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

Abstract

The first objective is to identify previously unknown factors or combinations of factors that can efficiently produce oocyte-like cells from pluripotent stem cells, etc. The second objective is to provide oocyte-like cells and an efficient method for producing them. [Solution] A method for producing egg-like cells, which includes a step of introducing a factor into a cell, wherein the factor includes DLX6 or a nucleic acid encoding DLX6, and egg-like cells derived from human pluripotent stem cells and expressing PADI6.

Description

[Technical Field]

[0001] The present invention relates to a method for producing an oocyte-like cell, and to an oocyte-like cell. [Background technology]

[0002] In order to efficiently produce egg-like cells, methods have been developed in which specific transcription factors or combinations of these factors are introduced into cells that have the ability to differentiate into egg-like cells, such as pluripotent stem cells, to induce the cells to become egg-like cells.

[0003] It is known that introducing four transcription factors consisting of FIGLA, NOBOX, LHX8, and TBPL2 into cells such as pluripotent stem cells can induce them to become immature oocytes (see Patent Document 1). It has also been reported that the combined overexpression of ZNF281, LHX8, and SOHLH1 promotes the formation of egg-like cells from pluripotent stem cells (see Patent Document 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 7302865 [Patent Document 2] International Publication No. 2023 / 192939 Summary of the Invention [Problem to be solved by the invention]

[0005] The first problem to be solved by the present invention is to identify a previously unknown factor or combination of factors that can be introduced into cells such as pluripotent stem cells to efficiently produce oocyte-like cells. The second problem to be solved by the present invention is to provide oocyte-like cells and an efficient method for producing them. [Means for solving the problem]

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

[0007] In the production method of the present invention, the cells into which the factors are introduced are cells that do not substantially express PADI6, and the oocyte-like cells obtained by introducing the factors can 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 method may be such that the above steps are carried out two or more times.

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

[0011] In another aspect of the present invention, the factors may further comprise 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 factors 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, and TBX3.

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

[0013] The cells of the present invention may be at least one type selected from the group consisting of pluripotent stem cells and primordial germ cell-like cells.

[0014] The present invention also provides oocyte-like cells derived from human pluripotent stem cells and expressing PADI6. The oocyte-like cells of the present invention are obtained by introducing a factor into the human pluripotent stem cells, the factor including DLX6, FIGLA, or a nucleic acid encoding either or both, and the human pluripotent stem cells do not substantially express PADI6. [Effects of the Invention]

[0015] According to the present invention, by introducing a previously unknown factor or a combination thereof into pluripotent stem cells, etc., the cells can be induced to become oocyte-like cells. The oocyte-like cells obtained by the present invention have properties similar to oocytes and / or eggs, such as expressing multiple maternal effect genes, typified by PADI6, and possessing ooplasm. [Brief explanation of the drawings]

[0016] [Figure 1]1 is a diagram showing the percentage of PADI6-expressing cells obtained in Example 1. The vertical axis shows the introduced factor, and the horizontal axis shows the percentage of PADI6-expressing cells. [Figure 2] 1 is a diagram showing the percentage of PADI6-expressing cells obtained in Example 2. The vertical axis shows the factor introduced and the number of times of introduction, and the horizontal axis shows the percentage of PADI6-expressing cells. [Figure 3] These are micrographs of cells obtained under the "21 factors x 3" condition in Example 2. "PADI6" is a fluorescent image showing PADI6 expression, and "bright field + PADI6" is a superimposed image of the fluorescent image and bright field image. The scale bar in the figure indicates 100 μm. The arrow in the figure indicates a spherical PADI6-expressing cell. [Figure 4] 1 is a heat map showing the expression patterns of each gene in PADI6-expressing cells (PADI6+), cells that did not express PADI6 (PADI6-), and pluripotent stem cells (iPSCs) before the introduction of 18 factors obtained in Example 2. Among the genes in the figure, SOHLH1, NOBOX, FIGLA, TP63, PADI6, ZP3, and NPM2 belong to a group of genes known as maternal effect genes. [Figure 5] 1 is a diagram showing 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. DETAILED DESCRIPTION OF THE INVENTION

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

[0018] As used herein, 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 cell defined herein does not necessarily have to have a follicle structure.

