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

Abstract

The first challenge is to identify previously unknown factors or combinations of factors that can efficiently produce oocyte-like cells obtained from pluripotent stem cells, etc. The second challenge is to provide oocyte-like cells and efficient methods for producing them. [Solution] 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 oocyte-like cells derived from human pluripotent stem cells and expressing PADI6.

Description

Technical Field

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

Background Art

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

[0003] It is known that by introducing four types of transcription factors consisting of FIGLA, NOBOX, LHX8, and TBPL2 into cells such as pluripotent stem cells, they can be induced into 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 egg cell-like cells from pluripotent stem cells (see Patent Document 2).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] The first problem to be solved by the present invention is to identify factors or combinations thereof that have not been known heretofore among factors or combinations thereof that can efficiently produce egg-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 egg-like cells and an efficient method for producing them.

Means for Solving the Problems

[0006] The present inventors have discovered that oocyte-like cells can be efficiently produced by introducing previously unknown factors or combinations thereof into pluripotent stem cells, and have arrived at the present invention. That is, the present invention is a method for producing oocyte-like cells, comprising the step of introducing a factor into cells. The factor may include DLX6 or a nucleic acid encoding DLX6. The factor may further include FIGLA or a nucleic acid encoding FIGLA.

[0007] 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.

[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 and primordial germ cell-like cells.

[0014] Furthermore, the present invention provides oocyte-like cells derived from human pluripotent stem cells that express PADI6. The oocyte-like cells of the present invention are obtained by introducing a factor into the human pluripotent stem cells, the factor comprising DLX6, FIGLA, or nucleic acids encoding these, and the human pluripotent stem cells substantially do not express PADI6. [Effects of the Invention]

[0015] According to the present invention, by introducing previously unknown factors or combinations thereof into pluripotent stem cells, etc., cells can be induced into oocyte-like cells. The oocyte-like cells obtained by the present invention express multiple maternal effect genes, such as PADI6, and have oocyte cytoplasm, and thus possess properties similar to oocytes and / or oocytes. [Brief explanation of the drawing]

[0016] [Figure 1]It is a drawing showing the ratio of PADI6-expressing cells obtained in Example 1. The vertical axis indicates the introduced factors, and the horizontal axis indicates the ratio of PADI6-expressing cells. [Figure 2] It is a drawing showing the ratio of PADI6-expressing cells obtained in Example 2. The vertical axis indicates the introduced factors and the number of introduction times, and the horizontal axis indicates the ratio of PADI6-expressing cells. [Figure 3] It is a microscopic photograph of the cells obtained under the condition of "21 factors · 3 times" in Example 2. "PADI6" is a fluorescence image showing PADI6 expression, and "bright field + PADI6" is an image obtained by superimposing the fluorescence image and the bright field image. The scale bar in the figure indicates 100 μm. The arrow in the figure indicates PADI6-expressing cells having a spherical morphology. [Figure 4] It is a heat map 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. Among the genes in the figure, SOHLH1, NOBOX, FIGLA, TP63, PADI6, ZP3, and NPM2 belong to a group of genes called maternal effect genes. [Figure 5] It is a drawing showing the ratio of PADI6-expressing cells obtained in Example 3. The vertical axis indicates the introduced factors, and the horizontal axis indicates the ratio of PADI6-expressing cells.

Modes for Carrying Out the Invention

[0017] The present invention will be described in detail based on embodiments, but the present invention is not limited to these embodiments.

[0018] In the present specification, an oocyte-like cell refers to a cell having the properties of an oocyte and / or an egg. Oocytes include primary oocytes and secondary oocytes. Oocytes also include immature oocytes, and the oocyte-like cells defined in the present specification do not necessarily have to have a follicular structure.

[0019] One aspect of the oocyte-like cells defined in this specification is a cell having a part of the properties of an oocyte and / or an egg, and expressing at least PADI6. Further, in addition to PADI6, it may express maternal effect genes such as SOHLH1, NOBOX, TP63, ZP3, NPM2, etc. The oocyte-like cells of the present invention are cells that express genes different from the introduced factors by introducing specific factors into the cells, and the genes different from the introduced genes may be maternal effect genes. More specifically, the expressed genes may be one or more selected from PADI6, SOHLH1, NOBOX, TP63, ZP3, and NPM2.

