Methods and compositions for producing oogonium-like cells

Abstract

Provided herein are methods and compositions for differentiating induced pluripotent stem cells into oocyte-like cells by overexpressing transcription factors (e.g., ZNF281, LHX8, and / or SOHLH1).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 326,607, filed on April 1, 2022, under 35 U.S.C. §119(e), which is incorporated herein by reference in its entirety.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic sequence listing (H049870757WO00-SEQ-KVC.xml; size: 12,492 bytes; creation date: March 28, 2023) are incorporated herein by reference in their entirety.

[0005] background

[0006] Oogonia are specialized cells that, upon maturation, form primordial follicles in the female fetus. Oogonia proliferate through mitosis and then differentiate into oocytes that participate in sexual reproduction. Oogonia dysfunction underlies many forms of human female infertility, but effective methods for generating oogonia in vitro remain unclear.

[0007] Overview

[0008] 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 (e.g., 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) from PSCs in as little as four days. + oogonia-like cells).

[0009] Some aspects of the present disclosure provide a pluripotent stem cell (PSC) comprising: an engineered polynucleotide comprising an open reading frame encoding a protein selected from the group consisting of ZNF281, LHX8, and SOHLH1.

[0010] In some embodiments, the PSC comprises an engineered polynucleotide comprising an open reading frame encoding ZNF281.

[0011] In some embodiments, the PSC comprises an engineered polynucleotide comprising an open reading frame encoding LHX8.

[0012] In some embodiments, the PSC comprises an engineered polynucleotide comprising an open reading frame encoding SOHLH1.

[0013] In some embodiments, the PSC expresses or overexpresses: ZNF281; LHX8; SOHLH1; ZNF281 and LHX8; ZNF281 and SOHLH1; LHX8 and SOHLH1; or ZNF281, LHX8, and SOHLH1.

[0014] In some embodiments, the PSC further comprises an engineered polynucleotide comprising an open reading frame encoding a folliculogenesis-specific BHLH transcription factor (FIGLA) protein, optionally wherein the PSC expresses or overexpresses FIGLA.

[0015] In some embodiments, the PSC further comprises: an engineered polynucleotide containing an open reading frame encoding a distal less homeobox 5 (DLX5) protein; and an engineered polynucleotide containing an open reading frame encoding a hematopoietic expression homeobox (HHEX) protein, optionally wherein the PSC expresses or overexpresses the DLX5 protein and the HHEX protein.

[0016] In some embodiments, the PSC further comprises: an engineered polynucleotide containing an open reading frame encoding a DEAD-Box polypeptide-4 (DDX4) protein; an engineered polynucleotide containing an open reading frame encoding a missing azoospermia (DAZL) protein; and an engineered polynucleotide containing an open reading frame encoding a Boule homolog (BOLL) protein, optionally wherein the PSC expresses or overexpresses DDX4 protein, DAZL protein and BOLL protein.

[0017] In some embodiments, the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0018] In some embodiments, the heterologous promoter is an inducible promoter.

[0019] Other aspects of the present disclosure provide a PSC comprising: a protein selected from the group consisting of ZNF281, LHX8 and SOHLH1, wherein the protein is overexpressed.

[0020] In some embodiments, the PSC expresses or overexpresses: ZNF281; LHX8; SOHLH1; ZNF281 and LHX8; ZNF281 and SOHLH1; LHX8 and SOHLH1; or ZNF281, LHX8, and SOHLH1.

[0021] In some embodiments, the PSC further comprises FIGLA protein, optionally wherein the PSC expresses or overexpresses FIGLA.

[0022] In some embodiments, the PSC further comprises: a DLX5 protein and a HHEX protein, optionally wherein the PSC expresses or overexpresses the DLX5 protein and the HHEX protein.

[0023] In some embodiments, the PSC further comprises: a DDX4 protein, a DAZL protein, and a BOLL protein, optionally wherein the PSC expresses or overexpresses the DDX4 protein, the DAZL protein, and the BOLL protein.

[0024] In some embodiments, the PSC is a human PSC.

[0025] In some embodiments, the PSC is an induced PSC (iPSC).

[0026] In some embodiments, the PSC comprises 1-20 copies of an engineered polynucleotide comprising an open reading frame encoding a protein selected from ZNF281, LHX8, and SOHLH1. In some embodiments, the PSC comprises 8-10 copies of an engineered polynucleotide comprising an open reading frame encoding a protein selected from ZNF281, LHX8, and SOHLH1.

[0027] Some aspects of the present disclosure provide a composition comprising: a population of PSCs as described in any of the preceding paragraphs or elsewhere herein.

[0028] In some embodiments, the population comprises at least 2500 cells / cm 2 PSC.

[0029] Other aspects of the present disclosure provide 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.

[0030] In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding ZNF281.

[0031] In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding LHX8.

[0032] In some embodiments, PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding SOHLH1.

[0033] In some embodiments, the PSCs of the expanded population further comprise an engineered polynucleotide comprising an open reading frame encoding a FIGLA protein.

[0034] In some embodiments, the PSCs of the expanded population further comprise: an engineered polynucleotide comprising an open reading frame encoding a DLX5 protein; and an engineered polynucleotide comprising an open reading frame encoding a HHEX protein.

[0035] In some embodiments, the PSCs of the expanded population further comprise: an engineered polynucleotide comprising an open reading frame encoding a DDX4 protein; an engineered polynucleotide comprising an open reading frame encoding a DAZL protein; and an engineered polynucleotide comprising an open reading frame encoding a BOLL protein.

[0036] In some embodiments, the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0037] In some embodiments, the heterologous promoter is an inducible promoter.

[0038] In some embodiments, the population comprises 1x10 2 -1x10 7 PSCs.

[0039] In some embodiments, the population of PSCs is cultured for about 3-5 days, optionally about 4 days.

[0040] In some embodiments, the oogonia-like cells are DDX4 + .

[0041] In some embodiments, the oogonia-like cells independently have a diameter of about 20 microns to 180 microns.

