Methods and compositions for producing oogonia-like cells

Abstract

Oogonia dysfunction underlies many forms of human female infertility, yet efficient methods for the generation of oogonia in vitro remain elusive. Provided herein are methods and compositions for differentiating induced pluripotent stem cells into oogonia-like cells by overexpressing transcription factors such as ZNF281, LHX8 and / or SOHLH1.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 326,607, filed April 1, 2022, which is incorporated by reference herein in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (H049870757WO00-SEQ-KVC.xml, size: 12,492 bytes and creation date: March 28, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] background Oogonia are specialized cells that upon maturation form the primordial follicles in the female fetus. Oogonia proliferate through mitosis before differentiating into oocytes involved in sexual reproduction. Oogonia dysfunction underlies many forms of human female infertility, but efficient methods for the generation of oogonia in vitro remain elusive. Summary of the Invention [Means for solving the problem]

[0004] overview The present disclosure relates, at least in part, to methods and compositions for generating oogonia in vitro from pluripotent stem cells (PSCs). The present disclosure provides experimental data that unexpectedly demonstrates that overexpression of certain transcription factors, such as zinc finger protein 281 (ZNF281), LIM homeobox 8 (LHX8), and spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (SOHLH1), is sufficient to generate oogonia (e.g., DDX4+ oogonia-like cells) from PSCs in as little as four days.

[0005] Some aspects of the present disclosure provide pluripotent stem cells (PSCs) comprising a modified polynucleotide comprising an open reading frame encoding a protein selected from ZNF281, LHX8 and SOHLH1.

[0006] In some embodiments, the PSC comprises a modified polynucleotide comprising an open reading frame encoding ZNF281.

[0007] In some embodiments, the PSC comprises a modified polynucleotide comprising an open reading frame encoding LHX8.

[0008] In some embodiments, the PSC comprises a modified polynucleotide comprising an open reading frame encoding SOHLH1.

[0009] In some embodiments, the PSCs express or overexpress ZNF281, LHX8, SOHLH1, ZNF281 and LHX8, ZNF281 and SOHLH1, LHX8 and SOHLH1, or ZNF281, LHX8 and SOHLH1.

[0010] In some embodiments, the PSCs further comprise a modified polynucleotide comprising an open reading frame encoding a folliculogenesis-specific BHLH transcription factor (FIGLA) protein, and optionally, the PSCs express or overexpress FIGLA.

[0011] In some embodiments, the PSCs further comprise a modified polynucleotide comprising an open reading frame encoding a Distal-Less Homeobox 5 (DLX5) protein and a modified polynucleotide comprising an open reading frame encoding a Hematopoietic Expressed Homeobox (HHEX) protein, and optionally the PSCs express or overexpress the DLX5 protein and the HHEX protein.

[0012] In some embodiments, the PSC further comprises a modified polynucleotide comprising an open reading frame encoding a DEAD box polypeptide-4 (DDX4) protein, a modified polynucleotide comprising an open reading frame encoding a Deleted in AZoospermia (DAZL) protein, and a modified polynucleotide comprising an open reading frame encoding a Boule homolog (BOLL) protein, and optionally the PSC expresses or overexpresses the DDX4 protein, the DAZL protein, and the BOLL protein.

[0013] In some embodiments, the open reading frame of the modified polynucleotide is operably linked to a heterologous promoter.

[0014] In some embodiments, the heterologous promoter is an inducible promoter.

[0015] Another aspect of the present disclosure provides a PSC comprising a protein selected from ZNF281, LHX8 and SOHLH1, in which the protein is overexpressed.

[0016] In some embodiments, the PSCs express or overexpress ZNF281, LHX8, SOHLH1, ZNF281 and LHX8, ZNF281 and SOHLH1, LHX8 and SOHLH1, or ZNF281, LHX8 and SOHLH1.

[0017] In some embodiments, the PSCs further comprise a FIGLA protein, and optionally, the PSCs express or overexpress FIGLA.

[0018] In some embodiments, the PSCs further comprise a DLX5 protein and an HHEX protein, and optionally, the PSCs express or overexpress the DLX5 protein and the HHEX protein.

[0019] In some embodiments, the PSCs further comprise DDX4, DAZL and BOLL proteins, and optionally, the PSCs express or overexpress the DDX4, DAZL and BOLL proteins.

[0020] In some embodiments, the PSCs are human PSCs.

[0021] In some embodiments, the PSCs are induced PSCs (iPSCs).

[0022] In some embodiments, the PSC comprises 1 to 20 copies of a modified polynucleotide comprising an open reading frame encoding a protein selected from ZNF281, LHX8, and SOHLH1, In some embodiments, the PSC comprises 8 to 10 copies of a modified polynucleotide comprising an open reading frame encoding a protein selected from ZNF281, LHX8, and SOHLH1.

[0023] Some embodiments of the present disclosure provide a composition comprising a population of PSCs as described in any one of the above paragraphs or elsewhere herein.

[0024] In some embodiments, the population is at least 2500 cells / cm 2 Includes PSCs.

[0025] Another aspect of the disclosure provides a method comprising culturing a population of pluripotent stem cells (PSCs) in a culture medium to produce an expanded population of PSCs, and expressing a protein selected from ZNF281, LHX8 and SOHLH1 in the expanded population of PSCs to produce oogonia-like cells.

[0026] In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide comprising an open reading frame encoding ZNF281.

[0027] In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide comprising an open reading frame encoding LHX8.

[0028] In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide that includes an open reading frame encoding SOHLH1.

[0029] In some embodiments, the PSCs of the expanded population further comprise a modified polynucleotide comprising an open reading frame encoding a FIGLA protein.

[0030] In some embodiments, the PSCs of the expanded population further comprise a modified polynucleotide comprising an open reading frame encoding a DLX5 protein and a modified polynucleotide comprising an open reading frame encoding an HHEX protein.

[0031] In some embodiments, the PSCs of the expanded population further comprise a modified polynucleotide comprising an open reading frame encoding a DDX4 protein, a modified polynucleotide comprising an open reading frame encoding a DAZL protein, and a modified polynucleotide comprising an open reading frame encoding a BOLL protein.

[0032] In some embodiments, the open reading frame of the modified polynucleotide is operably linked to a heterologous promoter.

[0033] In some embodiments, the heterologous promoter is an inducible promoter.

[0034] In some embodiments, the population is 1×10 2 ~1×10 7 Includes PSCs.

[0035] In some embodiments, the population of PSCs is cultured for about 3-5 days, optionally for about 4 days.

[0036] In some embodiments, the oogonia-like cells are + It is.

[0037] In some embodiments, the oogonia-like cells independently have a diameter of about 20 micrometers to 180 micrometers.

[0038] Some aspects of the disclosure provide methods that include: (a) delivering to PSCs a modified polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from ZNF281, LHX8, and SOHLH1; (b) culturing the PSCs in a feeder-free, serum-free culture medium to produce an expanded population of PSCs; and (c) culturing the expanded population of PSCs in a series of induction media comprising an inducer to produce DDX4+ oogonia-like cells.

[0039] In some embodiments, the modified polynucleotide is a transposon and delivering further comprises delivering a transposase to the PSC.

[0040] In some embodiments, the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

[0041] In some embodiments, the feeder-free, serum-free culture medium of (b) comprises a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

[0042] In some embodiments, the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

[0043] In some embodiments, the ECM protein is selected from laminin, collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

[0044] In some embodiments, the feeder-free, serum-free culture medium of (b) comprises a growth factor selected from recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor beta (rh TGFβ).

[0045] In some embodiments, the culturing in (b) is for about 6 to about 24 hours.

[0046] In some embodiments, the PSCs of the expanded population of (c) are cultured at a density of about 2,000 cells / cm2 to about 3,000 cells / cm2.

[0047] In some embodiments, the culturing of (c) comprises culturing the PSCs in a first induction medium, culturing the PSCs in a second induction medium, culturing the PSCs in a third induction medium, and culturing the PSCs in a fourth induction medium.

[0048] In some embodiments, the first induction medium comprises one or more of B-27, L-alanyl-L-glutamine, an inducer (e.g., doxycycline), activin A, a glycogen synthase kinase (GSK) 3 inhibitor, and a selective FGFR1 and FGFR3 inhibitor.

[0049] In some embodiments, the second induction medium comprises one or more of B-27, an inducer (eg, doxycycline), a small molecule inhibitor of tankyrase (TNKS), and human bone morphogenetic protein 4 (hBMP4).

[0050] In some embodiments, the third induction medium comprises one or more of B-27, an inducer (eg, doxycycline), a small molecule inhibitor of tankyrase, stem cell factor (SCF), and epidermal growth factor (EGF).

[0051] In some embodiments, the fourth induction medium comprises one or more of B-27, an inducer (eg, doxycycline), a small molecule inhibitor of tankyrase, hBMP4, SCF, and EGF.

[0052] Some aspects of the disclosure provide oogonia-like cells produced by the methods described in any one of the above paragraphs or elsewhere herein.

