Method for producing egg-like cell, and egg-like cell

Abstract

[Problem] The first problem addressed by the present invention is to specify a previously unknown factor or a combination of previously unknown factors among factors or combinations thereof capable of efficiently producing egg-like cells obtained from pluripotent stem cells or the like. The second problem addressed by the present invention is to provide an egg-like cell and a method for efficiently producing same. [Solution] This method for producing an egg-like cell involves a step for introducing a factor into a cell, wherein the factor includes DLX6 or a nucleic acid encoding DLX6. This egg-like cell is derived from a human pluripotent stem cell and expresses PADI6.

Description

Method for producing oocyte-like cells and oocyte-like cells

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

[0002] In order to efficiently produce oocyte-like cells, methods have been developed to introduce specific transcription factors or combinations thereof into cells having the ability to differentiate into oocyte-like cells, such as pluripotent stem cells, and induce the cells into oocyte-like cells.

[0003] It is known that introduction of four types of transcription factors consisting of FIGLA, NOBOX, LHX8, and TBPL2 into cells such as pluripotent stem cells can induce immature oocytes (see Patent Document 1). In addition, it has been reported that overexpression of a combination of ZNF281, LHX8, and SOHLH1 promotes the formation of oocyte-like cells from pluripotent stem cells (see Patent Document 2).

[0004] Japanese Patent No. 7302865 International Publication No. 2023 / 192939

[0005] The first problem to be solved by the present invention is to identify a factor or a combination of factors that has not been known so far among the factors or combinations thereof that can efficiently produce oocyte-like cells by introducing them into cells such as pluripotent stem cells. The second problem to be solved by the present invention is to provide oocyte-like cells and an efficient method for producing them.

[0006] The present inventors have found that oocyte-like cells can be efficiently produced by introducing a factor or a combination of factors that has not been known so far into pluripotent stem cells or the like, and have reached the present invention. That is, the present invention is a method for producing oocyte-like cells including a step of introducing a factor into a cell. The factor may include DLX6 or a nucleic acid encoding DLX6. Further, the factor may further include FIGLA or a nucleic acid encoding FIGLA.

[0007] In the production method of the present invention, the cell into which the factor is introduced is a cell that does not substantially express PADI6, and the oocyte-like cell obtained by introducing the factor may be a cell that expresses PADI6.

[0008] In another aspect of the present invention, the factor may further comprise one or more selected from the group consisting of DLX5, DYNLL1, HEY2, HOXD1, LHX8, PAX6, and nucleic acids encoding these proteins. In yet another aspect of the present invention, the factor may further comprise one or more selected from the group consisting of DLX5, DYNLL1, HEY2, HHEX, HOXD1, JARID2, JAZF1, LHX8, MESP1, NFKB2, NOBOX, PAX6, SOHLH1, SOHLH2, SOX30, STAT1, STAT3, SUB1, TBPL2, TBX3, and nucleic acids encoding these proteins.

[0009] In another aspect of the present invention, the above step may be performed two or more times.

[0010] Another aspect of the present invention is a method for producing oocyte-like cells, comprising the step of expressing a factor in cells. The factor may include DLX6. The factor may further include FIGLA.

[0011] In another aspect of the present invention, the factor may further include one or more selected from the group consisting of DLX5, DYNLL1, HEY2, HOXD1, LHX8, and PAX6. In yet another aspect of the present invention, the factor may further include one or more selected from the group consisting of DLX5, DYNLL1, HEY2, HHEX, HOXD1, JARID2, JAZF1, LHX8, MESP1, NFKB2, NOBOX, PAX6, SOHLH1, SOHLH2, SOX30, STAT1, STAT3, SUB1, TBPL2, and TBX3.

[0012] In the manufacturing method of the present invention, the cells into which the factor is introduced are cells that substantially do not express PADI6, and the oocyte-like cells obtained by introducing the factor may be cells that express PADI6.

[0013] The cells in this invention may be at least one selected from the group consisting of pluripotent stem cells, primordial germ cell-like cells, and epiblast-like cells. Furthermore, the cells in this invention may be human pluripotent stem cells.

[0014] Furthermore, the present invention provides oocyte-like cells obtained by expressing a factor in cells. The present invention also provides oocyte-like cells obtained by expressing a factor in cells. The factor may include DLX6. The factor may further include FIGLA.

[0015] In another aspect of the present invention, the factor may further include one or more selected from the group consisting of DLX5, DYNLL1, HEY2, HOXD1, LHX8, and PAX6. In yet another aspect of the present invention, the factor may further include one or more selected from the group consisting of DLX5, DYNLL1, HEY2, HHEX, HOXD1, JARID2, JAZF1, LHX8, MESP1, NFKB2, NOBOX, PAX6, SOHLH1, SOHLH2, SOX30, STAT1, STAT3, SUB1, TBPL2, and TBX3.