[0042] Some aspects of the present disclosure provide a method comprising: (a) delivering an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from the group consisting of ZNF281, LHX8, and SOHLH1 to a PSC; (b) culturing the PSC in a feeder-free, serum-free medium to produce an expanded population of PSCs; and (c) culturing the expanded population of PSCs in a series of induction medium comprising an inducing agent to produce DDX4 + Oogonia-like cells.

[0043] In some embodiments, the engineered polynucleotide is a transposon, and the delivering further comprises delivering a transposase to the PSC.

[0044] In some embodiments, the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

[0045] In some embodiments, the feeder-free, serum-free medium of (b) comprises a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

[0046] In some embodiments, the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

[0047] In some embodiments, the ECM protein is selected from laminin, collagen IV, heparin sulfate proteoglycans, and entactin / nidogen.

[0048] In some embodiments, the feeder-free, serum-free medium of (b) comprises a growth factor selected from recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ).

[0049] In some embodiments, the culturing of (b) continues for about 6 to about 24 hours.

[0050] In some embodiments, the PSCs in the expanded population of (c) are approximately 2,000 cells / cm 2 About 3,000 cells / cm 2 Density culture.

[0051] In some embodiments, the culturing of (c) comprises 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.

[0052] 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.

[0053] 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).

[0054] 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).

[0055] 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.

[0056] Some aspects of the disclosure provide oogonia-like cells produced by the methods of any of the preceding paragraphs or methods described elsewhere herein.

[0057] The details of one or more embodiments of the invention are set forth in the following description. Other features and advantages of the invention will be apparent from the following drawings and detailed description of several embodiments and the appended claims.

[0058] BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The drawings are not drawn to scale. In the drawings, each identical or nearly identical component shown in various figures is represented by a like numeral. For clarity, not every component is labeled in every figure. In the drawings:

[0060] Figure 1 Oogenesis scoring of TFs from scRNA-Seq is shown, where TF enrichment is scored based on the presence of the TF in cells expressing oocyte marker genes.

[0061] Figure 2A-2C We show that the three TFs drive high DDX4 expression when induced during germ cell formation. Figure 2A Shown are flow cytometry data for DDX4 expression in cells not expressing TF (upper left), ZNF281 (upper right), SOHLH1 (lower left), and LHX8 (lower right). Figure 2B The geometric mean expression of DDX4 fluorescence intensity compared to the control is shown. Figure 2C Shown are flow cytometry data of DDX4 expression in cells expressing the D3 combination, showing a high DDX4 oogonia-like signature.

[0062] Figure 3A-3B The D3 TF combination is shown to drive the formation of cells with an oocyte-like transcriptome. Figure 3A RNA-Seq gene expression patterns, shown as log2 fold changes compared to human induced pluripotent stem cell (hiPSC) controls, are shown for select oogenesis signature genes. Figure 3B Shown is a cell type classification analysis (TROM) in which the transcriptome of D3 oogonia-like cells was compared to a reference ovarian atlas, showing statistically significant similarity (defined as greater than TROM=12) to antral and secondary oocytes in vivo.

[0063] Figure 4 Shown is a schematic representation of the method for forming high DDX4-expressing cells from stem cells.

[0064] Figure 5 The TF combinations tested for oogonia formation are shown. The percentage of cells expressing DDX4 and the percentage of cells expressing NPM2 are shown.

[0065] Detailed Description

[0066] Oogonia are essential cells involved in reproduction. Oogonia dysfunction underlies many forms of human female infertility (Garg et al., 2015). Therefore, there is an unmet need for methods to develop methods for generating oogonia and oogonia-like cells. For example, such cells may form the basis for therapeutic interventions for human female infertility. It should be understood that the term "oogonia-like cells" encompasses cells that express oogonia-specific markers (e.g., DDX4) and exhibit other characteristics of natural oogonia.

[0067] Current methods for generating DDX4-positive human oogonia-like cells in vitro (e.g., 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 gonadal cells (mFGCs) to form xenogeneic reconstructed ovaries (xrOvaries), which induce a small population of human DDX4+ oogonia-like cells when grown for more than about 120 days (Yamashiro et al., 2018). While this method is capable of producing DDX4+ oogonia-like cells, it requires a great deal of technical expertise, expensive culture techniques, and a lengthy differentiation period, making the method intractable for most high-throughput applications. Aspects of the present disclosure relate to a method for inducing DDX4+ oogonia-like cells from stem cells within 4 days using direct transcription factor overexpression combined with growth factor culture.

[0068] Oogonia-like cells

[0069] Some aspects of the present disclosure provide oogonia-like cells and methods for producing such cells. Oogonia are small diploid cells that form primordial follicles in female fetuses after maturation. Oogonia are formed in large quantities from primordial germ cells by mitosis in the early stages of fetal development. During human development, primordial germ cells differentiate into oogonia, which further proliferate by mitosis. After proliferation, oogonia differentiate into primary oocytes by asymmetric division. A daughter cell produced by asymmetric division of primary oocytes becomes an oocyte through the process of oogenesis. The oocyte produced by oogenesis eventually undergoes meiosis and participates in sexual reproduction (Sathananthan et al., 2006).

[0070] Oogonia and oocytes produced by oogenesis express the gene DEAD-Box polypeptide-4 (DDX4) (e.g., UniProtKB-Q9NQI0 (DDX4_HUMAN)), which is a putative marker gene for female germ cells (Danny et al., 2021). Therefore, in some embodiments, the oogonia-like cells produced by the methods provided herein are DDX4+ oogonia-like cells (i.e., cells expressing DHX4 protein).