[0053] The details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will become apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component illustrated in various figures is represented by the same numeral. For clarity, not every component is depicted in every drawing. [Brief description of the drawings]

[0055] [Figure 1] Shown is the oocyte-sequence scoring of TFs from scRNA-Seq, where TF enrichment is scored based on the presence of TFs in cells expressing oocyte marker genes. [Figure 2A] Figure 2 shows that three TFs drive high DDX4 expression when induced during germ cell formation. Flow cytometry data of DDX4 expression in cells expressing no TF (top left), ZNF281 (top right), SOHLH1 (bottom left) and LHX8 (bottom right) are shown. [Figure 2B] Figure 1 shows that three TFs drive high DDX4 expression when induced during germ cell formation. Geometric mean expression of DDX4 fluorescence intensity compared to control is shown. [Figure 2C] Figure 2 shows that the three TFs drive high DDX4 expression when induced during germ cell formation. Figure 2 shows flow cytometry data of DDX4 expression in cells expressing the D3 combination, displaying high DDX4 oogonia-like properties. [Figure 3A] Figure 2 shows that the D3 TF combination drives the formation of cells with an oocyte-like transcriptome. RNA-Seq gene expression patterns shown as log2 fold change compared to human induced pluripotent stem cell (hiPSC) controls for selected oogenesis signature genes. [Figure 3B] We show that D3 TF combinations drive the formation of cells with oocyte-like transcriptomes. We show cell type classification analysis (TROM) in which the transcriptomes of D3 oogonia-like cells are compared to a reference ovarian atlas, showing statistically significant similarity (defined as TROM=>12) to in vivo antral and secondary oocytes. [Figure 4] FIG. 1 shows a schematic diagram of a method for generating high DDX4 expressing cells from stem cells. [Diagram 5] Figure 2 shows the examination of combinations of TFs for oogonia formation. The percentage of cells expressing DDX4 and the percentage of cells expressing NPM2 are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] Detailed Description Oogonia are essential cells involved in reproduction. Oogonia dysfunction underlies many forms of human female infertility (Garg et al. 2015). Accordingly, there is an unmet need to develop methods for the generation of oogonia and oogonia-like cells. Such cells may, for example, be the basis for therapeutic intervention of human female infertility. It is to be understood that the term "oogonia-like cells" encompasses cells that express oogonia-specific markers, such as DDX4, and exhibit other characteristics of naturally occurring oogonia cells.

[0057] Current methods for generating DDX4-positive human oogonia-like cells in vitro, such as from human induced pluripotent stem cells (hiPSCs), are laborious, expensive, and time-consuming. Current methods utilize co-culture of induced human primordial germ cell-like cells (hPGCLCs) and mouse E12.5 fetal gonad cells (mFGCs) to form xenogeneic reconstituted ovaries (xrOvaries), which induce a small population of human DDX4+ oogonia-like cells when grown for approximately 120 days (Yamashiro et al. 2018). While this approach can generate DDX4+ oogonia-like cells, it requires extensive technical expertise, costly culture techniques, and lengthy differentiation periods, making such methods difficult to handle for most high-throughput applications. Aspects of the present disclosure relate to a method for inducing DDX4+ oogonia-like cells from stem cells in 4 days using direct transcription factor overexpression in parallel with growth factor culture.

[0058] Oogonia-like cells Some aspects of the present disclosure provide oogonia-like cells and methods for producing such cells. Oogonia are small diploid cells that mature to form primordial follicles in female fetuses. Oogonia are formed in large numbers by mitosis from primordial germ cells early in fetal development. During human development, primordial germ cells differentiate into oogonia, which further proliferate through mitosis. After proliferation, oogonia differentiate into primary oocytes through asymmetric division. One daughter cell produced through the asymmetric division of the primary oocyte becomes an oocyte through the process of oogenesis. Oocytes, which are finally produced through oogenesis, undergo meiosis and are involved in sexual reproduction (Sathananthan et al. 2006).

[0059] Oogonia and oocytes produced through oogenesis express the gene DEAD box polypeptide 4 (DDX4) (e.g., UniProtKB-Q9NQI0(DDX4_HUMAN)), a putative marker gene for female germ cells (Danny et al. 2021). Thus, in some embodiments, the oogonia-like cells produced by the methods provided herein are DDX4+ oogonia-like cells (i.e., cells that express the DHX4 protein).

[0060] Oogonia-like cells have other characteristics that distinguish them from non-oogonia-like cells, including, but not limited to, size. For example, oogonia-like cells can have a diameter of about 20 micrometers (μm) to about 180 μm. In some embodiments, oogonia-like cells can have a diameter of 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 16 ... 0 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 120 μm, about 80 μm to about 100 μm, about 100 μm to about 180 μm, about 100 μm to about 160 μm, about 100 μm to about 140 μm, or about 100 μm to about 120 μm.

[0061] pluripotent stem cells The oogonia-like cells provided herein are differentiated from pluripotent stem cells, which are cells that have the ability to self-renew by division and develop into the three primary germ cell layers (e.g., ectoderm, endoderm, and mesoderm) of the early embryo, i.e., into all cells of the adult body, but not into extraembryonic tissues such as the placenta (Shi et al. 2017).

[0062] Non-limiting examples of pluripotent stem cells include induced pluripotent cells (iPSCs), "true" embryonic stem cells (ESCs) derived from embryos, embryonic stem cells generated by somatic cell nuclear transfer (ntESCs) and embryonic stem cells from unfertilized eggs (parthenogenetic embryonic stem cells or pESCs). In some embodiments, the pluripotent cells are human pluripotent cells.

[0063] In some embodiments, the pluripotent stem cells are embryonic stem cells, such as human embryonic stem cells. "Embryonic stem cells" is a general term that refers to pluripotent stem cells that are generated using embryos or eggs, rather than genetically reprogrammed cells from the body. As used herein, "ESCs" encompass true ESCs, ntESCs, and pESCs.

[0064] In other embodiments, the pluripotent stem cells are induced pluripotent stem cells, such as human induced pluripotent stem cells. iPSCs can be derived from skin or blood cells that have been reprogrammed back to an embryonic-like pluripotent state, allowing for the generation of an unlimited source of cells that can become any human cell type.

[0065] Some aspects of the present disclosure provide PSCs comprising a protein selected from ZNF281, LHX8 and SOHLH1, wherein the protein is expressed or overexpressed. In some embodiments, the protein is expressed at a level at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50% or at least 100% higher than a control level. In some embodiments, the control level is an endogenous level of the protein, such as in a spontaneously occurring pluripotent stem cell. In some embodiments, the PSC comprises ZNF281. In some embodiments, the PSC expresses or overexpresses ZNF281. In some embodiments, the PSC comprises LHX8. In some embodiments, the PSC expresses or overexpresses LHX8. In some embodiments, the PSC comprises SOHLH1. In some embodiments, the PSC expresses or overexpresses SOHLH1.

[0066] Data provided herein show that combinatorial expression of two proteins selected from ZNF281, LHX8 and SOHLH1 results in a 10-50 fold increase in the efficiency of DDX4+ oogonial cell-like production compared to a control, optionally the control being the efficiency of DDX4+ oogonial cell-like production in PSCs expressing only one of ZNF281, LHX8 or SOHLH1. In some embodiments, the PSCs comprise ZNF281 and LHX8. In some embodiments, the PSCs express or overexpress ZNF281 and LHX8. In some embodiments, the PSCs comprise ZNF281 and SOHLH1. In some embodiments, the PSCs express or overexpress ZNF281 and SOHLH1. In some embodiments, the PSCs comprise LHX8 and SOHLH1. In some embodiments, the PSCs express or overexpress LHX8 and SOHLH1.

[0067] The data provided herein also show that combinatorial expression of ZNF281, LHX8 and SOHLH1 in PSCs results in a 100-1000 fold increase in the efficiency of DDX4+ oogonial cell-like production compared to a control, optionally the control being the efficiency of DDX4+ oogonial cell-like production in PSCs expressing only one of ZNF281, LHX8 or SOHLH1. In some embodiments, the PSCs comprise ZNF281, LHX8 and SOHLH1. In some embodiments, the PSCs express or overexpress ZNF281, LHX8 and SOHLH1.

[0068] The data provided herein further show that combinatorial expression of FIGLA, ZNF281, LHX8 and SOHLH1 in PSCs results in an approximately 10-fold increase in the efficiency of DDX4+ oogonial cell-like production compared to a control, optionally the control being the efficiency of DDX4+ oogonial cell-like production in PSCs expressing only ZNF281, LHX8 and SOHLH1. In some embodiments, the producing cells express the oocyte-like marker NPM2. In some embodiments, the PSCs further comprise FIGLA. In some embodiments, the PSCs further express or overexpress FIGLA.

[0069] The data provided herein further demonstrate that combinatorial expression of FIGLA, DLX5, HHEX, ZNF281, LHX8 and SOHLH1 in PSCs results in increased efficiency of DDX4+ oogonial cell-like production compared to a control, optionally the control being the efficiency of DDX4+ oogonial cell-like production in PSCs expressing only ZNF281, LHX8 and SOHLH1. In some embodiments, the PSCs further comprise DLX5. In some embodiments, the PSCs further express or overexpress DLX5. In some embodiments, the PSCs further comprise HHEX. In some embodiments, the PSCs further express or overexpress HHEX.

[0070] The data provided herein further indicates that combinatorial expression of DDX4, DAZL, BOLL, FIGLA, DLX5, HHEX, ZNF281, LHX8 and SOHLH1 in PSCs results in increased efficiency of DDX4+ oogonial cell-like production compared to a control, optionally the control being the efficiency of DDX4+ oogonial cell-like production in PSCs expressing only ZNF281, LHX8 and SOHLH1. In some embodiments, the PSCs further comprise DDX4. In some embodiments, the PSCs further express or overexpress DDX4. In some embodiments, the PSCs further comprise DAZL. In some embodiments, the PSCs further express or overexpress DAZL. In some embodiments, the PSCs further comprise BOLL. In some embodiments, the PSCs further express or overexpress BOLL.

[0071] Surprisingly, combinatorial expression of ZNF281, LHX8 and SOHLH1, and combinatorial expression of only one of HHEX, DLX5, DAZL, DDX4, BOLL or DAZL, did not show increased efficiency of DDX4+ oogonocyte-like production in PSCs.