[0016] In the oocyte-like cells of the present invention, the cells into which the factor is introduced are cells that substantially do not express PADI6, and the oocyte-like cells obtained by introducing the factor may be cells that express PADI6.

[0017] In the oocyte-like cells of the present invention, the cells may be at least one selected from the group consisting of pluripotent stem cells, primordial germ cell-like cells, and epiblast-like cells.

[0018] Furthermore, the present invention provides oocyte-like cells derived from human pluripotent stem cells that express PADI6.

[0019] According to the present invention, by introducing previously unknown factors or combinations of factors into pluripotent stem cells, etc., it is possible to induce cells to become oocyte-like cells. The oocyte-like cells obtained by the present invention express multiple maternal effect genes, such as PADI6, and have properties similar to oocytes and / or eggs, such as having oocyte cytoplasm.

[0020] This figure shows the percentage of PADI6-expressing cells obtained in Example 1. The vertical axis shows the introduced factors, and the horizontal axis shows the percentage of PADI6-expressing cells. This figure shows the percentage of PADI6-expressing cells obtained in Example 2. The vertical axis shows the introduced factors and the number of introductions, and the horizontal axis shows the percentage of PADI6-expressing cells. This is a micrograph of cells obtained under the condition of "21 factors, 3 times" in Example 2. "PADI6" is a fluorescence image showing PADI6 expression, and "Brightfield + PADI6" is an image of the fluorescence image and brightfield image superimposed. The scale bar in the figure represents 100 μm. The arrows in the figure indicate spherical PADI6-expressing cells. This is a heatmap showing the expression patterns of each gene in PADI6-expressing cells (PADI6+) obtained by introducing 18 factors in Example 2, cells that did not express PADI6 (PADI6-), and pluripotent stem cells (iPSCs) before introduction. Of the genes shown in the figure, SOHLH1, NOBOX, FIGLA, TP63, PADI6, ZP3, and NPM2 belong to a group of genes called maternal effect genes. This figure shows the percentage of PADI6-expressing cells obtained in Example 3. The vertical axis shows the introduced factor, and the horizontal axis shows the percentage of PADI6-expressing cells.

[0021] The present invention will be described in detail based on embodiments, but the present invention is not limited to these embodiments.

[0022] In this specification, oocyte-like cells mean cells that have the properties of oocytes and / or oocytes. Oocytes include primary oocytes and secondary oocytes. Oocytes also include immature oocytes, and oocyte-like cells as defined herein do not necessarily have a follicular structure.

[0023] One embodiment of an oocyte-like cell as defined herein is a cell having some of the properties of an oocyte and / or an egg, and expressing at least PADI6. Furthermore, in addition to PADI6, it may also express maternal effect genes such as SOHLH1, NOBOX, TP63, ZP3, and NPM2. The oocyte-like cell of the present invention is a cell that expresses a gene different from the introduced factor by introducing a specific factor into the cell, and the gene different from the introduced factor may be a maternal effect gene, and more specifically, the expressed gene may be one or more selected from PADI6, SOHLH1, NOBOX, TP63, ZP3, and NPM2.

[0024] One embodiment of the oocyte-like cells of the present invention expresses at least PADI6, has a larger diameter than the cells before introduction, and exhibits a spherical morphology. The diameter of the oocyte-like cells can range from about 20 micrometers (μm) to about 180 μm. In some embodiments, the diameter of the oocyte-like cells is approximately 20 μm to 160 μm, approximately 20 μm to 140 μm, approximately 20 μm to 120 μm, approximately 20 μm to 100 μm, approximately 40 μm to 180 μm, approximately 40 μm to 160 μm, approximately 40 μm to 140 μm, approximately 40 μm to 120 μm, approximately 40 μm to 100 μm, approximately 60 μm to 180 μm, approximately 60 μm to 160 μm, approximately 60 μm to 140 μm, approximately 60 μm to 120 μm, approximately 60 μm to 100 μm, approximately 80 μm to 180 μm, approximately 80 μm to 160 μm, approximately 80 μm to 140 μm, and approximately 80 μm to 180 μm.

[0025] The cells used in the present invention are selected from the group consisting of pluripotent stem cells, primordial germ cell-like cells, and epiblast-like cells. Conventional manufacturing methods included the steps of inducing human pluripotent stem cells into primordial germ cell-like cells and inducing primordial germ cell-like cells into oocyte-like cells (see Non-Patent Literature 1). As shown in the examples described later, the present invention succeeds in directly inducing pluripotent stem cells into oocyte-like cells. Therefore, those skilled in the art will understand that oocyte-like cells can be produced using primordial germ cell-like cells in the same way as when pluripotent stem cells are used.

[0026] Non-patent document 1: Yamashiro et al., Generation of human oogonia from induced pluripotent stem cells in vitro. Science 362,356-360(2018) DOI:10.1126 / science.aat1674

[0027] Pluripotent stem cells are cells that possess both the ability to differentiate into various cell types 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 Muse cells derived from bone marrow stem cells.