[0071] There are other features of oogonium-like cells that distinguish them from non-oogonium-like cells, including, but not limited to, size. For example, the diameter of an oogonium-like cell can be from about 20 micrometers (μm) to about 180 μm. In some embodiments, the diameter of an oogonium-like cell is from about 20 μm to about 160 μm, from about 20 μm to about 140 μm, from about 20 μm to about 120 μm, from about 20 μm to about 100 μm, from about 40 μm to about 180 μm, from about 40 μm to about 160 μm, from about 40 μm to about 140 μm, from about 40 μm to about 120 μm, from about 40 μm to about 100 μm, from about 60 μm to about 180 μm, from about 60 μm to about 160 μm. m, about 60 μm to about 140 μm, about 60 μm to about 120 μm, about 60 μm to about 100 μm, about 80 μm to about 180 μm, about 80 μm to about 160 μm, about 80 μm to about 140 μm, about 80 μm to about 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.

[0072] Pluripotent stem cells

[0073] The oogonia-like cells provided herein are differentiated from pluripotent stem cells. Pluripotent stem cells are cells with the following abilities: self-renewal by division and the ability to develop into the three major germ cell layers of the early embryo (e.g., ectoderm, endoderm, and mesoderm), and therefore can develop into all cells of the human body, excluding extraembryonic tissues such as the placenta (Shi et al., 2017).

[0074] Non-limiting examples of pluripotent stem cells include induced pluripotent cells (iPSCs), "true" embryonic stem cells (ESCs) derived from embryos, embryonic stem cells made 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.

[0075] In some embodiments, the pluripotent stem cell is an embryonic stem cell, such as a human embryonic stem cell. "Embryonic stem cell" is a general term for pluripotent stem cells made using embryos or eggs, rather than cells genetically reprogrammed from the body. As used herein, "ESC" includes true ESC, ntESC and pESC.

[0076] 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, enabling the development of an unlimited source of any type of human cell.

[0077] Some aspects of the present disclosure provide a PSC comprising: a protein selected from ZNF281, LHX8, and SOHLH1, wherein the protein is expressed or overexpressed. In some embodiments, the expression level of the protein is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or at least 100% higher than the control level. In some embodiments, the control level is the endogenous level of the protein, such as in a naturally 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.

[0078] The data provided herein show that the combined expression of two proteins selected from ZNF281, LHX8 and SOHLH1 results in a 10-50 fold increase in the efficiency of DDX4+ oogonia-like cell generation compared to a control, optionally wherein the control is the efficiency of DDX4+ oogonia-like cell generation in a PSC expressing only one of ZNF281, LHX8 or SOHLH1. In some embodiments, the PSC comprises ZNF281 and LHX8. In some embodiments, the PSC expresses or overexpresses ZNF281 and LHX8. In some embodiments, the PSC comprises ZNF281 and SOHLH1. In some embodiments, the PSC expresses or overexpresses ZNF281 and SOHLH1. In some embodiments, the PSC comprises LHX8 and SOHLH1. In some embodiments, the PSC expresses or overexpresses LHX8 and SOHLH1.

[0079] The data provided herein also show that the combined expression of ZNF281, LHX8 and SOHLH1 in PSCs results in a 100-1000-fold increase in the efficiency of DDX4+ oogonia-like cell generation compared to a control, optionally wherein the control is the efficiency of DDX4+ oogonia-like cell generation in a PSC expressing only one of ZNF281, LHX8 or SOHLH1. In some embodiments, the PSC comprises ZNF281, LHX8 and SOHLH1. In some embodiments, the PSC expresses or overexpresses ZNF281, LHX8 and SOHLH1.

[0080] The data provided herein further demonstrate that the combined expression of FIGLA, ZNF281, LHX8, and SOHLH1 in PSCs results in an approximately 10-fold increase in the efficiency of DDX4+ oogonia-like cell generation compared to a control, optionally wherein the control is the efficiency of DDX4+ oogonia-like cell generation in PSCs expressing only ZNF281, LHX8, and SOHLH1. In some embodiments, the cells generated express NPM2, an oocyte-like marker. In some embodiments, the PSCs further comprise FIGLA. In some embodiments, the PSCs further express or overexpress FIGLA.

[0081] The data provided herein further demonstrate that the combined expression of FIGLA, DLX5, HHEX, ZNF281, LHX8, and SOHLH1 in PSCs results in an increased efficiency of DDX4+ oogonia-like cell generation compared to a control, optionally wherein the control is the efficiency of DDX4+ oogonia-like cell generation in a PSC expressing only ZNF281, LHX8, and SOHLH1. In some embodiments, the PSC further comprises DLX5. In some embodiments, the PSC further expresses or overexpresses DLX5. In some embodiments, the PSC further comprises HHEX. In some embodiments, the PSC further expresses or overexpresses HHEX.

[0082] The data provided herein further demonstrate that the combined expression of DDX4, DAZL, BOLL, FIGLA, DLX5, HHEX, ZNF281, LHX8, and SOHLH1 in a PSC results in an increased efficiency of DDX4+ oogonia-like cell generation compared to a control, optionally wherein the control is the efficiency of DDX4+ oogonia-like cell generation in a PSC expressing only ZNF281, LHX8, and SOHLH1. In some embodiments, the PSC further comprises DDX4. In some embodiments, the PSC further expresses or overexpresses DDX4. In some embodiments, the PSC further comprises DAZL. In some embodiments, the PSC further expresses or overexpresses DAZL. In some embodiments, the PSC further comprises BOLL. In some embodiments, the PSC further expresses or overexpresses BOLL.

[0083] Surprisingly, expression of only one of HHEX, DLX5, DAZL, DDX4, BOLL or DAZL in combination with ZNF281, LHX8 and SOHLH1 did not show increased efficiency of DDX4+ oogonia-like cell generation in PSCs.

[0084] Transcription Factors

[0085] In some embodiments, the oogonium-like cells provided herein 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 transcription rate). Differentiation is the process by which uncommitted cells or partially-committed cells are directed to a specialized cell fate. Aspects of the present disclosure relate to the differentiation of uncommitted pluripotent stem cells to oogonium-like cell fate. In some embodiments, the transcription factor is selected from ZNF281, LHX8 and SOHLH1. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress ZNF281. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress LHX8. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress SOHLH1. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress ZNF281 and LHX8. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are engineered to express or overexpress ZNF281 and SOHLH1. In some embodiments, pluripotent stem cells (such as hPSCs or hiPSCs) are engineered to express or overexpress LHX8 and SOHLH1. In some embodiments, pluripotent stem cells (such as hPSCs or hiPSCs) are engineered to express or overexpress ZNF281, LHX8, and SOHLH1.