[0072] Transcription factors The oogonia-like cells provided herein, in some embodiments, are differentiated from pluripotent stem cells by expressing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) transcription factors (i.e., proteins that control the rate of transcription). Differentiation is the process by which uncommitted or partially committed cells commit to a specialized cell fate. Aspects of the present disclosure relate to the differentiation of uncommitted pluripotent stem cells to an oogonia-like cell fate. In some embodiments, the transcription factors are selected from ZNF281, LHX8, and SOHLH1. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress ZNF281. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress LHX8. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress SOHLH1. In some embodiments, pluripotent stem cells such as hPSCs or hiPSCs are modified to express or overexpress ZNF281 and LHX8. In some embodiments, pluripotent stem cells such as hPSCs or hiPSCs are modified to express or overexpress ZNF281 and SOHLH1. In some embodiments, pluripotent stem cells such as hPSCs or hiPSCs are modified to express or overexpress LHX8 and SOHLH1. In some embodiments, pluripotent stem cells such as hPSCs or hiPSCs are modified to express or overexpress ZNF281, LHX8 and SOHLH1.

[0073] A cell "expresses" a particular protein if it is possible to detect levels of the protein in the cell (e.g., using a known protein assay). A cell "overexpresses" a particular protein if the levels of the protein are higher (e.g., at least 5%, at least 10%, or at least 20% higher) than the levels of protein expressed from an endogenous, naturally occurring polynucleotide encoding the protein (e.g., a modified polynucleotide encoding the protein).

[0074] In some embodiments, the transcription factor is selected from FIGLA, DLX5, and HHEX. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress FIGLA. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress DLX5. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress HHEX. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress FIGLA and DLX5. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress FIGLA and HHEX. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress DLX5 and HHEX. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress FIGLA, DLX5, and HHEX. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress ZNF281, LHX8, SOHLH1, and FIGLA. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are modified to express or overexpress ZNF281, LHX8, SOHLH1, FIGLA, DLX5, and HHEX.

[0075] In some embodiments, pluripotent stem cells such as hPSCs or hiPSCs are modified to express or overexpress DDX4. In some embodiments, pluripotent stem cells such as hPSCs or hiPSCs are modified to express or overexpress DAZL. In some embodiments, pluripotent stem cells such as hPSCs or hiPSCs are modified to express or overexpress BOLL. In some embodiments, pluripotent stem cells such as hPSCs or hiPSCs are modified to express or overexpress DDX4 and DAZL. In some embodiments, pluripotent stem cells such as hPSCs or hiPSCs are modified to express or overexpress DDX4 and BOLL. In some embodiments, pluripotent stem cells such as hPSCs or hiPSCs are modified to express or overexpress DAZL and BOLL. In some embodiments, pluripotent stem cells such as hPSCs or hiPSCs are modified to express or overexpress DDX4, DAZL and BOLL.

[0076] In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are engineered to express or overexpress ZNF281, LHX8, SOHLH1, FIGLA, DLX5, HHEX, DDX4, DAZL and BOLL.

[0077] Modified Polynucleotides and Polypeptides The pluripotent stem cells of the present disclosure, in some embodiments, comprise modified polynucleotides. Modified polynucleotides are non-naturally occurring nucleic acids (e.g., at least two nucleotides covalently linked together, in some cases containing phosphodiester bonds, referred to as a phosphodiester backbone). Modified polynucleotides include recombinant and synthetic nucleic acids. Recombinant nucleic acids are molecules constructed by linking nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or combinations thereof) from two different organisms (e.g., human and mouse). Synthetic nucleic acids are molecules that have been amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that have been chemically modified or otherwise modified, provided that they are capable of base pairing (binding) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids include molecules resulting from any of the above replications.

[0078] Modified polynucleotides can be DNA (e.g., genomic DNA, cDNA, or a combination of genomic DNA and cDNA), RNA, or hybrid molecules, e.g., the nucleic acid can contain any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural) and any combination of two or more bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine.

[0079] In some embodiments, the polynucleotide is complementary DNA (cDNA), which is synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.

[0080] The modified polynucleotides of the present disclosure can be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the nucleic acids are produced using GIBSON ASSEMBLY® cloning (see, e.g., Gibson, DG et al. Nature Methods, 343-345, 2009 and Gibson, DG et al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). In GIBSON ASSEMBLY®, typically three enzyme activities are used in a single tube reaction: 5' exonuclease activity, 3' extension activity of DNA polymerase, and DNA ligase activity. The 5' exonuclease activity degrades the 5' end sequence to expose complementary sequences for annealing. Gaps on the annealed domains are then filled by the polymerase activity. The nicks are then sealed by DNA ligase to covalently link the DNA fragments together. The overlapping sequences of the adjoining fragments are much longer than those used in Golden Gate assembly, resulting in a higher percentage of correct assembly. Other methods of producing modified polynucleotides can be used in accordance with the present disclosure.

[0081] In some embodiments, the modified polynucleotide comprises a promoter operably linked to the open reading frame. A promoter is a nucleotide sequence (e.g., ATG) to which RNA polymerase binds in coordination with initial transcription. A promoter is typically located directly upstream (5' end) of the transcription start site. In some embodiments, the promoter is a heterologous promoter. A heterologous promoter is not naturally associated with the open reading frame to which it is operably linked.

[0082] In some embodiments, the promoter is an inducible promoter. An inducible promoter can be regulated in vivo, for example, by a chemical agent, temperature, or light. An inducible promoter allows, for example, temporal and / or spatial control of gene expression. Inducible promoters for use according to the present disclosure include any inducible promoter described herein or known to one of skill in the art. Examples of inducible promoters include, but are not limited to, chemically / biochemically regulated promoters and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems including the tetracycline repressor protein (tetR), the tetracycline operator sequence (tetO) and the tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters from the rat glucocorticoid receptor, human estrogen receptor and the steroid / retinoid / thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (a protein that binds and sequesters metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters) and light-regulated promoters (e.g., light-responsive promoters from plant cells). In some embodiments, the inducible promoter is a tetracycline-inducible promoter. In some embodiments, the inducible promoter is a doxycycline-inducible promoter. In other embodiments, the promoter is a constitutive promoter (in vivo active, unregulated).

[0083] An open reading frame is a contiguous stretch of codons that begins with a start codon (e.g., ATG), ends with a stop codon (e.g., TAA, TAG, or TGA), and encodes a polypeptide, such as a protein. An open reading frame is operably linked to a promoter if that promoter controls transcription of the open reading frame.

[0084] Vectors used for delivery of modified polynucleotides include minicircles, plasmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes. Transposon-based systems such as the piggyBac™ system (e.g., Chen et al. Nature Communications. 2020; 11(1): 3446) are also contemplated herein.

[0085] The pluripotent stem cells, in some embodiments, comprise a modified polynucleotide comprising an open reading frame encoding a protein selected from ZNF281, LHX8, and SOHLH1. In some embodiments, the modified polynucleotide comprises an open reading frame encoding ZNF281. In some embodiments, the modified polynucleotide comprises an open reading frame encoding LHX8. In some embodiments, the modified polynucleotide comprises an open reading frame encoding SOHLH1.

[0086] In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding ZNF281 and a modified polynucleotide comprising an open reading frame encoding LHX8. In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding ZNF281 and a modified polynucleotide comprising an open reading frame encoding SOHLH1. In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding LHX8 and a modified polynucleotide comprising an open reading frame encoding SOHLH1. In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding ZNF281, a modified polynucleotide comprising an open reading frame encoding LHX8 and a modified polynucleotide comprising an open reading frame encoding SOHLH1.

[0087] In some embodiments, a modified polynucleotide coding for an open reading frame encoding zinc finger protein 281 (ZNF281) (e.g., UniprotKB Accession No. Q9Y2X9) can be used to [ka] It encodes a protein comprising the sequence

[0088] In some embodiments, a modified polynucleotide coding for LIM homeobox 8 (LHX8) (e.g., UniprotKB Accession No. Q68G74) is [ka] It encodes a protein comprising the sequence

[0089] In some embodiments, the modified polynucleotide coding for spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (SOHLH1) (e.g., UniprotKB Accession No. Q6IUP1) comprises an open reading frame encoding: [ka] It encodes a protein comprising the sequence

[0090] The pluripotent stem cells, in some embodiments, further comprise a modified polynucleotide comprising an open reading frame encoding a protein selected from FIGLA, DLX5, and HHEX. In some embodiments, the modified polynucleotide comprises an open reading frame encoding FIGLA. In some embodiments, the modified polynucleotide comprises an open reading frame encoding DLX5. In some embodiments, the modified polynucleotide comprises an open reading frame encoding HHEX.

[0091] In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding FIGLA and a modified polynucleotide comprising an open reading frame encoding DLX5. In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding FIGLA and a modified polynucleotide comprising an open reading frame encoding HHEX. In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding DLX5 and a modified polynucleotide comprising an open reading frame encoding HHEX. In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding FIGLA, a modified polynucleotide comprising an open reading frame encoding DLX5 and a modified polynucleotide comprising an open reading frame encoding HHEX.

[0092] The modified polynucleotide coding for the open reading frame encoding the follicle formation specific BHLH transcription factor (FIGLA) (e.g., UniprotKB Accession No. Q6QHK4) can, in some embodiments, be [ka] It encodes a protein comprising the sequence

[0093] In some embodiments, the modified polynucleotide coding for Distal-Less Homeobox 5 (DLX5) (e.g., UniprotKB Accession No. P56178) comprises an open reading frame encoding: [ka] It encodes a protein comprising the sequence

[0094] The modified polynucleotide coding for the open reading frame encoding the hematopoietic expressed homeobox (HHEX) (e.g., UniprotKB Accession No. Q03014) can, in some embodiments, be [ka] It encodes a protein comprising the sequence

[0095] The pluripotent stem cells, in some embodiments, further comprise a modified polynucleotide comprising an open reading frame encoding a protein selected from DDX4, DAZL, and BOLL. In some embodiments, the modified polynucleotide comprises an open reading frame encoding DDX4. In some embodiments, the modified polynucleotide comprises an open reading frame encoding DAZL. In some embodiments, the modified polynucleotide comprises an open reading frame encoding BOLL.