[0028] In this 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.

[0029] One embodiment of the present invention is a method for producing oocyte-like cells using human pluripotent stem cells. When oocyte-like cells are obtained by introducing factors into human pluripotent stem cells, the human pluripotent stem cells into which the factors have been introduced can be induced to differentiate into oocyte-like cells without passing through primordial germ cell-like cells or epiblast-like cells. In other words, this embodiment does not include the step of obtaining primordial germ cell-like cells and / or epiblast-like cells from pluripotent stem cells.

[0030] In another embodiment of the present invention, the cells may be primordial germ cell-like cells (PGCLCs) and / or epiblast-like cells (EpiLCs) derived from pluripotent stem cells. Methods known are employed to induce these cells from pluripotent stem cells.

[0031] The cells used in this invention substantially do not express maternal effect genes such as PADI6. 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 only to 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.

[0032] The cell population used in the present invention does not contain, or contains very few, cells expressing maternal effect genes such as PADI6. In one embodiment of the present invention, a cell population containing oocyte-like cells expressing maternal effect genes such as PADI6 is obtained by introducing a specific factor into the cells included in the cell population.

[0033] The present invention provides a method for producing oocyte-like cells, comprising the step of introducing a factor into cells. The factor may be a transcription factor, a protein thereof, or a nucleic acid encoding such a protein. That is, one embodiment of the present invention may be a method for producing oocyte-like cells, comprising the step of introducing a protein such as a transcription factor, or a nucleic acid encoding such a protein.

[0034] One embodiment of the present invention is a method for producing oocyte-like cells, comprising the step of introducing one or more factors selected from the group consisting of DLX6, FIGLA, DLX5, DYNLL1, HEY2, HOXD1, LHX8, and PAX6, or nucleic acids encoding said factors, into cells.

[0035] Another embodiment of the present invention is a method for producing oocyte-like cells, comprising the step of introducing DLX6 or a nucleic acid encoding DLX6 and FIGLA or a nucleic acid encoding FIGLA into cells. The combination of factors introduced in this embodiment is the two factors DLX6 and FIGLA. This embodiment is superior to the prior art in that it can induce differentiation of cells such as pluripotent stem cells into oocyte-like cells with the minimum combination of factors.

[0036] Furthermore, in this embodiment, in addition to DLX6 and FIGLA, another factor or nucleic acid encoding such factor may be introduced into the cells. In one embodiment, one or more selected from DLX5, DYNLL1, HEY2, HOXD1, LHX8, PAX6, and nucleic acids encoding these proteins may be further introduced into the cells. In another embodiment, one or more selected from DLX5, DYNLL1, HEY2, HHEX, HOXD1, JARID2, JAZF1, LHX8, MESP1, NFKB2, NOBOX, PAX6, SOHLH1, SOHLH2, SOX30, STAT1, STAT3, SUB1, TBPL2, TBX3, and nucleic acids encoding these proteins may be further introduced into the cells.

[0037] DLX5 is an abbreviation for Homeobox protein DLX-5 and belongs to the homeobox family, which has a homeobox domain. Human DLX5 is disclosed as UniprotKB P56178 "DLX5_HUMAN", and mouse DLX5 is disclosed as UniprotKB P70396 "DLX5_MOUSE".

[0038] DLX6 is an abbreviation for Homeobox protein DLX-6 and belongs to the homeobox family, which has a homeobox domain. Human DLX6 is disclosed as UniprotKB P56179 "DLX6_HUMAN", and mouse DLX6 is disclosed as Uniprot KB P70397 "DLX6_MOUSE".

[0039] DYNLL1 is an abbreviation for Dynein light chain 1, cytoplasmic. It is a light chain that constitutes cytoplasmic dynein, an abundant microtubule motor protein widely involved in intracellular transport, and is known to be involved in transcriptional regulation. Human DYNLL1 is disclosed as UniprotKB P63167 "DYL1_HUMAN", and mouse DYNLL1 is disclosed as UniprotKB P63168 "DYL1_MOUSE".

[0040] FIGLA is an abbreviation for Factor in the germline alpha and is known as a basic helix-loop-helix (bHLH) transcription factor that regulates multiple oocyte-specific genes (including genes involved in follicular formation and genes encoding the zona pellucida (ZP1, ZP2, and ZP3)). Human FIGLA is disclosed as UniprotKB Q6QHK4 "FIGLA_HUMAN" and mouse FIGLA is disclosed as UniprotKB O55208 "FIGLA_MOUSE".

[0041] HEY2 is an abbreviation for Hairy / enhancer-of-split related with YRPW motif protein 2 and is known as a bHLH type transcription factor. Human HEY2 is disclosed as Q9UBP5 "HEY2_HUMAN" and mouse HEY2 is disclosed as Q9QUS4 "HEY2_MOUSE".