[0086] A cell "expresses" a particular protein if the level of the protein in the cell is detectable (e.g., using a known protein assay). A cell "overexpresses" a particular protein (e.g., an engineered polynucleotide encoding the protein) if the level of the protein is greater (e.g., at least 5% greater, at least 10% greater, or at least 20% greater) than the level of the protein expressed by an endogenous, naturally occurring polynucleotide encoding the protein.

[0087] In some embodiments, the transcription factor is selected from FIGLA, DLX5 and HHEX. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress FIGLA. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress DLX5. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress HHEX. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress FIGLA and DLX5. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress FIGLA and HHEX. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress DLX5 and HHEX. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress FIGLA, DLX5 and HHEX. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress ZNF281, LHX8, SOHLH1, and FIGLA. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress ZNF281, LHX8, SOHLH1, FIGLA, DLX5, and HHEX.

[0088] In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress DDX4. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress DAZL. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress BOLL. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress DDX4 and DAZL. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress DDX4 and BOLL. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress DAZL and BOLL. In some embodiments, pluripotent stem cells (e.g., hPSC or hiPSC) are engineered to express or overexpress DDX4, DAZL, and BOLL.

[0089] In some embodiments, pluripotent stem cells (eg, hPSCs or hiPSCs) are engineered to express or overexpress ZNF281, LHX8, SOHLH1, FIGLA, DLX5, HHEX, DDX4, DAZL, and BOLL.

[0090] Engineered polynucleotides and peptides

[0091] In some embodiments, the pluripotent stem cells of the present disclosure include engineered polynucleotides. Engineered polynucleotides are nucleic acids that do not exist in nature (e.g., at least two nucleotides are covalently linked together, and in some cases, contain phosphodiester bonds, referred to as phosphodiester backbones). Engineered polynucleotides include recombinant nucleic acids and synthetic nucleic acids. Recombinant nucleic acids are molecules constructed by connecting nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or combinations thereof) from two different organisms (e.g., humans and mice). Synthetic nucleic acids are molecules that are amplified or chemically synthesized or otherwise synthesized. Synthetic nucleic acids include those nucleic acids that are chemically modified or otherwise modified but can be base paired (bound) with naturally occurring nucleic acid molecules. Recombinant nucleic acids and synthetic nucleic acids also include those molecules produced by replication of any of the above.

[0092] Engineered polynucleotides can include DNA (e.g., genomic DNA, cDNA, or a combination of genomic DNA and cDNA), RNA, or hybrid molecules, for example, wherein the nucleic acid comprises any combination of (e.g., artificial or natural) deoxyribonucleotides and ribonucleotides, and any combination of two or more bases (including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine).

[0093] In some embodiments, the polynucleotide is complementary DNA (cDNA).cDNA is synthesized from a single-stranded RNA (eg, messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.

[0094] The engineered polynucleotides of the present disclosure can be generated using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, nucleic acids are generated using GIBSON Clonal generation (see, eg, 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). GIBSON Three enzyme activities are usually used in a single tube reaction: 5' exonuclease, 3' extension activity of DNA polymerase and DNA ligase activity. The 5' exonuclease activity digests the 5' end sequence (chew back) and exposes the complementary sequence for annealing. The polymerase activity then fills the gap on the annealed domain. The DNA ligase then seals the nick and covalently connects the DNA fragments together. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate Assembly, thus resulting in a higher percentage of correct assembly. According to the present disclosure, other methods for producing engineered polynucleotides can be used.

[0095] In some embodiments, the engineered polynucleotide comprises a promoter operably connected to an open reading frame. A promoter is a nucleotide sequence (e.g., ATG) to which RNA polymerase binds to initiate transcription. A promoter is generally located directly upstream of the transcription start site (at the 5' end of the transcription start site). In some embodiments, a promoter is a heterologous promoter. A heterologous promoter is not naturally associated with the open reading frame to which it is operably connected.

[0096] In some embodiments, the promoter is an inducible promoter. An inducible promoter can be regulated in vivo by, for example, chemicals, temperature, or light. An inducible promoter can achieve, for example, temporal and / or spatial control of gene expression. Inducible promoters used according to the present disclosure include any inducible promoters described herein or known to those of ordinary skill in the art. Examples of inducible promoters include, but are not limited to, chemically / biochemically regulated and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems, which include tetracycline repressor protein (tetR), tetracycline operator sequence (tetO), and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptor, and promoters from the steroid / retinoic acid / thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters from metallothionein (proteins that bind and sequester metal ions) genes derived from yeast, mouse, and human), pathogenicity-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 (active in vivo, not regulated).

[0097] An open reading frame is a continuous segment of codons that begins with a start codon (e.g., ATG) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide, e.g., a protein. An open reading frame is operably linked to a promoter if the promoter regulates the transcription of the open reading frame.

[0098] Vectors for delivering engineered polynucleotides include minicircles, plasmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes. Transposon-based systems, such as piggyBac, are also contemplated herein. TM system (e.g. Chen et al. Nature Communications. 2020; 11(1): 3446).

[0099] In some embodiments, the pluripotent stem cell comprises an engineered polynucleotide comprising an open reading frame encoding a protein selected from ZNF281, LHX8, and SOHLH1. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding ZNF281. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding LHX8. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding SOHLH1.