[0096] In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding DDX4 and a modified polynucleotide comprising an open reading frame encoding DAZL. In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding DDX4 and a modified polynucleotide comprising an open reading frame encoding BOLL. In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding DAZL and a modified polynucleotide comprising an open reading frame encoding BOLL. In some embodiments, the pluripotent stem cells comprise a modified polynucleotide comprising an open reading frame encoding DDX4, a modified polynucleotide comprising an open reading frame encoding DAZL and a modified polynucleotide comprising an open reading frame encoding BOLL.

[0097] In some embodiments, a modified polynucleotide coding for DEAD box polypeptide 4 (DDX4) (e.g., UniprotKB Accession No. Q9NQI0) is [ka] It encodes a protein comprising the sequence

[0098] In some embodiments, a modified polynucleotide coding for Deleted in AZoospermia (DAZL) (e.g., UniprotKB Accession No. Q92904) is [ka] It encodes a protein comprising the sequence

[0099] A modified polynucleotide coding for the open reading frame encoding Boule homolog (BOLL) (e.g., UniprotKB Accession No. Q8N9W6) is, in some embodiments, [ka] It encodes a protein comprising the sequence

[0100] The number of copies of the modified polynucleotide delivered to the PSC can vary. In some embodiments, the PSC comprises 1-20 copies of the modified polynucleotide. For example, the PSC can comprise 1-15, 1-10, 2-10, 2-15, 2-10, 5-20, 5-15, or even 5-10 copies of the modified polynucleotide. In some embodiments, the PSC comprises 8-10 copies of the modified polynucleotide. More than 20 copies are also contemplated herein.

[0101] Methods for Producing Oogonia-Like Cells Methods of producing oogonia-like cells provided herein, in some aspects, include culturing a population of pluripotent stem cells (PSCs) in a culture medium to produce an expanded population of PSCs, and expressing a protein selected from ZNF281, LHX8, and SOHLH1 in the expanded population of PSCs to produce oogonia-like cells.

[0102] In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide comprising an open reading frame encoding ZNF281. In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide comprising an open reading frame encoding LHX8. In some embodiments, the PSCs of the expanded population comprise a modified polynucleotide comprising an open reading frame encoding SOHLH1. In some embodiments, the PSCs of the expanded population further comprise a modified polynucleotide comprising an open reading frame encoding a FIGLA protein. In some embodiments, the PSCs of the expanded population further comprise a modified polynucleotide comprising an open reading frame encoding a DLX5 protein and a modified polynucleotide comprising an open reading frame encoding a HHEX protein. In some embodiments, the PSCs of the expanded population further comprise a modified polynucleotide comprising an open reading frame encoding a DDX4 protein, a modified polynucleotide comprising an open reading frame encoding a DAZL protein, and a modified polynucleotide comprising an open reading frame encoding a BOLL protein.

[0103] In some embodiments, the open reading frame of the modified polynucleotide is operably linked to a heterologous promoter.

[0104] In some embodiments, the heterologous promoter is an inducible promoter, non-limiting examples of which are provided elsewhere herein.

[0105] The starting population was approximately 1 × 10 2 ~1×10 10 , about 1×10 2 ~1×10 9 , about 1×10 2 ~1×10 8 Or about 1 x 10 2 ~1×10 7 In some embodiments, the population comprises about 1 x 10 PSCs. 3 ~1×10 8 Or about 1 x 10 3 ~1×10 7 In some embodiments, the population comprises about 1 x 10 PSCs. 4 ~1×10 7 Or about 1 x 10 5 ~1×10 6 In some embodiments, the population comprises about 1 x 10 PSCs. 1 PSCs, approximately 1 x 10 2 PSCs, approximately 1 x 10 3 PSCs, approximately 1 x 10 4 PSCs, approximately 1 x 10 5 PSCs, approximately 1 x 10 6 PSCs, approximately 1 x 10 7 PSCs, approximately 1 x 10 8 PSCs, approximately 1 x 10 9 PSCs or approximately 1 x 10 10 Includes PSCs.

[0106] In some embodiments, the population of PSCs is cultured for about 2 to about 6 days, about 2 to about 5 days, about 2 to about 4 days, about 3 to about 6 days, about 3 to about 5 days, or about 3 to about 4 days. In some embodiments, the population of PSCs is cultured for about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days.

[0107] Some methods of the disclosure provide methods comprising: (a) delivering to PSCs a modified polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from ZNF281, LHX8 and SOHLH1 (or selected from ZNF281, LHX8, SOHLH1, FIGLA, DLX5, HHEX, DDX4, DAZL and BOLL); (b) culturing the PSCs in a feeder-free serum-free culture medium to produce an expanded population of PSCs; and (c) culturing the expanded population of PSCs in a series of induction media comprising an inducer to produce DDX4+ oogonia-like cells. In some embodiments, the series of induction media comprises a first, second, third and fourth induction media.

[0108] In some embodiments, the PSCs are cultured in a feeder-free serum-free culture medium for about 6 to about 24 hours. For example, the PSCs may be cultured in a feeder-free serum-free culture medium for about 6 to about 12 hours. In some embodiments, the PSCs are cultured in a feeder-free serum-free culture medium for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.

[0109] In some embodiments, the expanded population of PSCs comprises at least 5×10 3 For example, an expanded population (e.g., upon induction) may contain at least 1 x 10 PSCs. 4 , at least 1 × 10 5 , at least 1 × 10 6 Or at least 1 × 10 7 In some embodiments, the expanded population of PSCs may comprise about 5×10 3 Approximately 1 × 10 PSCs 7 Includes PSCs.

[0110] In some embodiments, the expanded population of PSCs comprises about 2,000 cells / cm. 2to approximately 3,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 500 cells / cm. 2 ~10000 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 1000 cells / cm. 2 ~9500 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 1500 cells / cm. 2 ~9000 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 2000 cells / cm. 2 ~8500 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 2500 cells / cm. 2 ~8000 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 3000 cells / cm. 2 ~7500 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 3500 cells / cm. 2 ~7000 pieces / cm 2 In some embodiments, the population is cultured at a density of PSCs of 4000 cells / cm. 2 ~6500 pieces / cm 2 In some embodiments, the expanded population of PSCs comprises about 4500 PSCs / cm. 2 ~6000 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 5000 cells / cm. 2 ~5500 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of at least 500 cells / cm. 2 PSCs, at least 1000 pieces / cm 2 PSC, at least 1500 pieces / cm 2 PSCs, at least 2000 pieces / cm 2 PSC, at least 2500 pieces / cm 2PSCs, at least 3000 pieces / cm 2 PSC, at least 3500 particles / cm 2 PSCs, at least 4000 pieces / cm 2 PSCs, at least 4500 particles / cm 2 PSCs, at least 5000 pieces / cm 2 PSC, at least 5500 particles / cm 2 PSCs, at least 6000 pieces / cm 2 PSCs, at least 6500 particles / cm 2 PSCs, at least 7000 / cm 2 PSCs, at least 7500 particles / cm 2 PSCs, at least 8000 pieces / cm 2 PSCs, at least 8500 particles / cm 2 PSCs, at least 9000 particles / cm 2 PSCs, at least 9500 particles / cm 2 PSC or at least 10,000 particles / cm 2 PSCs are cultured at a density of 100x.

[0111] In some embodiments, the PSCs of the expanded population are cultured for 8 days or less, 7 days or less, 6 days or less, 5 days or less, or 4 days or less. For example, the PSCs of the expanded population can be cultured for about 2 to about 8 days, about 2 to about 7 days, about 2 to about 6 days, about 2 to about 5 days, about 2 to about 4 days, about 3 to about 8 days, about 3 to about 7 days, about 3 to about 6 days, about 3 to about 5 days, or about 3 to about 4 days. In some embodiments, the PSCs of the expanded population are cultured for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days.

[0112] In some embodiments, the PSCs of the expanded population are cultured in the first induction medium for about 6 to about 36 hours. For example, the PSCs can be cultured in the first induction medium for about 6 to about 24 hours, about 6 to about 18 hours, about 6 to about 12 hours, 12 to about 36 hours, about 12 to about 24 hours, about 12 to about 18 hours, 18 to about 36 hours, or about 18 to about 24 hours. In some embodiments, the PSCs are cultured in the first induction medium for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours.

[0113] In some embodiments, the PSCs of the expanded population are cultured in the second induction medium for about 6 to about 36 hours. For example, the PSCs can be cultured in the second induction medium for about 6 to about 24 hours, about 6 to about 18 hours, about 6 to about 12 hours, 12 to about 36 hours, about 12 to about 24 hours, about 12 to about 18 hours, 18 to about 36 hours, or about 18 to about 24 hours. In some embodiments, the PSCs are cultured in the second induction medium for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours.

[0114] In some embodiments, the PSCs of the expanded population are cultured in the third induction medium for about 6 to about 36 hours. For example, the PSCs can be cultured in the third induction medium for about 6 to about 24 hours, about 6 to about 18 hours, about 6 to about 12 hours, 12 to about 36 hours, about 12 to about 24 hours, about 12 to about 18 hours, 18 to about 36 hours, or about 18 to about 24 hours. In some embodiments, the PSCs are cultured in the third induction medium for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours.

[0115] In some embodiments, the PSCs of the expanded population are cultured in the fourth induction medium for about 6 to about 36 hours. For example, the PSCs can be cultured in the fourth induction medium for about 6 to about 24 hours, about 6 to about 18 hours, about 6 to about 12 hours, 12 to about 36 hours, about 12 to about 24 hours, about 12 to about 18 hours, 18 to about 36 hours, or about 18 to about 24 hours. In some embodiments, the PSCs are cultured in the fourth induction medium for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours.