[0019] One embodiment of an oocyte-like cell as defined herein is a cell that has some of the properties of an oocyte and / or an egg and expresses at least PADI6. Furthermore, in addition to PADI6, the oocyte-like cell may 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. The gene different from the introduced gene may be a maternal effect gene. More specifically, the expressed gene may be one or more selected from PADI6, SOHLH1, NOBOX, TP63, ZP3, and NPM2.

[0020] In one embodiment, the egg-like cells of the present invention express at least PADI6, have a larger diameter than the cells before the introduction, and exhibit a spherical morphology. The diameter of the egg-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, or about 80 μm to about 180 μm.

[0021] The cells used in the present invention may be pluripotent stem cells and / or primordial germ cell-like cells induced from pluripotent stem cells. Conventional production methods include the steps of inducing human pluripotent stem cells into primordial germ cell-like cells and inducing the primordial germ cell-like cells into oocyte-like cells (see Non-Patent Document 1). As shown in the Examples below, the present invention has succeeded 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 as well as 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 have the ability to differentiate into various cells and the ability to self-proliferate. 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 the present invention, pluripotent stem cells derived from mammals such as humans, mice, rats, cows, pigs, horses, sheep, rabbits, dogs, and cats, as well as birds, reptiles, amphibians, and fish are used. The pluripotent stem cells may be derived from humans.

[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 a factor into human pluripotent stem cells, the factor-introduced human pluripotent stem cells can be induced to differentiate into oocyte-like cells without first becoming primordial germ cell-like cells. That is, this embodiment does not include a 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) induced from pluripotent stem cells. These cells can be induced from pluripotent stem cells using known methods.

[0027] The cells used in the present invention do not substantially express maternal effect genes such as PADI6. Here, "substantially not expressing" means that the mRNA and / or protein present in the cells is below the detection limit, or even if detected, the expression of these genes is so low that they cannot exert their functions. 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 almost no 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 cells contained in the cell population.

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

[0030] One embodiment of the present invention is a method for producing an egg-like cell, comprising the step of introducing into a cell one or more factors selected from the group consisting of DLX6, FIGLA, DLX5, DYNLL1, HEY2, HOXD1, LHX8, and PAX6, or nucleic acids encoding such factors.

[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. In this embodiment, the combination of factors introduced is the two factors DLX6 and FIGLA. This embodiment is advantageous over the prior art in that it can induce differentiation of cells such as pluripotent stem cells into oocyte-like cells using a minimal combination of factors.

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

[0033] DLX5 is an abbreviation for Homeobox protein DLX-5, and belongs to the homeobox family, which contains 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 contains a homeobox domain. Human DLX6 is disclosed as UniprotKB P56179 "DLX6_HUMAN," and mouse DLX6 is disclosed as UniprotKB P70397 "DLX6_MOUSE."

[0035] DYNLL1 (Dynein light chain 1, cytoplasmic) is a light chain of cytoplasmic dynein, an abundant microtubule motor protein widely involved in intracellular transport and 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 folliculogenesis 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 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 contains 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 has 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, an abbreviation for Juxtaposed with another zinc finger protein 1, is a transcription factor with a C2H2-type zinc finger, also known as TAK1-interacting protein 27 (TIP27) and zinc finger protein 802 (ZNF802). Human JAZF1 is listed as UniprotKB Q86VZ6 "JAZF1_HUMAN" and mouse JAZF1 is listed 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 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 follicle 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 family, which has a paired domain, 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 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 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, a transcription factor with 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 as UniprotKB P42224 "STAT1_HUMAN", and mouse STAT1 is disclosed as 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 as UniprotKB P40763 "STAT3_HUMAN", and mouse STAT3 is disclosed as 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 contains an open reading frame encoding the above-mentioned protein. 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 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.