[0100] In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding ZNF281 and an engineered polynucleotide containing an open reading frame encoding LHX8. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding ZNF281 and an engineered polynucleotide containing an open reading frame encoding SOHLH1. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding LHX8 and an engineered polynucleotide containing an open reading frame encoding SOHLH1. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding ZNF281, an engineered polynucleotide containing an open reading frame encoding LHX8, and an engineered polynucleotide containing an open reading frame encoding SOHLH1.

[0101] In some embodiments, an engineered polynucleotide comprising an open reading frame encoding zinc finger protein 281 (ZNF281) (e.g., UniprotKB Accession No. Q9Y2X9) encodes a protein comprising the following sequence:

[0102] (SEQ ID NO:1)

[0103] In some embodiments, an engineered polynucleotide comprising an open reading frame encoding LIM homeobox 8 (LHX8) (e.g., UniprotKB Accession No. Q68G74) encodes a protein comprising the following sequence:

[0104] MQILSRCQGLMSEECGRTTALAAGRTRKGAGEEGLVSPEGAGDEDSCSSSAPLSPSSSPRSMASGSGCPPGKCVCNSCGLEIVDKYLLKVNDLCWHVRCLSCSVCRTSLGRHTSCYIKDKDIFCKLDYFRRYGTRCSRCGRHIHSTDWVRRAKGNVYHLACFACFSCKRQLSTGEEFALV EEKVLCRVHYDCMLDNLKREVENGNGISVEGALLTEQDVNHPKPAKRARTSFTADQLQVMQAQFAQDNNPDAQTLQKLAERTGLSRRVIQVWFQNCRARHKKHVSPNHSSSTPVTAVPPSRLSPPMLEEMAYSAYVPQDGTMLTALHSYMDAHSPTTLGLQPLLPHSMTQLPISHT(SEQ ID NO:2)

[0105] In some embodiments, the engineered polynucleotide comprising an open reading frame encoding spermatogenesis and oogenesis specific basic helix-loop-helix 1 (SOHLH1) (e.g., UniprotKB Accession No. Q6IUP1) encodes a protein comprising the following sequence:

[0106] MASGGHERANEDYRVSGITGCSKTPQPETQDSLQTSSQSSALCTAPVAAANLGPSLRRNVVSERERRRRISLSCEHLRALLPQFDGRREDMASVLEMSVYFLQLAHSMDPSWEQLSVPQPPQEMWHMWQGDVLQVTLANQIADSKPDSGIAKPSAVSRVQDPPCFGMLDTDQSQATERES ELLERPSSCPGHRQSALSFSEPESSSLGPGLPPWIPHSWQPATPEASDIVPGGSHQVASLAGDPESSGMLAEEANLVLASVPDARYTTGAGSDVVDGAPFLMTTNPDWWLGSVEGRGGPALARSSPVDGAEPSFIGDPELCSQELQAGPGELWGLDFGSPGLALKDEADSIFPDFFP(SEQ ID NO:3)

[0107] In some embodiments, the pluripotent stem cell further comprises an engineered polynucleotide comprising an open reading frame encoding a protein selected from FIGLA, DLX5 and HHEX. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding FIGLA. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding DLX5. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding HHEX.

[0108] In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding FIGLA and an engineered polynucleotide containing an open reading frame encoding DLX5. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding FIGLA and an engineered polynucleotide containing an open reading frame encoding HHEX. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding DLX5 and an engineered polynucleotide containing an open reading frame encoding HHEX. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding FIGLA, an engineered polynucleotide containing an open reading frame encoding DLX5, and an engineered polynucleotide containing an open reading frame encoding HHEX.

[0109] In some embodiments, the engineered polynucleotide comprising an open reading frame encoding a folliculogenesis-specific BHLH transcription factor (FIGLA) (e.g., UniprotKB Accession No. Q6QHK4) encodes a protein comprising the following sequence:

[0110] MDPAPGVLDPRAAPPALLGTPQAEVLEDVLREQFGPLPQLAAVCRLKRLPSGGYSSTENLQLVLERRRVANAKERERIKNLNRGFARLKALVPFLPQSRKPSKVDILKGAT EYIQVLSDLLEGAKDSKKQDPDEQSYSNNSSESHTSSARQLSRNITQHISCAFGLKNEEEGPWADGGSGEPAHACRHSVMSTTEIISPTRSLDRFPEVELLSHRLPQV(SEQ ID NO:4)

[0111] In some embodiments, an engineered polynucleotide comprising an open reading frame encoding distal less homeobox 5 (DLX5) (e.g., UniprotKB Accession No. P56178) encodes a protein comprising the following sequence:

[0112] MTGVFDRRRVPSIRSGDFQAPFQTSAAMHHPSQESPTLPESSATDSDYYSPTGGAPHGYCSPTSASYGKALNPYQYQYHGVNGSAGSYPAKAYADYSYASSYHQYGGAYNRVPSATNQPEKEVTEPEVRMVNGKPKKVRKPRTIYSS FQLAALQRRFQKTQYLALPERAELAASLGLTQTQVKIWFQNKRSKIKKIMKNGEMPPEHSPSSSDPMACNSPQSPAVWEPQGSSRSLSHHPHAHPPTSNQSPASSYLENSASWYTSAASSINSHLPPPGSLQHPLALASGTLY(SEQ IDNO: 5)

[0113] In some embodiments, an engineered polynucleotide comprising an open reading frame encoding a hematopoietic expression homeobox (HHEX) (e.g., UniprotKB Accession No. Q03014) encodes a protein comprising the following sequence:

[0114] MQYPHPGPAAGAVGVPLYAPTPLLQPAHPTPFYIEDILGRGPAAPTPAPTLPSPNSSFTSLVSPYRTPVYEPTPIHPAFSHHSAAALAAAYGPGGFGGPLYPFPRTVNDYTHALLRHDPLGKPLLWSPFLQRPLHKR KGGQVRFSNDQTIELEKKFETQKYLSPPERKRLAKMLQLSERQVKTWFQNRRAKWRRLKQENPQSNKKEELESLDSSCDQRQDLPSEQNKGASLDSSQCSPSPASQEDLESEISEDSDQEVDIEGDKSYFNAG(SEQ ID NO: 6).