[0116] In some embodiments, the PSCs are incubated for at least 6 hours. In some embodiments, after incubation, the medium is removed from the plate and the plate is washed with DMEM / F12. In some embodiments, Medium #1 (Table 1) is added at 250 μL / cm 2In some embodiments, the iPSCs are incubated for 18 hours before removing medium #1 and adding medium #2 (Table 1). In some embodiments, the iPSCs are incubated for 24 hours before removing medium #2 and adding medium #3 (Table 1). In some embodiments, the iPSCs are incubated for 18 hours before removing medium #3 and adding medium #4 (Table 1). In some embodiments, after incubating the iPSCs with medium #4, there are DDX4+ oogonia-like cells in the plate.

[0117] Transfection methods The modified polynucleotides of the present disclosure may be delivered to the PSCs using any one or more transfection methods, including chemical transfection methods, viral transduction methods, and electroporation methods.

[0118] In some embodiments, the modified polynucleotide is delivered in a vector. A vector is any vehicle, such as a virus or a plasmid, used to transfer a desired polynucleotide into a host cell, such as a PSC. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is not a naturally occurring viral vector. The viral vector can be derived from adeno-associated virus (AAV), adenovirus, herpes simplex virus, lentivirus, retrovirus, varicella virus, smallpox virus, hepatitis B virus, cytomegalovirus, JC polyoma virus, BK polyoma virus, monkeypox virus, varicella zoster virus, Epstein-Barr virus, human herpes virus type 7, Kaposi's sarcoma associated herpes virus, or human parvovirus B19. Other viral vectors are encompassed by the present disclosure.

[0119] In some embodiments, the viral vector is an AAV vector. AAV is a small non-enveloped virus that packages a single-stranded linear DNA genome of approximately 5 kb length, adapted for use as a gene transfer vehicle (Samulski, RJ et al., Annu Rev Virol. 2014; 1(1):427-51). The coding region of AAV is flanked by inverted terminal repeats (ITRs), which act as DNA replication origins and serve as primary packaging signals (McLaughlin, SK et al. Virol. 1988; 62(6): 1963-73;Hauswirth, WW et al. 1977; 78(2):488-99). For this reason, AAV vectors typically contain ITR sequences. Both the positive and negative strands are packaged equally well into virions and are infectious (Zhong, L et al. Mol Ther. 2008; 16(2):290-5; Zhou, X et al. Mol Ther. 2008; 16(3):494-9; Samulski, RJ et al. Virol. 1987; 61(10):3096-101). In addition, a small deletion in one of the two ITRs allows packaging of a self-complementary vector, and the genome self-anneals after viral uncoating. This results in more efficient transduction of cells, but reduces the coding capacity by half (McCarty, DM et al. Mol Ther. 2008; 16(10): 1648-56; McCarty, DM et al. Gene Ther. 2001; 8(16): 1248-54).

[0120] In some embodiments, polynucleotides are delivered to cells using a transposon / transposase system. For example, the piggyBac™ transposon system can be used. The piggyBac™ transposon is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism (Woodard et al. 2015). During transposition, the piggyBac™ transposase recognizes transposon-specific inverted terminal repeats (ITRs) located at both ends of the transposon vector, efficiently displacing the contents from the original site and integrating it into the TTAA chromosomal site. The piggyBac™ transposon system facilitates efficient integration of polynucleotides into the cell genome.

[0121] Thus, in some embodiments, the method further comprises delivering a transposon comprising the modified polynucleotide to the PSC, and also delivering a transposase.

[0122] In some embodiments, modified polynucleotide is delivered to cell by electroporation.Electroporation is a physical transfection method that uses electric pulse to create temporary holes in cell membrane, through which modified polynucleotide can enter cell.See, for example, Chicaybam L et al. Front. Bioeng. Biotechnol., 23 January 2017.

[0123] After transfection, the modified polynucleotide may be integrated into the genome of the PSC. In some embodiments, the modified polynucleotide may further comprise an antibiotic resistance gene that confers resistance to an antibiotic used in the antibiotic drug selection process. In this way, a "pure" cell population containing the integrated modified polynucleotide may be obtained. In some embodiments, the cell population containing the integrated modified polynucleotide is selected using antibiotic drug selection. Antibiotic drug selection is a process in which a cell population is treated with an antibiotic such that only cells capable of surviving in the presence of the antibiotic remain in the population. Non-limiting examples of antibiotics that may be used for antibiotic drug selection include puromycin, blasticidin, geneticin, hygromycin, mycophenolic acid, zeocin, carbenicillin, kanemycin, ampicillin, and actinomycin.

[0124] Culture medium The methods provided herein, in some embodiments, include culturing PSCs in a feeder-free, serum-free culture medium. The culture medium can include, for example, a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., Corning® Matrigel® Matrix) (75 to 150 μl / cm 2 (coated with a lot-based diluted suspension of). In some embodiments, the solubilized basement membrane preparation comprises one or more extracellular matrix (ECM) proteins and one or more growth factors. For example, the ECM proteins can be selected from laminin, collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

[0125] In some embodiments, the culture medium further comprises one or more growth factors selected from, for example, recombinant human basic fibroblast growth factor (rh bFGF) (e.g., 80 ng / ml to 120 ng / ml) and recombinant human transforming growth factor beta (rh TGFβ) (e.g., 20 to 25 pM). In some embodiments, the culture medium further comprises rh bFGF and rh TGFβ. In some embodiments, the culture medium comprises mTeSR™ medium (STEMCELL Technologies).

[0126] In some embodiments, the first induction medium comprises one or more (e.g., 2, 3, 4 or more) of B-27 supplement (e.g., 90x to 110x), L-alanyl-L-glutamine (e.g., 1.8 mM to 2.2 mM), an inducer (e.g., doxycycline (e.g., 50 ng / ml to 2000 ng / ml)), activin A (e.g., 50 ng / ml to 150 ng / ml), a glycogen synthase kinase (GSK) 3 inhibitor (e.g., 2.8 μM to 3.2 μM), a selective FGFR1 and FGFR3 inhibitor (e.g., 90 nM to 110 nM), and a small molecule ROCK inhibitor (e.g., 8 μM to 12 μM). In some embodiments, the first induction medium comprises B-27, L-alanyl-L-glutamine, an inducer (e.g., doxycycline), activin A, a glycogen synthase kinase (GSK) 3 inhibitor, and a selective FGFR1 and FGFR3 inhibitor. For example, the first induction medium can comprise aRB27 medium, doxycycline, activin A, CHIR99021, and PD173074.

[0127] In some embodiments, the second induction medium comprises one or more (e.g., 2, 3, 4 or more) of B-27 supplement (e.g., 90x to 110x), an inducer (e.g., doxycycline (e.g., 50ng / ml to 2000ng / ml)), a small molecule inhibitor of tankyrase (TNKS) (e.g., 0.9μM to 1.1μM), and human bone morphogenetic protein 4 (hBMP4) (e.g., 20ng / ml to 250ng / ml). In some embodiments, the second induction medium comprises B-27, an inducer (e.g., doxycycline), a small molecule inhibitor of tankyrase (TNKS), and human bone morphogenetic protein 4 (hBMP4). For example, the second induction medium can comprise aRB27 medium, doxycycline, XAV939, and human bone morphogenetic protein 4 (hBMP4).

[0128] In some embodiments, the third induction medium comprises one or more (e.g., 2, 3, 4 or more) of B-27, an inducer (e.g., doxycycline), a small molecule inhibitor of tankyrase (e.g., 0.9 μM to 1.1 μM), stem cell factor (SCF) (e.g., 25 ng / ml to 200 ng / ml), and epidermal growth factor (EGF) (e.g., 25 ng / ml to 100 ng / ml). In some embodiments, the third induction medium comprises B-27 supplement (e.g., 90× to 110×), an inducer (e.g., doxycycline (e.g., 50 ng / ml to 2000 ng / ml)), a small molecule inhibitor of tankyrase (e.g., 0.9 μM to 1.1 μM), stem cell factor (SCF) (e.g., 25 ng / ml to 200 ng / ml), and epidermal growth factor (EGF) (e.g., 25 ng / ml to 100 ng / ml). For example, the third induction medium can include aRB27 medium, doxycycline, XAV939, SCF and EGF.

[0129] In some embodiments, the fourth induction medium comprises one or more (e.g., 2, 3, 4 or more) of B-27 supplement (90-110x), an inducer (e.g., doxycycline (e.g., 50ng / ml to 2000ng / ml)), a small molecule inhibitor of tankyrase (e.g., 0.9μM to 1.1μM), hBMP4 (e.g., 20ng / ml to 250ng / ml), SCF (e.g., 25ng / ml to 200ng / ml), and EGF (e.g., 25ng / ml to 100ng / ml). In some embodiments, the fourth induction medium comprises B-27, an inducer (e.g., doxycycline), a small molecule inhibitor of tankyrase, hBMP4, SCF, and EGF. For example, the fourth induction medium can comprise aRB27 medium, doxycycline, XAV939, hBMP4, SCF, and EGF.

[0130] As used herein, "aRB27 medium" includes modified RPMI, B-27™ supplement, minus or plus Vitamin A (Thermo Fisher), GlutaMAX™ supplement (Thermo Fisher), non-essential amino acids (NEAA), Primocin® (broad-spectrum antibiotic), and Y-27632 (a small molecule ROCK inhibitor).

[0131] GlutaMAX™ supplement contains L-alanyl-L-glutamine, an alternative dipeptide to L-glutamine.

[0132] Activin-A is a dimeric glycoprotein that belongs to the transforming growth factor-β (TGF-β) family.

[0133] CHIR99021 inhibits GSK3β (IC 50 = 6.7 nM) and GSK3α (IC 50CHIR99021 is an aminopyrimidine derivative that is a highly potent glycogen synthase kinase (GSK) 3 inhibitor that inhibits both vasopressin and vasopressin (vasopressin-1, vasopressin-2, and vasopressin-3) at elevated levels (T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T12, T14, T16, T18, T18, T20, T16, T18, T21, T18 ...