[0042] HHEX is an abbreviation for hematopoietically expressed homeobox and is known as a transcription factor belonging to the homeobox family. Human HHEX is disclosed as UniprotKB Q03014 "HHEX_HUMAN", and mouse HHEX is disclosed as UniprotKB P43120 "HHEX_MOUSE".

[0043] HOXD1 is an abbreviation for Homeobox protein Hox-D1 and belongs to the homeobox family, which has a homeobox domain. Human HOXD1 is disclosed as UniprotKB Q9GZZ0 "HXD1_HUMAN", and mouse HOXD1 is disclosed as UniprotKB Q01822 "HXD1_MOUSE".

[0044] JARID2 is an abbreviation of jumonji and AT-rich interaction domain containing 2. It is a transcription factor belonging to the α-ketoglutaric acid-dependent hydroxylase superfamily and having an AT-rich interaction domain. Human JARID2 is disclosed as UniprotKB Q92833 "JARD2_HUMAN", and mouse JARID2 is disclosed as UniprotKB Q62315 "JARD2_MOUSE", respectively.

[0045] JAZF1 is an abbreviation of Juxtaposed with another zinc finger protein 1. It is a transcription factor having a C2H2-type zinc finger and is also known as TAK1-interacting protein 27 (TIP27) and Zinc finger protein 802 (ZNF802). Human JAZF1 is disclosed as UniprotKB Q86VZ6 "JAZF1_HUMAN", and mouse JAZF1 is disclosed as UniprotKB Q80ZQ5 "JAZF1_MOUSE", respectively.

[0046] LHX8 is a member of the LIM homeobox family and is known as a transcription factor that plays an important role in embryonic development. Human LHX8 is disclosed as UniprotKB Q68G74 "LHX8_HUMAN", and mouse LHX8 is disclosed as UniprotKB O35652 "LHX8_MOUSE", respectively.

[0047] MESP1 is an abbreviation of Mesoderm posterior protein 1 and is known as a bHLH-type transcription factor. Human MESP1 is disclosed as UniprotKB Q9BRJ9 "MESP1_HUMAN", and mouse MESP1 is disclosed as UniprotKB P97309 "MESP1_MOUSE", respectively.

[0048] NFKB2 is an abbreviation for Nuclear factor NF-kappa-B p100 subunit and is a transcription factor belonging to the NF-κB family. Human NFKB2 is disclosed as UniprotKB Q00653 "NFKB2_HUMAN", and mouse NFKB2 is disclosed as UniprotKB Q9WTK5 "NFKB2_MOUSE".

[0049] NOBOX is an abbreviation for Newborn ovary homeobox protein and is known as a transcription factor that plays an important role in follicular formation. Human NOBOX is disclosed as UniprotKB O60393 "NOBOX_HUMAN", and mouse NOBOX is disclosed as Q8VIH1 "NOBOX_MOUSE".

[0050] PAX6 is an abbreviation for Paired box protein 6 and belongs to the Pax gene group, which has a paired domain that is a DNA-binding domain. Human PAX6 is disclosed as UniprotKB P26367 "PAX6_HUMAN", and mouse PAX6 is disclosed as UniprotKB P63015 "PAX6_MOUSE".

[0051] SOHLH1 is an abbreviation for Spermatogenesis- and oogenesis-specific basic helix-loop-helix-containing protein 1 and is known as a bHLH-type transcription factor. Human SOHLH1 is disclosed as UniprotKB Q5JUK2 "SOLH1_HUMAN", and mouse SOHLH1 is disclosed as UniprotKB Q6IUP1 "SOLH1_MOUSE".

[0052] SOHLH2 is an abbreviation for Spermatogenesis- and oogenesis-specific basic helix-loop-helix-containing protein 2, and is known as a bHLH-type transcription factor. Human SOHLH2 is disclosed as UniprotKB Q9NX45 "SOLH2_HUMAN", and mouse SOHLH2 is disclosed as UniprotKB Q9D489 "SOLH2_MOUSE".

[0053] SOX30 is an abbreviation for SRY-related HMG box transcription factor 30, and is a transcription factor that possesses an HMG-box motif. Human SOX30 is disclosed as UniprotKB O94993 "SOX30_HUMAN", and mouse SOX30 is disclosed as UniprotKB Q8CGW4 "SOX30_MOUSE".

[0054] STAT1 is an abbreviation for Signal transducer and activator of transcription 1, and is a transcription factor activated by cytokines. Human STAT1 is disclosed in UniprotKB P42224 "STAT1_HUMAN", and mouse STAT1 is disclosed in UniprotKB P42225 "STAT1_MOUSE".

[0055] STAT3 is an abbreviation for Signal transducer and activator of transcription 3, and is a transcription factor activated by cytokines. Human STAT3 is disclosed in UniprotKB P40763 "STAT3_HUMAN", and mouse STAT3 is disclosed in UniprotKB P42227 "STAT3_MOUSE".