[0115] In some embodiments, the pluripotent stem cells further comprise an engineered polynucleotide comprising an open reading frame encoding a protein selected from DDX4, DAZL, and BOLL. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding DDX4. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding DAZL. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding BOLL.

[0116] In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding DDX4 and an engineered polynucleotide containing an open reading frame encoding DAZL. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding DDX4 and an engineered polynucleotide containing an open reading frame encoding BOLL. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding DAZL and an engineered polynucleotide containing an open reading frame encoding BOLL. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding DDX4, an engineered polynucleotide containing an open reading frame encoding DAZL, and an engineered polynucleotide containing an open reading frame encoding BOLL.

[0117] In some embodiments, an engineered polynucleotide comprising an open reading frame encoding DEAD-Box polypeptide-4 (DDX4) (e.g., UniprotKB Accession No. Q9NQI0) encodes a protein comprising the following sequence:

[0118] (SEQ ID NO: 7)

[0119] In some embodiments, the engineered polynucleotide comprising an open reading frame encoding azoospermia deletion (DAZL) (e.g., UniprotKB Accession No. Q92904) encodes a protein comprising the following sequence:

[0120] MSTANPETPNSTISREASTQSSSAATSQGYILPEGKIMPNTVFVGGIDVRMDETEIRSFFARYGSVKEVKIITDRTGVSKGYGFVSFFNDVDVQKIVESQINFHGKKLKLGPAIRKQNLCAYHVQPRPLVFNHPPPPQFQNVWTNPNTE TYMQPTTTMNPITQYVQAYPTYPNSPVQVITGYQLPVYNYQMPPQWPVGEQRSYVVPPAYSAVNYHCNEVDPGAEVVPNECSVHEATPPSGNGPQKKSVDRSIQTVVSCLFNPENRLRNSVVTQDDYFKDKRVHHFRRSRAMLKSV(SEQ ID NO:8)

[0121] In some embodiments, an engineered polynucleotide comprising an open reading frame encoding Boule homolog (BOLL) (e.g., UniprotKB Accession No. Q8N9W6) encodes a protein comprising the following sequence:

[0122] MQTDSLSPSPNPVSPVPLNNPTSAPRYGTVIPNRIFVGGIDFKTNESDLRKFFSQYGSVKEVKIVNDRAGVSKGYGFVTFETQEDAQKILQEAEKLNYKDKKLNIGPAIRKQQVGIPRSSIMPAAGTMYLTTSTGYPYTYHNG VAYFHTPEVTSVPPPWPSRSVCSSPVMVAQPIYQQPAYHYQATTQYLPGQWQWSVPQPSASSAPFLYLQPSEVIYQPVEIAQDGGCVPPPLSLMETSVPEPYSDHGVQATYHQVYAPSAITMPAPVMQPEPIKTVWSIHY(SEQ ID NO:9)

[0123] The number of copies of the engineered polynucleotide delivered to the PSC may vary. In some embodiments, the PSC comprises 1-20 copies of the engineered polynucleotide. For example, the PSC may comprise 1-15, 1-10, 2-10, 2-15, 2-10, 5-20, 5-15, or 5-10 copies of the engineered polynucleotide. In some embodiments, the PSC comprises 8-10 copies of the engineered polynucleotide. More than 20 copies are also contemplated herein.

[0124] Methods for generating oogonia-like cells

[0125] In some aspects, the method of producing oogonia-like cells provided herein includes 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.

[0126] In some embodiments, the PSC of the expanded population comprises an engineered polynucleotide containing an open reading frame encoding ZNF281. In some embodiments, the PSC of the expanded population comprises an engineered polynucleotide containing an open reading frame encoding LHX8. In some embodiments, the PSC of the expanded population comprises an engineered polynucleotide containing an open reading frame encoding SOHLH1. In some embodiments, the PSC of the expanded population also comprises an engineered polynucleotide containing an open reading frame encoding FIGLA protein. In some embodiments, the PSC of the expanded population also comprises: an engineered polynucleotide containing an open reading frame encoding DLX5 protein; and an engineered polynucleotide containing an open reading frame encoding HHEX protein. In some embodiments, the PSC of the expanded population also comprises: an engineered polynucleotide containing an open reading frame encoding DDX4 protein; an engineered polynucleotide containing an open reading frame encoding DAZL protein; and an engineered polynucleotide containing an open reading frame encoding BOLL protein.

[0127] In some embodiments, the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0128] In some embodiments, the heterologous promoter is an inducible promoter, non-limiting examples of which are provided elsewhere herein.

[0129] The population (starting population) contains about 1x10 2 -1x10 10 , about 1x10 2 -1x10 9 , about 1x10 2 -1x10 8 or about 1x10 2 -1x10 7 In some embodiments, the population comprises about 1x10 3 -1x10 8 or about 1x10 3 -1x10 7 In some embodiments, the population comprises about 1x10 4 -1x10 7 or about 1x10 5 -1x10 6 In some embodiments, the population comprises about 1x101 PSC, about 1x10 2 PSC, about 1x10 3 PSC, about 1x10 4 PSC, about 1x10 5 PSC, about 1x10 6 PSC, about 1x10 7 PSC, about 1x10 8 PSC, about 1x10 9 PSCs or about 1x10 10 PSCs.

[0130] 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.

[0131] Some methods of the present disclosure provide methods comprising (a) delivering an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from the group consisting of ZNF281, LHX8, and SOHLH1 (or selected from the group consisting of ZNF281, LHX8, SOHLH1, FIGLA, DLX5, HHEX, DDX4, DAZL, and BOLL) to a PSC; (b) culturing the PSC in a feeder-free, serum-free medium to produce an expanded population of PSCs; and (c) culturing the expanded population of PSCs in a series of induction medium comprising an inducing agent to produce DDX4; + Oogonia-like cells. In some embodiments, the series of induction medium includes a first induction medium, a second induction medium, a third induction medium, and a fourth induction medium.