[0134] PD173074 is a selective FGFR1 and FGFR3 inhibitor (IC50 values ​​of approximately 5 nM, approximately 21.5 nM, approximately 100 nM, approximately 17600 nM and approximately 19800 nM for FGFR3, FGFR1, VEGFR2, PDGFR and c-Src, respectively, and >50000 nM for EGFR, InsR, MEK and PKC).

[0135] XAV939 is a potent small molecule inhibitor of tankyrase (TNKS) 1 and 2 (IC 50 = 11 and 4 nM) (Huang et al.). By inhibiting TNKS activity, XAV939 increases the protein levels of the axin-GSK3β complex and promotes the degradation of β-catenin in SW480 cells, thereby inhibiting downstream effects of the WNT pathway (Huang et al.).

[0136] Therapeutic Compositions and Methods of Use The present disclosure provides, in some embodiments, a therapeutic composition comprising the oogonia-like cells produced herein. In some embodiments, the composition further comprises a pharma- ceutically acceptable excipient. The composition, in some embodiments, is cryopreserved.

[0137] Such compositions may be administered to a subject, such as a human subject, using any suitable route of administration. Suitable routes of administration include parenteral routes, such as intravenous, intrathecal, intraparenchymal, intraventricular routes, etc. Suitable routes of administration include parenteral routes, such as intravenous, intrathecal, intraparenchymal, intraventricular injection, etc.

[0138] In some embodiments, the subject is a human subject. The subject may have an infertility disorder, such as Turner syndrome. The infertility disorder includes disorders in which germ cell development is severely affected in female fetuses. Blood sample analysis can be used to diagnose the infertility disorder. In some embodiments, the infertility disorder can include oogonia damage.

[0139] The composition may be administered to a subject in a therapeutically effective amount. The term "therapeutically effective amount" refers to the amount of oogonia necessary to confer a therapeutic effect on a subject, either alone or in combination with at least one other active agent. The effective amount varies depending on the route of administration, excipient use, and co-use of other active agents, as will be appreciated by those skilled in the art. The dosage depends on the subject being treated, including, for example, the strength of the individual's immune system or genetic predisposition. Suitable dosage ranges can be readily determined by those skilled in the art and may be on the order of micrograms for the polypeptides of the present disclosure. The dosage of the preparations disclosed herein may depend on the route of administration and vary depending on the size of the subject.

[0140] It is believed that a person skilled in the art can utilize the present invention to its fullest extent based on the above description. The following specific embodiments are therefore to be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications cited in this application are incorporated by reference for the purpose or subject matter referred to in this disclosure.

[0141] Additional Embodiments The present disclosure also relates to additional embodiments set forth in the following numbered paragraphs.

[0142] 1. A pluripotent stem cell (PSC) comprising a modified polynucleotide comprising an open reading frame encoding a protein selected from zinc finger protein 281 (ZNF281), LIM homeobox 8 (LHX8) and spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (SOHLH1).

[0143] 2. The PSC of paragraph 1, comprising a modified polynucleotide comprising an open reading frame encoding ZNF281.

[0144] 3. A PSC according to paragraph 1 or 2, comprising a modified polynucleotide comprising an open reading frame encoding LHX8.

[0145] 4. The PSC of any one of the above paragraphs, comprising a modified polynucleotide comprising an open reading frame encoding SOHLH1.

[0146] 5. A PSC according to any one of the above paragraphs which expresses or overexpresses ZNF281; LHX8; SOHLH1; ZNF281 and LHX8; ZNF281 and SOHLH1; LHX8 and SOHLH1; or ZNF281, LHX8 and SOHLH1.

[0147] 6. The PSC of any one of the above paragraphs, further comprising a modified polynucleotide comprising an open reading frame encoding a follicle formation-specific BHLH transcription factor (FIGLA) protein, and optionally expressing or overexpressing FIGLA.

[0148] 7. The PSC of paragraph 6, further comprising a modified polynucleotide comprising an open reading frame encoding a Distal-Less Homeobox 5 (DLX5) protein, and a modified polynucleotide comprising an open reading frame encoding a Hematopoietic Expressed Homeobox (HHEX) protein, and optionally expressing or overexpressing the DLX5 protein and the HHEX protein.

[0149] 8. The PSC of paragraph 7, further comprising a modified polynucleotide comprising an open reading frame encoding a DDX4 protein, a modified polynucleotide comprising an open reading frame encoding a DAZL protein, and a modified polynucleotide comprising an open reading frame encoding a BOLL protein, and optionally expressing or overexpressing the DDX4 protein, the DAZL protein and the BOLL protein.

[0150] 9. The PSC of any one of the above paragraphs, wherein the modified polynucleotide open reading frame is operably linked to a heterologous promoter.

[0151] 10. The PSC of paragraph 9, wherein the heterologous promoter is an inducible promoter.

[0152] 11. A pluripotent stem cell (PSC) comprising a protein selected from ZNF281, LHX8 and SOHLH1, wherein the protein is overexpressed.

[0153] 12. A PSC according to paragraph 11, which expresses or overexpresses ZNF281; LHX8; SOHLH1; ZNF281 and LHX8; ZNF281 and SOHLH1; LHX8 and SOHLH1; or ZNF281, LHX8 and SOHLH1.

[0154] 13. The PSC of paragraph 12, further comprising a FIGLA protein, and optionally expressing or overexpressing FIGLA.

[0155] 14. The PSC of paragraph 13, further comprising a DLX5 protein and an HHEX protein, optionally expressing or overexpressing the DLX5 protein and the HHEX protein.

[0156] 15. The PSC according to paragraph 14, further comprising DDX4 protein, DAZL protein and BOLL protein, and optionally expressing or overexpressing DDX4 protein, DAZL protein and BOLL protein.

[0157] 16. The PSCs of any one of the above paragraphs, which are human PSCs.

[0158] 17. The PSC according to any one of the above paragraphs, which is an induced PSC (iPSC).

[0159] 18. A PSC according to any one of the above paragraphs, comprising 1 to 20, optionally 8 to 10 copies of a modified polynucleotide comprising an open reading frame encoding a protein selected from ZNF281, LHX8 and SOHLH1.

[0160] 19. A composition comprising a population of PSCs as described in any one of the above paragraphs or elsewhere herein.

[0161] 20. The population should be at least 2500 cells / cm 2 19. The composition of paragraph 18, comprising a PSC of

[0162] 21. A method comprising culturing a population of pluripotent stem cells (PSCs) in a culture medium to produce an expanded population of PSCs; and expressing a protein selected from ZNF281, LHX8 and SOHLH1 in the expanded population of PSCs to produce oogonia-like cells.

[0163] 22. The method of paragraph 21, wherein the PSCs of the expanded population comprise a modified polynucleotide comprising an open reading frame encoding ZNF281.

[0164] 23. The method of paragraph 21 or 22, wherein the PSCs of the expanded population comprise a modified polynucleotide comprising an open reading frame encoding LHX8.

[0165] 24. The method of any one of paragraphs 21 to 23, wherein the PSCs of the expanded population comprise a modified polynucleotide comprising an open reading frame encoding SOHLH1.

[0166] 25. The method of any one of paragraphs 21 to 24, wherein the PSCs of the expanded population further comprise a modified polynucleotide comprising an open reading frame encoding a FIGLA protein.

[0167] 26. The method of paragraph 25, wherein the PSCs of the expanded population further comprise a modified polynucleotide comprising an open reading frame encoding a DLX5 protein and a modified polynucleotide comprising an open reading frame encoding an HHEX protein.

[0168] 27. The method of paragraph 26, wherein the PSCs of the expanded population further comprise a modified polynucleotide comprising an open reading frame encoding a DDX4 protein, a modified polynucleotide comprising an open reading frame encoding a DAZL protein, and a modified polynucleotide comprising an open reading frame encoding a BOLL protein.

[0169] 28. The method of any one of the above paragraphs, wherein the open reading frame of the modified polynucleotide is operably linked to a heterologous promoter.

[0170] 29. The method of any one of the above paragraphs, wherein the heterologous promoter is an inducible promoter.

[0171] 30. The population is 1 x 10 2 ~1×10 7 The method of any one of the above paragraphs, comprising a PSC.

[0172] 31. The method of any one of the above paragraphs, wherein the population of PSCs is cultured for about 3 to 5 days, optionally for about 4 days.

[0173] 32. The method of paragraph 31, wherein the population of PSCs is cultured for about days.

[0174] 33. Oogon-like cells express DDX4 + 3. The method of any one of the above paragraphs,

[0175] 34. The method of any one of the above paragraphs, wherein the oogonium-like cells independently have a diameter of about 20 micrometers to 180 micrometers.

[0176] 35. (a) delivering to a pluripotent stem cell (PSC) a modified polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from ZNF281, LHX8, and SOHLH1; (b) culturing the PSCs in a feeder-free, serum-free culture medium to generate an expanded population of PSCs; (c) Culturing the expanded population of PSCs in a series of induction media containing inducers to produce DDX4+ oogonia-like cells. The method includes:

[0177] 36. The method of paragraph 35, wherein the modified polynucleotide is a transposon and the delivery further comprises delivering a transposase to the PSC.

[0178] 37. The method of paragraph 35 or 36, wherein the inducible promoter is a chemically inducible promoter, optionally a doxycycline-inducible promoter.

[0179] 38. The method of any one of paragraphs 35 to 37, wherein the feeder-free, serum-free culture medium of (b) comprises a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

[0180] 39. The method of paragraph 38, wherein the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

[0181] 40. The method of paragraph 39, wherein the ECM protein is selected from laminin, collagen IV, heparan sulfate proteoglycan and entactin / nidogen.