[0020] One aspect of the oocyte-like cells of the present invention expresses at least PADI6, has a larger diameter compared to the cells before introduction, and shows a spherical morphology. The diameter of the oocyte-like cells can be from about 20 micrometers (μm) to about 180 μm. In some embodiments, the diameter of the oocyte-like cells is about 20 μm to about 160 μm, about 20 μm to about 140 μm, about 20 μm to about 120 μm, about 20 μm to about 100 μm, about 40 μm to about 180 μm, about 40 μm to about 160 μm, about 40 μm to about 140 μm, about 40 μm to about 120 μm, about 40 μm to about 100 μm, about 60 μm to about 180 μm, about 60 μm to about 160 μm, about 60 μm to about 140 μm, about 60 μm to about 120 μm, about 60 μm to about 100 μm, about 80 μm to about 180 μm, about 80 μm to about 160 μm, about 80 μm to about 140 μm, about 80 μm to about 180 μm.

[0021] The cells used in the present invention can be pluripotent stem cells and / or primordial germ cell-like cells derived from pluripotent stem cells. In conventional production methods, it 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 Document 1). As shown in the examples described later, in the present invention, we have succeeded in directly inducing pluripotent stem cells into oocyte-like cells. Therefore, those skilled in the art can understand that it is possible to produce oocyte-like cells when using primordial germ cell-like cells as well as when using pluripotent stem cells.

[0022] 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

[0023] 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.

[0024] 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.

[0025] 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 going through the primordial germ cell-like cell stage. In other words, this embodiment does not include the step of obtaining primordial germ cell-like cells from pluripotent stem cells.

[0026] In another embodiment of the present invention, the cells may be primordial germ cell-like cells (PGCLCs) derived from pluripotent stem cells. Methods known are employed to induce these cells from pluripotent stem cells.

[0027] 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 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.

[0028] 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.

[0029] 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 protein such as a transcription factor, 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.

[0030] 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.

[0031] 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 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.

[0032] Furthermore, in this embodiment, cells may be further selectively introduced with one or more selected from DLX5, DYNLL1, HEY2, HOXD1, LHX8, PAX6, and nucleic acids encoding these proteins. In another embodiment, cells may be further selectively introduced with 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.

[0033] 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".

[0034] 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".

[0035] 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".

[0036] 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".

[0037] 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".

[0038] 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".

[0039] 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".

[0040] JARID2 is an abbreviation for jumonji and AT-rich interaction domain containing 2. It is a transcription factor belonging to the α-ketoglutarate-dependent hydroxylase superfamily and possessing an AT-rich interaction domain. Human JARID2 is disclosed as UniprotKB Q92833 "JARD2_HUMAN", and mouse JARID2 is disclosed as UniprotKB Q62315 "JARD2_MOUSE".

[0041] JAZF1 is an abbreviation for Juxtaposed with another zinc finger protein 1, and is a transcription factor possessing a C2H2 type zinc finger, also known as TAK1-interacting protein 27 (TIP27) or Zinc finger protein 802 (ZNF802). Human JAZF1 is disclosed as UniprotKB Q86VZ6 "JAZF1_HUMAN", and mouse JAZF1 is disclosed as UniprotKB Q80ZQ5 "JAZF1_MOUSE".

[0042] 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".

[0043] MESP1 is an abbreviation for 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".

[0044] 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".

[0045] 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".

[0046] 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".

[0047] 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".

[0048] 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".

[0049] 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".

[0050] 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".

[0051] 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".

[0052] 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."

[0053] 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".

[0054] 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".

[0055] In one embodiment, the factor introduced into the cell is a nucleic acid. The nucleic acid introduced into the cell includes an open reading frame that encodes the protein described above. The nucleic acid encoding the factor is introduced alone or in combination with a promoter. The promoter 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 expresses or overexpresses the factor encoded by the nucleic acid.

[0056] 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.

[0057] 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.

[0058] Non-viral vectors include plasmid vectors and artificial chromosomes, with specific examples including plasmid vectors utilizing transposon systems and episomal vectors.

[0059] In one embodiment, the factor is introduced into cells by methods such as electroporation, microinjection, or lipofection. If the factor is a protein, it can be introduced into cells by a protein introduction domain or by fusing it with a cell membrane-permeable peptide.