[0132] In some embodiments, the PSC is cultured in a feeder-free, serum-free medium for about 6 to about 24 hours. For example, the PSC can be cultured in a feeder-free, serum-free medium for about 6 to about 12 hours. In some embodiments, the PSC is cultured in a feeder-free, serum-free 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.

[0133] In some embodiments, the expanded population of PSCs comprises at least 5×10 3 For example, the expanded population (e.g., upon induction) can contain at least 1x10 4, at least 1x10 5 , at least 1x10 6 or at least 1x10 7 In some embodiments, the expanded population of PSCs comprises about 5x10 3 PSC is about 1x10 7 PSCs.

[0134] In some embodiments, the PSCs of the expanded population are approximately 2,000 cells / cm 2 About 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 -10000pcs / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 1000 cells / cm 2 -9500pcs / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 1500 / cm 2 -9000pcs / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 2000 / cm 2 -8500pcs / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 2500 / cm 2 -8000pcs / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 3000 / cm 2 -7500pcs / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 3500 / cm 2 -7000pcs / cm 2 In some embodiments, the population comprises 4000 PSCs / cm 2 -6500pcs / cm 2 In some embodiments, the PSCs in the expanded population are approximately 4500 / cm 2 -6000pcs / cm 2 In some embodiments, the PSCs in the expanded population are cultured at a density of about 5000 / cm 2 -5500pcs / cm 2 In some embodiments, the expanded population of PSCs is cultured at a density of at least 500 cells / cm 2PSC, at least 1000 / cm 2 PSC, at least 1500 / cm 2 PSC, at least 2000 / cm 2 PSC, at least 2500 / cm 2 PSC, at least 3000 pieces / cm 2 PSC, at least 3500 / cm 2 PSC, at least 4000 / cm 2 PSC, at least 4500 / cm 2 PSC, at least 5000 pieces / cm 2 PSC, at least 5500 / cm 2 PSC, at least 6000 pieces / cm 2 PSC, at least 6500 / cm 2 PSC, at least 7000 pieces / cm 2 PSC, at least 7500 / cm 2 PSC, at least 8000 pieces / cm 2 PSC, at least 8500 / cm 2 PSC, at least 9000 / cm 2 PSC, at least 9500 / cm 2 PSC or at least 10,000 pcs / cm 2 PSCs were cultured at a density of

[0135] In some embodiments, the PSCs of the expanded population are cultured for no more than 8 days, no more than 7 days, no more than 6 days, no more than 5 days, or no more than 4 days. 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.

[0136] In some embodiments, the PSC of the expanded population is cultured in the first induction medium for about 6 to about 36 hours. For example, the PSC 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, about 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 PSC is 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.

[0137] In some embodiments, the PSC of the expanded population is cultured in the second induction medium for about 6 to about 36 hours. For example, the PSC 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, about 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 PSC is 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.

[0138] In some embodiments, the PSC of the expanded population is cultured in the third induction medium for about 6 to about 36 hours. For example, the PSC 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, about 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 PSC is 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.

[0139] In some embodiments, the PSC of the expanded population is cultured in the fourth induction medium for about 6 to about 36 hours. For example, the PSC 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, about 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 PSC is 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.

[0140] In some embodiments, the PSCs are incubated for at least 6 hours. In some embodiments, after incubation, the culture 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 2 In some embodiments, the iPSCs are incubated for 18 hours, then medium #1 is removed and medium #2 is added (Table 1). In some embodiments, the iPSCs are incubated for 24 hours, then medium #2 is removed and medium #3 is added (Table 1). In some embodiments, the iPSCs are incubated for 18 hours, then medium #3 is removed and medium #4 is added (Table 1). In some embodiments, after incubating the iPSCs with medium #4, DDX4+ oogonia-like cells are present in the plate.

[0141] Transfection method

[0142] The engineered polynucleotides of the present disclosure can be delivered to PSCs using one or more transfection methods, including chemical transfection methods, viral transduction methods, and electroporation.

[0143] In some embodiments, the engineered polynucleotide is delivered on a vector. A vector is any vehicle, such as a virus or plasmid, for transferring the desired polynucleotide to a host cell (e.g., 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 from adeno-associated virus (AAV), adenovirus, herpes simplex virus, lentivirus, retrovirus, varicella, smallpox virus, hepatitis B, cytomegalovirus, JC polyomavirus, BK polyomavirus, monkeypox virus, herpes zoster virus, Epstein-Barr virus, human herpes virus 7, Kaposi's sarcoma-associated herpes virus, or human parvovirus B19. The present disclosure also encompasses other viral vectors.

[0144] 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 about 5 kb and has been engineered for use as a gene transfer vector (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 serve as the starting point of DNA replication and act as the main packaging signal (McLaughlin, SK et al. Virol. 1988; 62 (6): 1963-73; Hauswirth, WW et al. 1977; 78 (2): 488-99). Therefore, AAV vectors generally include ITR sequences. Both positive and negative strands are packaged equally well into virions and are able to infect (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 self-complementary vectors in which the genome anneals to itself after viral uncoating. This results in more efficient transduction of cells, but the coding capacity is reduced by half (McCarty, DM et al. Mol Ther. 2008; 16 (10): 1648-56; McCarty, DM et al. Gene Ther. 2001; 8 (16): 1248-54).

[0145] In some embodiments, a transposon / transposase system is used to deliver a polynucleotide to a cell. For example, piggyBac TM Transposon system. piggyBac TM Transposons are mobile genetic elements that efficiently transpose between vectors and chromosomes via a “cut and paste” mechanism (Woodard et al., 2015). TM The transposase recognizes the transposon-specific inverted terminal repeats (ITRs) located at both ends of the transposon vector and efficiently moves the contents from the original site and integrates them into the TTAA chromosomal locus. piggyBac TM The transposon system facilitates the efficient integration of polynucleotides into the cellular genome.