[0056] SUB1 is a transcription factor also known as Activated RNA polymerase II transcriptional coactivator p15. Human SUB1 is disclosed as P53999 "TCP4_HUMAN," and mouse SUB1 is disclosed as P11031 "TCP4_MOUSE."

[0057] TBPL2 is an abbreviation for TATA-box binding protein like 2 and is known as a transcription factor that binds to the TATA box. Human TBPL2 is disclosed as UniprotKB Q6SJ96 "TBPL2_HUMAN", and mouse TBPL2 is disclosed as UniprotKB Q6SJ95 "TBPL2_MOUSE".

[0058] TBX3 is an abbreviation for T-box transcription factor 3 and is a transcription factor belonging to the T-box family. Human TBX3 is disclosed as UniprotKB O15119 "TBX3_HUMAN", and mouse TBX3 is disclosed as UniprotKB P70324 "TBX3_MOUSE".

[0059] In one embodiment, a nucleic acid encoding a factor is introduced into a cell. The nucleic acid introduced into the cell includes an open reading frame encoding the aforementioned protein. The nucleic acid encoding the factor is introduced either alone or in combination with a transcriptional regulatory sequence such as a promoter. The transcriptional regulatory sequence is not limited as long as it promotes the transcription of the nucleic acid encoding the factor within the cell into which it is introduced. The cell into which the nucleic acid has been introduced may express or overexpress the factor encoded by the nucleic acid.

[0060] In one embodiment, nucleic acids are introduced into cells using a vector designed to express or overexpress proteins within the cell. The vector may be a viral vector and / or a non-viral vector. When introducing multiple factors, the multiple factors may be incorporated into a single vector, or one or more genes may be incorporated into multiple vectors.

[0061] Examples of viral vectors include retroviral vectors, lentiviral vectors, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, Epstein-Barr virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, sylvisvirus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors.

[0062] Non-viral vectors include plasmid vectors and artificial chromosomes, with specific examples including plasmid vectors utilizing transposon systems and episomal vectors.

[0063] In one embodiment, the factor is introduced into cells by methods such as electroporation, microinjection, lipofection, gene gun, or calcium phosphate method. If the factor is a protein, a fusion protein obtained by fusing the factor with a protein introduction domain or a cell membrane-permeable peptide can be introduced into the cells.

[0064] In one embodiment, when a nucleic acid encoding a factor is introduced into a cell, the nucleic acid is not integrated into the cell's chromosome. If the nucleic acid is not integrated into the chromosome, the factor may be transiently expressed within the cell. In another embodiment, the nucleic acid encoding the factor is integrated into the cell's chromosome. If the nucleic acid is integrated into the chromosome, the factor may be stably expressed within the cell.

[0065] In this invention, the step of introducing the factor may be performed only once. In another embodiment, the step of introducing the factor may be performed multiple times. By introducing the factor multiple times, it may be possible to obtain 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.

[0066] Another embodiment of the present invention includes the steps of introducing a nucleic acid encoding a factor containing at least DLX6 into a cell, and expressing the factor containing at least DLX6 in the cell. This step may be the step of expressing a protein containing at least DLX6 in the cell by introducing the nucleic acid, etc., into the cell by the introduction step described above.

[0067] Another embodiment of the present invention includes the steps of introducing nucleic acids encoding a factor comprising at least DLX6 and FIGLA into cells, and expressing the factor comprising at least DLX6 and FIGLA in the cells. This step may be the step of expressing a protein comprising at least DLX6 and FIGLA in cells by introducing nucleic acids, etc., into cells by the introduction step described above.

[0068] A further embodiment of the present invention includes the steps of introducing nucleic acids into cells that encode one or more factors selected from TBPL2, SOHLH1, NOBOX, LHX8, and HHEX, in addition to DLX6 and FIGLA, and expressing the factors encoded by the introduced nucleic acids in the cells. This step may involve introducing nucleic acids into cells by the introduction step described above, thereby expressing in the cells a protein containing one or more factors selected from TBPL2, SOHLH1, NOBOX, LHX8, and HHEX, in addition to DLX6 and FIGLA.

[0069] In this invention, the step of expressing the factor in cells may be performed only 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 may be performed may be two, three, four, or five or more times.

[0070] 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.

[0071] The step of expressing a protein in cells may involve incubating cells into which the aforementioned expression vector 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 a protein in cells may involve incubating cells into which the expression vector has been introduced in the presence of that specific substance.

[0072] In one embodiment, a Tet-On system can be used to induce expression in the presence of a tetracycline antibiotic such as doxycycline. Specifically, rtTA (reverse tetracycline-controlled transactivator) is introduced into cells to regulate the expression of the factor. When inducing the expression of the factor, the cells are cultured in the presence of doxycycline, and rtTA binds to the tetO sequence in the presence of doxycycline, inducing the expression of the factor.

