ovarian somatic cells 5) A step of inducing the selected cells into pregranulosa cell-like cells, granulosa cell-like cells, and / or cumulus cell-like cells

[0079] Embodiment 2 1) A step of providing pluripotent stem cells 2) A step of inducing the pluripotent stem cells into mesodermal cells 3) A step of introducing a factor or a nucleic acid encoding the factor into the pluripotent stem cells induced into mesodermal cells 4) A step of inducing the introduced cells into pregranulosa cell-like cells, granulosa cell-like cells, and / or cumulus cell-like cells

[0080] Embodiment 3 1) A step of providing pluripotent stem cells 2) A step of introducing a factor or nucleic acid encoding the factor into the pluripotent stem cells 3) A step of selecting cells that express a marker gene for ovarian somatic cells 4) A step of inducing the selected cells into pregranulosa cell-like cells, granulosa cell-like cells, and / or cumulus cell-like cells

[0081] Embodiment 4 1) A step of providing pluripotent stem cells 2) A step of introducing a factor or a nucleic acid encoding the factor into the pluripotent stem cells 3) A step of inducing the introduced cells into pregranulosa cell-like cells, granulosa cell-like cells, and / or cumulus cell-like cells

[0082] Embodiment 5 1) A step of providing pluripotent stem cells 2) A step of inducing the pluripotent stem cells into mesodermal cells 3) A step of expressing factors in the pluripotent stem cells induced into mesodermal cells 4) A step of selecting cells that express marker genes for ovarian somatic cells 5) A step of inducing the selected cells into pregranulosa cell-like cells, granulosa cell-like cells, and / or cumulus cell-like cells

[0083] Embodiment 6 1) A step of providing pluripotent stem cells 2) A step of inducing the pluripotent stem cells into mesodermal cells 3) A step of expressing a factor in the pluripotent stem cells induced into mesodermal cells 4) A step of inducing the introduced cells into pregranulosa cell-like cells, granulosa cell-like cells, and / or cumulus cell-like cells

[0084] Embodiment 7 1) A step of providing pluripotent stem cells 2) A step of causing the pluripotent stem cells to express a factor in the cells 3) A step of selecting cells that express a marker gene for ovarian somatic cells 4) A step of inducing the selected cells to be pregranulosa cell-like cells, granulosa cell-like cells, and / or cumulus cell-like cells

[0085] Embodiment 8 1) A step of providing pluripotent stem cells; 2) A step of expressing a factor in the pluripotent stem cells; 3) A step of inducing the expressed cells to become pregranulosa cell-like cells, granulosa cell-like cells, and / or cumulus cell-like cells.

[0086] In embodiments 1 to 8 described above, the factor may include NR1H4 and / or WT1.

[0087] In embodiments 1 to 8 described above, the factors may include NR1H4, WT1, and TCF21.

[0088] In embodiments 1 to 8 described above, the factor includes NR1H4, WT1, and TCF21, and may further include one or more selected from the group consisting of SIX1, NR2F2, ETV5, and TOX3.

Claims

1. A method for producing ovarian somatic cell-like cells, comprising the step of introducing a factor or a nucleic acid encoding the factor into cells, wherein the factor comprises NR1H4 and / or WT1.

2. The method according to claim 1, wherein the cells are cells that substantially do not express FOXL2, and the ovarian somatic cell-like cells are FOXL2-expressing cells.

3. The method according to claim 2, wherein the ovarian somatic cell-like cells express a pregranulosa cell marker gene and / or a granulosa cell marker gene, and substantially do not express a theca cell marker gene.

4. The method according to claim 3, wherein the factor further comprises TCF21.

5. The method according to claim 3, wherein the factors include at least NR1H4, WT1, and TCF21.

6. The method according to claim 5, wherein the factor further comprises one or more selected from the group consisting of SIX1, NR2F2, ETV5, and TOX3.

7. The method according to claim 5, wherein the factor further comprises NR2F2.

8. The method according to claim 5, wherein the factor further comprises SIX1.

9. The method according to claim 5, wherein the above step is performed two or more times.

10. A method for producing ovarian somatic cell-like cells, comprising the step of expressing a factor in cells, wherein the factor comprises NR1H4 and / or WT1.

11. The method according to claim 10, wherein the cells are cells that substantially do not express FOXL2, and the ovarian somatic cell-like cells are FOXL2-expressing cells.

12. The method according to any one of claims 1 to 11, wherein the cells are pluripotent stem cells.

13. Ovarian somatic cell-like cells obtained by introducing a factor or nucleic acid encoding the factor into pluripotent stem cells, wherein the factor comprises NR1H4 and / or WT1, the pluripotent stem cells substantially do not express FOXL2, and the ovarian somatic cell-like cells express FOXL2.

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