[0056] In one embodiment, the nucleic acid is introduced into a cell using a vector designed to express or overexpress a protein in the cell. The vector may be a viral vector and / or a non-viral vector. When multiple factors are introduced, the multiple factors may be incorporated into a single vector, or one or more genes may be incorporated into multiple vectors.

[0057] Specific examples of viral vectors include retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, adeno-associated virus vectors, herpes virus vectors, Epstein-Barr virus vectors, vaccinia virus vectors, poxvirus vectors, poliovirus vectors, Silvis virus vectors, rhabdovirus vectors, paramyxovirus vectors, and orthomyxovirus vectors.

[0058] Non-viral vectors include plasmid vectors and artificial chromosomes, and specific examples include plasmid vectors that utilize a transposon system and episomal vectors.

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

[0060] In the present invention, the step of introducing a factor may be performed once. In another embodiment, the step of introducing a factor may be performed multiple times. By introducing a factor multiple times, it may be possible to obtain a large number of target cells. In this embodiment, the number of times of introducing a factor may be two, three, four, five or more times.

[0061] Another embodiment of the present invention is a method for producing a composition comprising at least D LX 6 into the cell, and LX and a step of expressing a factor including 6 in the cell. This step is carried out by introducing a nucleic acid or the like into the cell by the above-mentioned introducing step, thereby producing at least D LX 6 in a cell.

[0062] Another embodiment of the present invention is a method for producing a composition comprising at least D LXintroducing into the cell a nucleic acid encoding a factor, including 6 and FIGLA; LX and a step of expressing a factor including FIGLA in the cell. This step involves introducing a nucleic acid or the like into the cell by the above-mentioned introducing step, thereby producing at least D LX 6 and FIGLA-containing proteins in the cell.

[0063] Yet another embodiment of the present invention is a method for producing a composition comprising at least D LX The method includes the steps of introducing nucleic acids encoding one or more factors selected from TBPL2, SOHLH1, NOBOX, LHX8, and HHEX in addition to 6 and FIGLA into cells, and allowing the cells to express the factors encoded by the introduced nucleic acids. LX The step may be a step of expressing in the cell a protein comprising one or more selected from TBPL2, SOHLH1, NOBOX, LHX8, and HHEX in addition to FIGLA.

[0064] In the present invention, the step of expressing a factor in a cell may be performed once. In another embodiment, the step of expressing a factor in a cell may be performed multiple times. By expressing a factor multiple times, it may be possible to obtain a large number of target cells. In this embodiment, the step of expressing a factor may be performed two, three, four, five or more times.

[0065] In the above-described embodiment, the nucleic acid can be incorporated into an expression vector containing the nucleic acid and a promoter and then introduced into a cell. The promoter used here is not limited, but specific examples include promoters that are active in the cells into which they are introduced and promoters whose activity is induced in the presence of a specific substance. Examples of promoters that are active in the cells into which they are introduced include the CAG promoter, CMV promoter, EF1-α promoter, UbC promoter, and PGK promoter. Examples of promoters whose activity is induced in the presence of a specific substance include tetracycline-inducible promoters and estrogen-inducible promoters.

[0066] The step of expressing a protein in cells can be a step of incubating cells into which the above-mentioned expression vector has been introduced. Furthermore, when an expression vector incorporating a promoter whose activity is induced in the presence of a specific substance has been introduced, the step of expressing a protein in cells can be a step of incubating cells into which the expression vector has been introduced in the presence of the specific substance.

[0067] The factors of the present invention may be one or more selected from the above-mentioned proteins, nucleic acids encoding the proteins, as well as derivatives and / or modified forms of the factors, factors that have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology with the factors and that exhibit substantially the same action as the factors, factors in which one or more amino acids have been substituted, deleted, inserted, and / or added and that exhibit substantially the same action as the factors, and nucleic acids, DNAs, or RNAs encoding these factors, etc.