[0182] 41. The method according to any one of paragraphs 35 to 40, wherein the feeder-free, serum-free culture medium (b) comprises a growth factor selected from recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ).

[0183] 42. The method of any one of paragraphs 35 to 41, wherein the culturing in (b) is for about 6 to about 24 hours.

[0184] 43.(c) The PSCs in the expanded population have a density of about 2,000 cells / cm2 to about 3,000 cells / cm2. 2 43. The method of any one of paragraphs 35 to 42, wherein the cells are cultured at a density of

[0185] 44. The method of any one of paragraphs 35 to 43, wherein the culturing in (c) includes culturing the PSCs in a first induction medium, culturing the PSCs in a second induction medium, culturing the PSCs in a third induction medium, and culturing the PSCs in a fourth induction medium.

[0186] 45. The method of paragraph 44, wherein the first induction medium comprises one or more of B-27, L-alanyl-L-glutamine, an inducer (e.g., doxycycline), activin A, a glycogen synthase kinase (GSK) 3 inhibitor, and a selective FGFR1 and FGFR3 inhibitor.

[0187] 46. ​​The method of paragraph 44 or 45, wherein the second induction medium comprises one or more of B-27, an inducer (e.g., doxycycline), a small molecule inhibitor of tankyrase (TNKS), and human bone morphogenetic protein 4 (hBMP4).

[0188] 47. The method of any one of paragraphs 44 to 46, wherein the third induction medium comprises one or more of B-27, an inducer (e.g., doxycycline), a small molecule inhibitor of tankyrase, stem cell factor (SCF), and epidermal growth factor (EGF).

[0189] 48. The method of any one of paragraphs 44 to 47, wherein the fourth induction medium comprises one or more of B-27, an inducer (e.g., doxycycline), a small molecule inhibitor of tankyrase, hBMP4, SCF, and EGF.

[0190] 49. An oogonia-like cell produced by the method of any one of the above paragraphs. EXAMPLES

[0191] Working Example Example 1. Three-transcription factor cocktail for inducing DDX4+ oogonia-like cells from hiPSCs Current methods for generating DDX4+ human oogonia-like cells from human induced pluripotent stem cells (hiPSCs) are laborious, expensive, and time-consuming. For example, one such method utilizes co-culture of induced human primordial germ cell-like cells (hPGCLCs) and mouse E12.5 fetal gonad cells (mFGCs) to form a xenogeneic reconstituted ovary (xrOvary), which induces a small population of human DDX4+ oogonia-like cells when grown for approximately 120 days (Yamashiro et al., Science. 2018 Oct 19; 362(6412):356-360). Although this method can generate oogonia-like cells, it requires extensive rodent handling expertise, costly culture techniques, and a lengthy differentiation period. This example describes an efficient and reliable method for generating DDX4+ oogonia-like cells from hiPSCs in 4 days using direct transcription factor (TF) overexpression in parallel with growth factor culture.

[0192] The present disclosure relates to the identification of three transcription factors (ZNF281, LHX8, SOHLH1) that, when overexpressed individually or in combination, drive the formation of DDX4+ oogonia-like cells from hiPSCs.

[0193] A computational algorithm was developed to predict transcription factors involved in human oocyte differentiation to identify three transcription factors (ZNF281, LHX8, and SOHLH1) (Kramme et al. Cell Reports Methods, 100082, 2021). Utilizing the predicted set of 53 transcription factors, we generated a combinatorial pool of TFs across 53 individual hiPSC lines, each carrying one TF, and 42 million unique combinations. Following TF induction, we identified a subset of TFs that drive oocyte-specific gene expression signatures via bulk RNA sequencing (RNA-seq) and single-cell RNA-seq (Figure 1). We then utilized the DDX4-tdTomato hiPSC reporter line to induce human germ cell (hPGCLC) formation using cytokines in parallel with individual TF overexpression. We identified three TFs (ZNF281, LHX8, and SOHLH1) that, when individually overexpressed in hiPSCs, showed the production of a small population of high DDX4-expressing cells (Figure 2A-2B). A combinatorial hiPSC line, designated DDX4-3 (D3), was then constructed, carrying integrated expression vectors of all three TFs, which, when induced during hPGCLC formation, drove a higher yield of DDX4+ cells than individual TF expression (Figure 2C). This high DDX4+ population driven by the three TFs was characterized by bulk RNA-Seq, which showed that overexpression of the combination of the three TFs drove the upregulation of key oocyte-regulated genes (Figure 3A). Through transcriptome comparison with the Human Ovarian Atlas reference, the D3 combination was shown to drive a potent oocyte-like transcriptome signature, as observed in statistically significant TROM classification when compared to secondary and antral oocytes (Figure 3B). Taken together, these results demonstrate that direct overexpression of these three TFs, individually and in combination, is sufficient to generate DDX4+ oogonia-like cells from hiPSCs in 4 days in parallel with hPGCLC differentiation.

[0194] This TF-based high DDX4-expressing cell differentiation protocol is highly scalable and cost-effective (Figure 4). Briefly, TFs were integrated individually or in combination into female hiPSCs as doxycycline-inducible gene expression cassettes using the piggyBac transposase system. Antibiotic drug selection was used to achieve a pure cell population containing the integrated expression cassette. TF-containing hiPSCs were then grown feeder-free in serum-free culture mTeSR™ medium on Corning® Matrigel® matrix.

[0195] To induce oogonia formation, cells were plated at 2,500 cells / cm on Corning® Matrigel® matrix-coated plates in mTeSR™ medium supplemented with approximately 8-10 μM Y-27623 and 0.5-3 μg / mL doxycycline. 2 hiPSCs were seeded at a density of 100-200 μg / ml. After approximately 6 hours, the medium was removed and the plates were briefly washed with DMEM / F12. The medium was then replaced with early mesoderm induction medium (medium #1) listed in Table 1. After approximately 18 hours in medium #1, the medium was removed, the plates were briefly washed again with DMEM / F12, and hPGCLC induction medium was added for 24 hours (medium #2 listed in Table 1). After 24 hours, the medium was replaced with medium #3 and incubated again for 24 hours. Finally, medium #3 was replaced with medium #4 and incubated for 24 hours, after which high DDX4 expressing cells were present in the cell pool. At this point, DDX4+ expressing cells were isolated using sorting techniques and used for downstream analysis.

[0196] FIG. 5 shows the examination of combinations of TFs for oogonia formation. The percentage of cells expressing DDX4 and the percentage of cells expressing NPM2 are shown. For oogonia induction, individual independent expression of SOHLH1, ZNF281 or LHX8 (referred to herein as D3 or D3 combo) all show a yield of DDX4+ cells. Combinatorial overexpression of all three shows a 100-1000 fold increase in DDX4+ oogonia production efficiency. Combinations of two, especially the combination in which LHX8 is present, show a 10-50 fold increase compared to individual overexpression. In addition, the addition of FIGLA to the D3 combo increased the yield 10-fold compared to D3 alone and also induced NPM2+ oocyte-like formation. Similarly, the addition of DLX5, HHEX and FIGLA to the D3 combination was found to show an increase in DDX4+ yield compared to D3 alone (combination referred to herein as D3N3). Finally, additional overexpression of DDX4, DAZL and BOLL in the D3N3 combination showed increased DDX4+ compared to D3 alone (a combination referred to herein as DNR3). D3+HHEX, D3+DLX5, D3+DAZL, D3+DDX4, D3+BOLL and D3+DAZL, DDX4, BOLL did not show increased DDX4+ yield compared to D3 alone.

[0197] Methods and Materials iPSC culture iPSCs were cultured in mTESR1 medium (Stemcell Technologies) on standard polystyrene plates coated with hESC-qualified Corning® Matrigel® matrix. Medium was changed daily. Passages were performed using TRYPLE (Gibco). After each passage, iPSCs were treated with approximately 8-12 μM Y-27632 (Ambeed) for 24 hours. Mycoplasma testing was performed by PCR every 3 months, and all tested cells were negative.

[0198] TF plasmid construction TF cDNAs were synthesized as full-length transcripts or obtained from ORFeome (The ORFeome Colobration, Nat Methods. 13, 191-192 (2016)) as Gateway entry clones. They were cloned into doxycycline-inducible PiggyBac expression plasmids (Addgene #175503) using MegaGate (Kramme et al., STAR Protoc. 2, 100907 (2021)). Final expression constructs were verified by Sanger sequencing, which was also used to determine the barcode sequence for each TF.

[0199] Integration of TF plasmids into hiPSCs An expression plasmid containing TF cDNA under the control of a doxycycline-inducible promoter was integrated into iPSCs using PiggyBac transposase. To perform the integration, approximately 50-100 fmol of TF cDNA plasmid, approximately 150-250 ng PiggyBac transposase expression plasmid, and approximately 100,000-200,000 iPSCs were combined using Lonza P3 buffer and electroporated using a Lonza Nucleofector 4D. After electroporation, cells were seeded in 24-well plates in mTeSR™ Plus medium + approximately 8-12 μM Y-27632. 48 hours after electroporation, selection with the appropriate agent (typically puromycin) was initiated and continued for approximately 3-5 days. Cells were then passaged for approximately 3 days without drug selection to allow for non-integrated plasmid loss. Finally, cells were passaged again under drug selection to generate a pure selected integrant pool. The presence and approximate copy number of integrated TF plasmid was confirmed by qPCR on genomic DNA. In oogonia, hiPSC pools and no single cell selected clones were used. The average copy number was 8-10.