[0073] The factors of the present invention may include not only the proteins described above and the nucleic acids encoding said proteins, but also derivatives and / or modifications 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 said factors, factors in which one or more amino acids are substituted, deleted, inserted, and / or added and performing substantially the same function as said factors, and one or more selected nucleic acids, DNA, or RNA encoding these factors, etc.

[0074] Furthermore, another embodiment of the present invention may include the steps of growing cells and selecting cells. The step of growing cells involves culturing a population of cells such as pluripotent stem cells and primordial germ cell-like cells and growing these cells. The culture conditions are not limited as long as they are conditions that allow these cells to grow. In the step of growing cells, the population of cells such as pluripotent stem cells and primordial germ cell-like cells is grown in a cell growth medium. The medium can be any medium that is suitable for the growth of the population of cells such as pluripotent stem cells and primordial germ cell-like cells.

[0075] In the cell selection step, cells into which the factor has been introduced are selected from a cell population that also contains other cells. 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. Therefore, in one embodiment, even if the proportion of PADI6-expressing cells obtained by introducing the factor is low, the selection step can yield a cell population with a higher proportion of PADI6-expressing cells. Thus, it is not essential that the cell population containing PADI6-expressing cells obtained by introducing the factor has a high proportion of PADI6 cells before selection; a proportion of 10% or less, 1% or less, or even 0.1% or less may be acceptable.

[0076] The step of selecting cells may be the step of separating the target cells from other cells. In one example of the cell selection step, the target cells are selected by using a reporter gene. That is, one embodiment of the manufacturing method of the present invention may be a method for producing oocyte-like cells, which includes the steps of introducing the above-mentioned factors and a reporter gene, and selecting cells using the expression of the reporter gene as an indicator.

[0077] In another embodiment, the method for producing oocyte-like cells may include the steps of introducing a reporter gene into cells, expressing the aforementioned factors in the cells, and selecting cells based on the expression of the reporter gene.

[0078] One embodiment of the oocyte-like cells of the present invention is an oocyte-like cell obtained by introducing the above-mentioned factors into cells, or by expressing the above-mentioned factors in cells, and which is an oocyte-like cell into which a reporter gene has been introduced.

[0079] A specific example of a reporter gene is a nucleic acid encoding a fusion protein, which is a marker for oocyte-like cells, i.e., a maternal effect gene such as PADI6, linked to a fluorescent protein reporter. Since cells induced to be oocyte-like express the fusion protein, oocyte-like cells and other cells can be separated by fluorescence-activated cell sorting (FACS) using the presence of the fluorescent protein in the cell as an indicator.

[0080] Examples of fluorescent proteins include, but are not limited to, green fluorescent protein (GFP), its variants (EGFP, YFP, CFP, BFP, etc.), red fluorescent protein (RFP, mCherry, tdTomato, etc.), and their derivatives, variants, or functionally equivalent fluorescent proteins.

[0081] In another embodiment, the step of selecting cells may be to select target cells based on drug resistance genes to drugs to which the cells are sensitive. For example, by introducing an expression vector containing factors 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 target cells can be obtained.

[0082] One embodiment of the manufacturing method of the present invention may be a method for producing oocyte-like cells, comprising the steps of introducing the above-mentioned factors and drug resistance genes, and selecting cells based on drug sensitivity as an indicator.

[0083] In another embodiment, there may be a method for producing oocyte-like cells that includes the steps of introducing a drug resistance gene into cells, expressing the aforementioned factors in the cells, and selecting cells based on drug sensitivity.

[0084] One embodiment of the oocyte-like cells of the present invention is an oocyte-like cell obtained by introducing the above-mentioned factors into cells, or by expressing the above-mentioned factors in cells, and may be an oocyte-like cell into which a drug resistance gene has been introduced.

[0085] Examples of drug resistance genes include, but are not limited to, those such as the neomycin resistance gene (neoR), chloramphenicol resistance gene (cat), hygromycin resistance gene (hygR), puromycin resistance gene (pac), zeosin resistance gene (Sh ble), and blastosidine resistance gene (bsd), or derivatives, mutants, or equivalent genes with similar functions.

[0086] Furthermore, the present invention also encompasses a method for producing oocytes by co-culturing the aforementioned oocyte-like cells with ovarian somatic cells, and oocytes obtained by co-culturing the aforementioned oocyte-like cells with ovarian somatic cells.

[0087] 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.

[0088] The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0089] Example 1: We attempted to produce oocyte-like cells by introducing a factor into human iPS cells (802-3G strain, ReproCELL, Inc.) and using PADI6 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 PADI6, and a donor vector containing a homology arm with a nucleotide sequence adjacent to the gene region, and containing the genes for PADI6, 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 PADI6.

[0090] Furthermore, according to the method described in Patent Document 1, a vector was constructed to overexpress FIGLA and one transcription factor selected from DLX6, DLX5, PAX6, LHX8, HEY2, HOXD1, DYNLL1, and other transcription factors in iPS cells. Specifically, a CAG promoter and an instability domain (DD) were introduced into a PiggyBAC vector to obtain a PB-CAG-DD vector. Then, the combination of the two transcription factors described above was introduced into the PB-CAG-DD vector using the In-Fusion HD Cloning Kit (Takara Bio Inc.).