[0068] In yet another embodiment of the present invention, the method may include a step of expanding cells and a step of selecting cells. The step of expanding cells is a step of culturing a population of cells such as pluripotent stem cells, primordial germ cell-like cells, etc., and expanding these cells. The culture conditions are not limited as long as they allow these cells to expand.

[0069] In the cell selection step, factor-introduced cells are selected from a cell population containing other cells. The cell population obtained by selecting factor-introduced cells has a higher proportion of target cells compared to the cell population before selection. Therefore, in one embodiment, even if the proportion of PADI6-expressing cells obtained by factor introduction is low, a cell population with a higher proportion of PADI6-expressing cells can be obtained by the selection step. Therefore, it is not essential that the proportion of PADI6 cells in the cell population containing PADI6-expressing cells obtained by factor introduction be high before selection; a proportion of 10% or less, 1% or less, or 0.1% or less is acceptable.

[0070] The cell selection step can be a step of separating the target cells from other cells. For example, by introducing a nucleic acid encoding a fusion protein combining a gene that serves as an oocyte marker with a fluorescent protein reporter, the cells induced to become oocytes can be separated from other cells by fluorescence-activated cell sorting (FACS).

[0071] In another embodiment, the step of selecting cells may be a step of selecting the target cells based on a drug resistance gene for a drug to which the cells are sensitive. For example, an expression vector containing a factor and a drug resistance gene is introduced into cells, and the resulting cell population is incubated in a medium containing the drug, thereby obtaining a cell population with a high proportion of the target cells.

[0072] The present invention also includes a method for producing ova by co-culturing the above-mentioned ova-like cells with ovarian somatic cells, and ova obtained by co-culturing the above-mentioned ova-like cells with ovarian somatic cells. [Example]

[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: Human iPS cells (strain 802-3G, ReproCELL) were transfected with factors to generate oocyte-like cells using PADI6 gene expression as an indicator. Human iPS cells were lipofected with a CRISPR-Cas9 vector that cleaves the region near the termination codon of the PADI6-encoding gene and a donor vector containing homologous arms flanking the gene region, PADI6, the red fluorescent protein tdTomato, and a drug resistance gene between the 5' and 3' arms. This vector generated iPS cells in which the tdTomato gene was inserted downstream of the PADI6-encoding gene. Furthermore, vectors were constructed to overexpress FIGLA and a transcription factor selected from DLX6, DLX5, PAX6, LHX8, HEY2, HOXD1, DYNLL1, and other transcription factors in iPS cells according to the method described in Patent Document 1. Specifically, a CAG promoter and a destabilization domain (DD) were introduced into a PiggyBAC vector to generate the PB-CAG-DD vector. Next, the combination of the two types of transcription factors described above was introduced into the PB-CAG-DD vector using the In-Fusion HD Cloning Kit (Takara Bio Inc.).

[0075] iPS cells were plated at 4.0 × 10 cells / well in a 24-well plate containing 500 μL of medium (Stemfit® AK02N, Ajinomoto Healthy Supply Co., Ltd.). 4The cells were seeded and cultured for two days. Lipofection solution 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)) was added to each well and cultured for two 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 PADI6-expressing cells was counted using an Attune NxT flow cytometer (Thermo Fisher Scientific). The expression level of cells without factor transfection (negative control) was set as the threshold.

[0076] The results are shown in Figure 1. PADI6-expressing cells were obtained when FIGLA and one factor selected from DLX6, DLX5, PAX6, LHX8, HEY2, HOXD1, NC, and DYNLL1 were introduced into iPS cells. In particular, it was confirmed that the use of FIGLA and DLX6 resulted in significantly higher levels of PADI6-expressing cells than other combinations of transcription factors.

[0077] Example 2: Human iPS cells were transfected with multiple transcription factors to attempt 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 is referred to as the "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 is referred to as the "18 factors").