[0200] Protocol for oogonia induction via TF overexpression hiPSCs containing integrated TF expression plasmids were cultured on Corning® Matrigel® matrix in mTeSR™ medium. For monolayer induction, hiPSCs were dissociated into single cells using StemPro™ Accutase™ cell dissociation reagent and grown at 2,500–3,000 cells / cm. 2 The cells were seeded onto Corning® Matrigel® matrix or vitronectin XF coated plates in mTeSR™ medium + about 8-10 μM Y-27632 and about 0.5-3 μg / ml doxycycline at a density of 1000 μg / ml for about 6 hours. The medium was then removed, washed with dPBS or DMEM / F12, and replaced with aRB27 medium #1. After about 12-18 hours of induction, medium #1 was removed, washed with dPBS or DMEM / F12, and replaced with medium #2. After about 24 hours, medium #2 was removed and replaced with medium #3. After about 24 hours, medium #3 was replaced with medium #4. After about 24 hours in medium #4 or additionally after about 2 more days of culture in medium #4 (day 6 of the protocol), oogonia were harvested for use. Oogonia-like cells were isolated via the DDX4 reporter. In addition, oogonia can be generated with similar efficiency compared to monolayer protocols by embryoid body formation via methods established in Yamashiro et al. Science, 362(6412), 356-360, Kobayashi et al., Stem Cell Reports, 9(3), 999-1015, Stem Cell Reports, 9(3), 999-1015, Murase et al., The EMBO Journal, 1-25, 2020 and Mitsunaga et al., Proceedings of the National Academy of Sciences of the United States of America, 114(46), E9913-E9922.

[0201] Protocol for expansion of oogonia-like cells after isolation Expansion of sorted oogon-like cells can be performed via FACS isolation of DDX4+ cells from step 6 on approximately day 4 or 6. Isolated oogonial cells are plated onto Corning® Matrigel® matrix-coated plates in S-CM medium as described by Kobayashi et al. 2022 with Y-27632 and doxycycline. Half of the medium is changed every 2-3 days and cells can be expanded, passaged and re-purified as needed.

[0202] [Table 1]

[0203] [Table 2]

[0204] [Table 3]

[0205] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may include the entire document.

[0206] The indefinite articles "a" and "an," as used in the specification and claims, should be understood to mean "at least one," unless clearly indicated to the contrary.

[0207] It is also to be understood that in any method claimed herein that includes two or more steps or actions, unless clearly indicated to the contrary, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described.

[0208] In the claims and the foregoing specification, transitional phrases such as "comprise," "include," "carry," "have," "contain," "involve," "hold," "comprise," and the like, are all to be understood as open-ended, i.e., to mean inclusive, but not exclusive. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedure.

[0209] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0210] When a range of values ​​is provided, each value therebetween, including the upper and lower limits of the range, is specifically contemplated and described herein.

Claims

1. Pluripotent stem cells (PSCs) containing modified polynucleotides including an open reading frame encoding a protein selected from zinc finger protein 281 (ZNF281), LIM homeobox 8 (LHX8), and spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (SOHLH1).

2. The PSC according to claim 1, expressing or overexpressing ZNF281; LHX8; SOHLH1; ZNF281 and LHX8; ZNF281 and SOHLH1; LHX8 and SOHLH1; or ZNF281, LHX8 and SOHLH1.

3. Further comprising a modified polynucleotide containing an open reading frame encoding a follicle-specific BHLH transcription factor (FIGLA) protein, or Further comprising a modified polynucleotide containing an open reading frame encoding the follicle formation-specific BHLH transcription factor (FIGLA) protein, wherein FIGLA is expressed or overexpressed. The PSC according to claim 1.

4. A modified polynucleotide comprising an open reading frame encoding a distal-less homeobox 5 (DLX5) protein, and a modified polynucleotide comprising an open reading frame encoding a hematopoietic expression homeobox (HHEX) protein, or further comprising such a modified polynucleotide. A modified polynucleotide comprising an open reading frame encoding a distal-less homeobox 5 (DLX5) protein, and a modified polynucleotide comprising an open reading frame encoding a hematopoietic expression homeobox (HHEX) protein, wherein the DLX5 protein and the HHEX protein are expressed or overexpressed. The PSC according to claim 3.

5. A modified polynucleotide containing an open reading frame encoding the DDX4 protein, a modified polynucleotide containing an open reading frame encoding the DAZL protein, and a modified polynucleotide containing an open reading frame encoding the BOLL protein, or A modified polynucleotide comprising an open reading frame encoding the DDX4 protein, a modified polynucleotide comprising an open reading frame encoding the DAZL protein, and a modified polynucleotide comprising an open reading frame encoding the BOLL protein, wherein the DDX4 protein, the DAZL protein, and the BOLL protein are expressed or overexpressed. The PSC according to claim 4.

6. The PSC according to claim 1, wherein the open reading frame of the modified polynucleotide is operably linked to a heterogeneous promoter or an inducible promoter.

7. A pluripotent stem cell (PSC) comprising a protein selected from ZNF281, LHX8, and SOHLH1, wherein the protein is overexpressed in the pluripotent stem cell (PSC).

8. The PSC according to claim 7, expressing or overexpressing ZNF281; LHX8; SOHLH1; ZNF281 and LHX8; ZNF281 and SOHLH1; LHX8 and SOHLH1; or ZNF281, LHX8 and SOHLH1.

9. Further containing FIGLA protein, or Further containing FIGLA protein, expressing or overexpressing FIGLA, The PSC according to claim 8.

10. Further comprising DLX5 protein and HHEX protein, or Further comprising DLX5 protein and HHEX protein, expressing or overexpressing the DLX5 protein and the HHEX protein, The PSC as described in claim 9.

11. Further comprising DDX4 protein, DAZL protein and BOLL protein, or Further comprising DDX4 protein, DAZL protein and BOLL protein, expressing or overexpressing the DDX4 protein, DAZL protein and BOLL protein, The PSC according to claim 10.

12. The PSC according to claim 1, which is a human PSC or an induced PSC (iPSC).

13. The PSC according to claim 1, comprising 1 to 20 or 8 to 10 copies of the modified polynucleotide, which includes the open reading frame encoding the protein selected from ZNF281, LHX8, and SOHLH1.

14. A composition comprising a population of PSCs as described in claim 1, or a population of PSCs as described in claim 1 at a concentration of at least 2,500 per cm².

15. A method comprising culturing a population of pluripotent stem cells (PSCs) in a culture medium to produce an expanded population of PSCs, and expressing a protein selected from ZNF281, LHX8, and SOHLH1 in the PSCs of the expanded population to produce oogonia-like cells.

16. The method according to claim 15, wherein the PSC of the expanded population comprises a modified polynucleotide including an open reading frame encoding a protein selected from ZNF281, LHX8, and SOHLH1.

17. The method according to claim 15, wherein the PSC of the expanded population further comprises a modified polynucleotide containing an open reading frame encoding the FIGLA protein.

18. The method according to claim 17, wherein the PSC of the expanded population further comprises a modified polynucleotide including an open reading frame encoding the DLX5 protein, and a modified polynucleotide including an open reading frame encoding the HHEX protein.

19. The method according to claim 18, wherein the PSC of the expanded population further comprises a modified polynucleotide including an open reading frame encoding the DDX4 protein, a modified polynucleotide including an open reading frame encoding the DAZL protein, and a modified polynucleotide including an open reading frame encoding the BOLL protein.

20. The method according to claim 15, wherein the open reading frame of the modified polynucleotide is operably linked to a heterologous promoter or an inducible promoter.

21. The group comprises 1 × 10² to 1 × 10⁷ PSCs, and / or The aforementioned PSC population is cultured for approximately 3 to 5 days, or approximately 4 days. The method according to claim 15.

22. The aforementioned oogonia-like cells are DDX4 ＋ is, and / or The oogonia-like cells have a diameter of approximately 20 micrometers to 180 micrometers. The method according to claim 15.

23. (a) Delivering a modified polynucleotide to pluripotent stem cells (PSCs) that includes an inducible promoter operably linked to an open reading frame encoding a protein selected from ZNF281, LHX8, and SOHLH1, (b) Culturing the PSCs in a feeder-free, serum-free culture medium to produce an expanded population of PSCs, (c) Culturing the expanded population of PSCs in a series of induction media containing an inducer to produce DDX4+ oogonia-like cells. A method that includes this.

24. The method according to claim 23, wherein the modified polynucleotide is a transposon, and the delivery further comprises delivering a transposase to the PSC.

25. The method according to claim 23, wherein the inducible promoter is a chemically inducible promoter, or optionally a doxycycline-inducible promoter.

26. (b) The feeder-free serum-free culture medium is A soluble basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, and / or Includes growth factors selected from recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ), The method according to claim 23.

27. The method according to claim 26, wherein the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

28. The method according to claim 27, wherein the ECM protein is selected from laminin, collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

29. The method according to claim 23, wherein the culture in (b) is carried out for about 6 to about 24 hours.

30. (c) The culture is (i) Culturing the PSCs at a density of approximately 2,000 cells / cm² to approximately 3,000 cells / cm², and / or (ii) Culturing the PSC in a first induction medium, culturing the PSC in a second induction medium, culturing the PSC in a third induction medium, and culturing the PSC in a fourth induction medium. The method according to claim 23, including the method described in claim 23.

31. The first induction medium comprises B-27, L-alanyl-L-glutamine, an inducer, activin A, a glycogen synthase kinase (GSK) 3 inhibitor, and one or more selective FGFR1 and FGFR3 inhibitors. The second induction medium comprises one or more of B-27, an inducer, a low-molecular-weight inhibitor of tankirase (TNKS), and human bone morphogenetic protein 4 (hBMP4). The third induction medium comprises one or more of B-27, an inducer, a low-molecular-weight inhibitor of tankirase, stem cell factor (SCF), and epidermal growth factor (EGF). The fourth induction medium comprises one or more of B-27, an inducer, a low-molecular-weight tankylase inhibitor, hBMP4, SCF, and EGF. The method according to claim 30.

32. The method according to claim 31, wherein the inducer in the first inducement medium, the second inducement medium, the third inducement medium, and / or the fourth inducement medium comprises doxycycline.

33. Ooocyte-like cells produced by the method described in claim 23.

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