[0091] iPS cells were placed in a 24-well plate containing 500 μL of culture medium (Stemfit® AK02N, Ajinomoto Healthy Supply Co., Ltd.) with a capacity 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 expanded. On the eighth day of culture, the number of cells expressing PADI6 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.

[0092] The results are shown in Figure 1. When FIGLA and one factor selected from DLX6, DLX5, PAX6, LHX8, HEY2, HOXD1, NC, and DYNLL1 were introduced into iPS cells, PADI6-expressing cells were obtained. In particular, FIGLA and DLX6 were found to produce PADI6-expressing cells at extremely high levels compared to other combinations of transcription factors.

[0093] Example 2: Multiple transcription factors were introduced into human iPS cells, and attempts were made to produce oocyte-like cells using PADI6 gene expression as an indicator. The transcription factors used were FIGLA, DLX6, DLX5, DYNLL1, HEY2, HHEX, JARID2, JAZF1, LHX8, MESP1, NFKB2, NOBOX, PAX6, SOHLH1, SOHLH2, SOX30, STAT1, STAT3, SUB1, TBPL2, and TBX3 (hereinafter, this combination of transcription factors will be referred to as "21 factors") and FIGLA, DLX5, DYNLL1, HEY2, HHEX, JARID2, LHX8, MESP1, NOBOX, PAX6, SOHLH1, SOHLH2, SOX30, STAT1, STAT3, SUB1, TBPL2, and TBX3 (hereinafter, this combination of transcription factors will be referred to as "18 factors").

[0094] Vectors were constructed using the same method as in Example 1 and introduced into human iPS cells. Factor 21 was introduced into cells under two conditions: on day 2 of culture, a lipofection solution containing a vector incorporating the gene encoding Factor 21 was added to the culture medium (hereinafter referred to as "Factor 21, 1st time"), or on days 2, 4, and 6 of culture, a lipofection solution containing the vector was added to the culture medium (hereinafter referred to as "Factor 21, 3rd time"). Factor 18 was introduced into cells on day 2 of culture by adding a lipofection solution containing a vector incorporating the gene encoding Factor 18 to the culture medium. In all conditions, expression was induced by changing to a medium containing Shield1 two days after introduction. On day 8 of culture, the number of cells expressing PADI6 was measured using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0095] The results are shown in Figure 2. Compared to the 18 factors that did not include DLX6, a large number of PADI6-expressing cells were observed in the 21 factors including FIGLA and DLX6. In particular, PADI6-expressing cells were obtained with an extremely high probability in the 21 factors, 3-step condition. Furthermore, when the morphology of the cells obtained in the 21 factors, 3-step condition was observed using a fluorescence microscope, spherical cells expressing PADI6 were found, as indicated by the arrows in Figure 3.

[0096] Furthermore, RNA-seq analysis was performed on PADI6-expressing cells (PADI6+) and non-PADI6-expressing cells (PADI6-) obtained by separating cells introduced with 18 factors using a flow cytometer, as well as on iPS cells (iPSCs) before factor introduction, to examine the expression levels of each gene. The results are shown in Figure 4. In addition to PADI6 and FIGLA, NOBOX, and SOHLH1, which were used for introduction, PADI6-expressing cells were found to express maternal effect genes such as TP63, ZP3, and NPM2.

[0097] Example 3: Multiple transcription factors were introduced into human iPS cells, and the production of oocyte-like cells was attempted using PADI6 gene expression as an indicator. The transcription factors compared were seven factors: FIGLA, DLX6, HHEX, LHX8, NOBOX, SOHLH1, and TBPL2, and six factors obtained by removing one of the seven factors, for a total of seven combinations. A vector was constructed using the same method as in Example 1 and introduced into human iPS cells. Similar to Example 1, Shield1 was added once on day 2 of culture to induce expression, and the number of cells expressing PADI6 was measured on day 8 of culture using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0098] The results are shown in Figure 5. PADI6-expressing cells were confirmed at a high rate in cells into which all seven factors were introduced. Furthermore, PADI6-expressing cells were confirmed at a similar rate to those in the case of the seven factors with one of the following removed: HHEX, LHX8, NOBOX, SOHLH1, and TBPL2. However, PADI6-expressing cells were hardly confirmed in the case of the six factors with either FIGLA or DLX6 removed from the seven factors, suggesting that FIGLA and DLX6 are important factors in inducing human iPS cells into oocyte-like cells.