[0078] Vectors were constructed using the same method as in Example 1 and transfected into human iPS cells. The 21 factors were transfected into cells by adding a Lipofection solution containing vectors incorporating genes encoding the 21 factors to the medium on day 2 of culture (hereinafter referred to as "21 factors, once") or by adding a Lipofection solution containing the vectors to the medium on days 2, 4, and 6 of culture (hereinafter referred to as "21 factors, three times"). The 18 factors were transfected into cells by adding a Lipofection solution containing vectors incorporating genes encoding the 18 factors to the medium on day 2 of culture. In both conditions, expression was induced by changing the medium to contain Shield1 two days after transfection. On day 8 of culture, the number of cells expressing PADI6 was counted using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0079] The results are shown in Figure 2. A higher number of PADI6-expressing cells were observed with the 21 factors, including FIGLA and DLX6, compared to the 18 factors without DLX6. In particular, PADI6-expressing cells were obtained with an extremely high probability under the 21 factors × 3 conditions. Furthermore, when the morphology of the cells obtained under the 21 factors × 3 conditions was observed using a fluorescence microscope, PADI6-expressing spherical cells were found, as indicated by the arrows in Figure 3.

[0080] We also performed RNA-seq analysis on PADI6-expressing cells (PADI6+) and non-PADI6-expressing cells (PADI6-) obtained by separating the 18-factor-transfected cells using a flow cytometer, as well as iPSCs (iPSCs) before factor transfection, to examine the expression levels of each gene. The results are shown in Figure 4. In addition to PADI6 and the genes used for transfection, FIGLA, NOBOX, and SOHLH1, the 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 an attempt was made to produce oocyte-like cells using PADI6 gene expression as an indicator. Seven transcription factor combinations were compared: FIGLA, DLX6, HHEX, LHX8, NOBOX, SOHLH1, and TBPL2, plus six other factors obtained by omitting one of the seven factors. A vector was constructed and introduced into human iPS cells using the same method as in Example 1. Shield1 was added once on day 2 of culture to induce expression, as in Example 1, and the number of cells expressing PADI6 was counted 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 confirmed with a high probability in cells transfected with the seven factors. Furthermore, when HHEX, LHX8, NOBOX, SOHLH1, or TBPL2 were removed from the seven factors, PADI6-expressing cells were confirmed at the same level as when the seven factors were removed. However, when FIGLA or DLX6 was removed from the seven factors, PADI6-expressing cells were hardly detected, suggesting that FIGLA and DLX6 are important factors in inducing human iPS cells to oocyte-like cells.

Claims

1. introducing the factor into a cell (except in vivo in humans), The factor comprises DLX6 or a nucleic acid encoding DLX6, and FIGLA or a nucleic acid encoding FIGLA; the cells are human pluripotent stem cells; A method for producing egg-like cells.

2. the cell does not express PADI6, The method of claim 1, wherein the egg-like cells are PADI6-expressing cells.

3. The method of claim 2, 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.

4. The method of claim 2, 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.

5. The method of claim 2 , wherein said steps are performed two or more times.

6. expressing factors including at least DLX6 and FIGLA in a cell (except in vivo in humans); the cells are human pluripotent stem cells; A method for producing egg-like cells.

7. the cell does not express PADI6, The method of claim 6, wherein the egg-like cells are PADI6-expressing cells.

8. These are egg-like cells obtained by introducing factors into cells, The factor comprises DLX6 or a nucleic acid encoding DLX6, and FIGLA or a nucleic acid encoding FIGLA; the cells are human pluripotent stem cells; Oocyte-like cells.

9. An oocyte-like cell obtained by expressing a factor in a cell, the factors include DLX6 and FIGLA, the cells are human pluripotent stem cells; Oocyte-like cells.

10. The cell does not express PADI6, The egg-like cell described in claim 8 or 9, wherein the egg-like cell is a PADI6-expressing cell.

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