[0060] 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.

[0061] Another embodiment of the present invention includes the steps of introducing a nucleic acid encoding a factor containing at least DXL6 into a cell, and expressing the factor containing at least DXL6 in the cell. This step may be the step of expressing a protein containing at least DXL6 in the cell by introducing the nucleic acid, etc., into the cell by the introduction step described above.

[0062] Another embodiment of the present invention includes the steps of introducing a nucleic acid encoding a factor comprising at least DXL6 and FIGLA into a cell, and expressing the factor comprising at least DXL6 and FIGLA in the cell. This step may be the step of expressing a protein comprising at least DXL6 and FIGLA in the cell by introducing the nucleic acid, etc., into the cell by the introduction step described above.

[0063] 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 at least DXL6 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 DXL6 and FIGLA.

[0064] 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 quantity of the 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.

[0065] 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.

[0066] 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.

[0067] The factors of the present invention may be not only the proteins described above, the nucleic acids encoding the 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 the factors, factors in which one or more amino acids are substituted, deleted, inserted, and / or added and performing substantially the same function as the factors, and one or more selected nucleic acids, DNA, or RNA encoding these factors, etc.

[0068] 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 or 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.

[0069] 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.

[0070] The step of selecting cells may involve separating the target cells from other cells. For example, by introducing nucleic acids that encode a fusion protein linking a gene that serves as a marker for oocytes with a fluorescent protein reporter, cells induced to resemble oocytes can be separated from other cells using fluorescence-activated cell sorting (FACS).

[0071] 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.

[0072] 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. [Examples]

[0073] The present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0074] Example 1: We attempted to produce oocyte-like cells by introducing factors into human iPS cells (802-3G strain, ReproCELL, Inc.) and using PADI6 gene expression as an indicator. 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, were lipofectioned into human iPS cells to produce iPS cells in which the gene encoding tdTomato was inserted downstream of the gene encoding PADI6. Furthermore, according to the method described in Patent Document 1, a vector was constructed that overexpresses 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. Next, the combination of the two transcription factors mentioned above was introduced into the PB-CAG-DD vector using the In-Fusion HD cloning kit (Takara Bio Inc.).

[0075] 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. 4Cells 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.

[0076] 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.

[0077] 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").

[0078] 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).

[0079] 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-factor, 3-step test. In particular, PADI6-expressing cells were obtained with an extremely high probability in the 21-factor, 3-step test condition. Furthermore, when the morphology of the cells obtained in the 21-factor, 3-step test condition was observed using a fluorescence microscope, spherical cells expressing PADI6 were found, as indicated by the arrows in Figure 3.

[0080] 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.

[0081] 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).

[0082] The results are shown in Figure 5. PADI6-expressing cells were observed at a high rate in cells into which all seven factors were introduced. Furthermore, PADI6-expressing cells were observed at a similar rate to those observed with the seven factors, even when one of the following six factors was removed: HHEX, LHX8, NOBOX, SOHLH1, and TBPL2. However, PADI6-expressing cells were hardly observed with the six factors, even when FIGLA or DLX6 was removed from the seven factors, suggesting that FIGLA and DLX6 are important factors in inducing human iPS cells into oocyte-like cells.

Claims

1. The process includes the step of introducing the factor into the cells, A method for producing oocyte-like cells, wherein the factor comprises DLX6 or a nucleic acid encoding DLX6.

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

3. The method according to claim 2, wherein the factor further comprises FIGLA or a nucleic acid encoding FIGLA.

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

7. The step includes expressing a factor containing at least DXL6 in cells, A method for producing oocyte-like cells.

8. The aforementioned cells are cells that substantially do not express PADI6, The method according to claim 7, wherein the oocyte-like cells are PADI6-expressing cells.

9. The method according to any one of claims 1 to 8, wherein the cells are at least one selected from the group consisting of pluripotent stem cells and primordial germ cell-like cells.

10. Oocyte-like cells derived from human pluripotent stem cells that express PADI6.

11. The oocyte-like cell according to claim 10 is obtained by introducing the factor into the human pluripotent stem cell, The aforementioned factor includes DLX6, FIGLA, or nucleic acids encoding these, Oocyte-like cells in which the aforementioned human pluripotent stem cells substantially do not express PADI6.

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