[0099] The present invention encompasses the following embodiments: [Embodiment 1] A method for producing oocyte-like cells, comprising the step of introducing a factor into cells, wherein the factor comprises DLX6 or a nucleic acid encoding DLX6, and FIGLA or a nucleic acid encoding FIGLA. [Embodiment 2] A method for producing oocyte-like cells, comprising the step of expressing a factor containing DLX6 and FIGLA in cells. [Embodiment 3] The method according to embodiment 1 or 2, wherein the cells are cells that substantially do not express PADI6, and the oocyte-like cells are PADI6-expressing cells. [Embodiment 4] The method according to any one of embodiments 1 to 3, wherein the factor further comprises one or more selected from the group consisting of DLX5, DYNLL1, HEY2, HOXD1, LHX8, PAX6, and nucleic acids encoding these proteins. [Aspect 5] The method according to any one of aspects 1 to 3, wherein the factor further comprises one or more selected from the group consisting of DLX5, DYNLL1, HEY2, HHEX, HOXD1, JARID2, JAZF1, LHX8, MESP1, NFKB2, NOBOX, PAX6, SOHLH1, SOHLH2, SOX30, STAT1, STAT3, SUB1, TBPL2, TBX3, and nucleic acids encoding these proteins. [Aspect 6] The method according to any one of aspects 1 to 5, wherein the cell is at least one selected from the group consisting of pluripotent stem cells, primordial germ cell-like cells, and epiblast-like cells. [Aspect 7] The method according to any one of aspects 1 to 6, wherein the step is performed two or more times. [Aspect 8] The method according to any one of aspects 1 to 7, wherein the step does not include a step performed in vivo on a human. [Aspect 9] Oocyte-like cells obtained by introducing a factor into cells, wherein the factor comprises DLX6 or a nucleic acid encoding DLX6, and FIGLA or a nucleic acid encoding FIGLA. [Aspect 10] Oocyte-like cells obtained by expressing a factor in cells, wherein the factor comprises DLX6 and FIGLA. [Aspect 11] Oocyte-like cells according to aspect 9 or 10, wherein the cells are cells that do not express PADI6, and the oocyte-like cells are PADI6-expressing cells.[Aspect 12] An oocyte-like cell according to any one of aspects 9 to 11, wherein the cell is at least one selected from the group consisting of pluripotent stem cells, primordial germ cell-like cells, and epiblast-like cells. [Aspect 13] An oocyte-like cell according to any one of aspects 9 to 12, wherein the cell is introduced with a reporter gene and / or a drug resistance gene. [Aspect 14] An oocyte-like cell according to any one of aspects 9 to 13, derived from human pluripotent stem cells and expressing PADI6. [Aspect 15] An oocyte-like cell derived from human pluripotent stem cells and expressing PADI6.

Claims

1. A method for producing oocyte-like cells, comprising the step of introducing a factor into cells (except when performed in vivo in humans), wherein the factor comprises DLX6 or a nucleic acid encoding DLX6, and FIGLA or a nucleic acid encoding FIGLA.

2. A method for producing oocyte-like cells, comprising the step of expressing factors including DLX6 and FIGLA in cells (except when performed in vivo in humans).

3. The method according to claim 1 or 2, wherein the cells are cells that substantially do not express PADI6, and the oocyte-like cells are PADI6-expressing cells.

4. The method according to any one of claims 1 to 3, wherein the factor further comprises one or more selected from the group consisting of DLX5, DYNLL1, HEY2, HOXD1, LHX8, PAX6, and nucleic acids encoding these proteins.

5. The method according to any one of claims 1 to 3, wherein the factor further comprises one or more selected from the group consisting of DLX5, DYNLL1, HEY2, HHEX, HOXD1, JARID2, JAZF1, LHX8, MESP1, NFKB2, NOBOX, PAX6, SOHLH1, SOHLH2, SOX30, STAT1, STAT3, SUB1, TBPL2, TBX3, and nucleic acids encoding these proteins.

6. The method according to any one of claims 1 to 5, wherein the cells are at least one selected from the group consisting of pluripotent stem cells, primordial germ cell-like cells, and epiblast-like cells.

7. The method according to any one of claims 1 to 5, wherein the cells are human pluripotent stem cells.

8. The method according to any one of claims 1 to 7, wherein the above step is performed two or more times.

9. Oocyte-like cells obtained by introducing a factor into cells, wherein the factor comprises DLX6 or a nucleic acid encoding DLX6, and FIGLA or a nucleic acid encoding FIGLA.

10. Oocyte-like cells obtained by expressing a factor in cells, wherein the factor includes DLX6 and FIGLA.

11. The oocyte-like cell according to claim 9 or 10, wherein the cell is a cell that does not express PADI6, and the oocyte-like cell is a PADI6-expressing cell.

12. The oocyte-like cell according to any one of claims 9 to 11, wherein the cell is at least one selected from the group consisting of pluripotent stem cells, primordial germ cell-like cells, and epiblast-like cells.

13. The oocyte-like cell according to any one of claims 9 to 11, wherein the cell is a human pluripotent stem cell.

14. Oocyte-like cells derived from human pluripotent stem cells and expressing PADI6.

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