[0146] Thus, in some embodiments, the method further comprises delivering a transposon comprising the engineered polynucleotide to the PSC, and also delivering a transposase.

[0147] In some embodiments, the engineered polynucleotide is delivered to the cell using electroporation. Electroporation is a physical transfection method that uses electric pulses to create temporary holes in the cell membrane through which the engineered polynucleotide can enter the cell. See, e.g., Chicaybam L et al. Front. Bioeng. Biotechnol., 23 January 2017.

[0148] After transfection, the engineered polynucleotide can be integrated into the genome of the PSC. In some embodiments, the engineered polynucleotide may also include an antibiotic resistance gene to confer resistance to the antibiotic used in the antibiotic drug selection process. In this way, a "pure" cell population containing the integrated engineered polynucleotide can be obtained. In some embodiments, antibiotic drug selection is used to select a cell population containing the integrated engineered polynucleotide. Antibiotic drug selection is the process of treating a cell population with an antibiotic so that only cells that can survive in the presence of the antibiotic will remain in the population. Non-limiting examples of antibiotics that can be used for antibiotic drug selection include: puromycin, blasticidin, geneticin, hygromycin, mycophenolic acid, bleomycin, carbenicillin, kanamycin, ampicillin and actinomycin.

[0149] Culture medium

[0150] In some embodiments, the methods provided herein include culturing PSCs in a feeder-free, serum-free medium. The medium may include, for example, a dissolved basement membrane preparation extracted from an Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g. Matrix (per cm 2 75 to 150 μl of suspension based on batch dilution coating). 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 protein can be selected from laminin, collagen IV, heparin sulfate proteoglycans, and paranectin / nidogen.

[0151] In some embodiments, the culture medium further comprises one or more growth factors, for example, selected from recombinant human basic fibroblast growth factor (rh bFGF) (e.g., 80 ng / ml to 120 ng / ml) and recombinant human transforming growth factor β (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 TM Culture medium (STEMCELL Technologies).

[0152] 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., 1.8mM to 2.2mM), inducer (e.g., doxycycline (e.g., 50ng / ml to 2000ng / ml)), activin A (e.g., 50ng / ml to 150ng / ml), glycogen synthase kinase (GSK) 3 inhibitor (e.g., 2.8μM to 3.2μM), selective FGFR1 and FGFR3 inhibitor (e.g., 90nM to 110nM) and small molecule ROCK inhibitor (e.g., 8μM to 12μM). In some embodiments, the first induction medium comprises B-27, L-alanyl-L-glutamine, inducer (e.g., doxycycline), activin A, glycogen synthase kinase (GSK) 3 inhibitor and selective FGFR1 and FGFR3 inhibitor. For example, the first induction medium can include aRB27 medium, doxycycline, activin A, CHIR99021, and PD173074.

[0153] 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 tank turbinase (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 tank turbinase (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).

[0154] 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), a stem cell factor (SCF) (e.g., 25 ng / ml to 200 ng / ml), and an epidermal growth factor (EGF) (e.g., 25 ng / ml to 100 ng / ml). In some embodiments, the third induction medium comprises a B-27 supplement (e.g., 90X to 110X), 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), a stem cell factor (SCF) (e.g., 25 ng / ml to 200 ng / ml), and an epidermal growth factor (EGF) (e.g., 25 ng / ml to 100 ng / ml). For example, the third induction medium may comprise aRB27 medium, doxycycline, XAV939, SCF, and EGF.

[0155] 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., 50 ng / ml to 2000 ng / ml)), a small molecule inhibitor of tankyrase (e.g., 0.9 μM to 1.1 μM), hBMP4 (e.g., 20 ng / ml to 250 ng / ml), SCF (e.g., 25 ng / ml to 200 ng / ml), and EGF (e.g., 25 ng / ml to 100 ng / 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.

[0156] The "aRB27 medium" used in this article includes Advanced RPMI, B-27 TM Supplements, without vitamin A (Thermo Fisher) or with vitamin A, GlutaMAX TM Supplements (Thermo Fisher), Non-essential Amino Acids (NEAA), (broad-spectrum antibiotic) and Y-27632 (small molecule ROCK inhibitor).

[0157] GlutaMAX TM The supplement contains L-alanyl-L-glutamine, which is a dipeptide substitute for L-glutamine.

[0158] Activin-A is a dimeric glycoprotein that belongs to the transforming growth factor-β (TGF-β) family.

[0159] CHIR99021 is an aminopyrimidine derivative that is a very potent glycogen synthase kinase (GSK) 3 inhibitor, inhibiting GSK3β (IC 50 =6.7 nM) and GSK3α (IC 50 =10 nM). GSK3 is a serine / threonine kinase that is a key inhibitor of the WNT pathway; therefore, CHIR99021 acts as a WNT activator.

[0160] PD173074 is a selective FGFR1 and FGFR3 inhibitor (IC50 values ​​for FGFR3, FGFR1, VEGFR2, PDGFR and c-Src are approximately 5nM, approximately 21.5nM, approximately 100nM, approximately 17600nM and approximately 19800nM, respectively, and IC50 values ​​for EGFR, InsR, MEK and PKC are >50000nM).

[0161] XAV939 is a potent small molecule inhibitor of tankyrase (TNKS) 1 and 2 (IC 50 By inhibiting TNKS activity, XAV939 increased the protein level of axin-GSK3β complex and promoted the degradation of β-catenin in SW480 cells (Huang et al.), thereby inhibiting the downstream effects of WNT pathway.

[0162] Therapeutic compositions and methods of use

[0163] In some embodiments, the present disclosure provides a therapeutic composition comprising the oogonia-like cells produced herein. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition is cryopreserved.

[0164] Such compositions can be administered to a subject, such as a human subject, using any suitable route of administration. Suitable routes of administration include, for example, parenteral routes, such as intravenous, intrathecal, parenchymal, or intraventricular routes. Suitable routes of administration include, for example, parenteral routes, such as intravenous, intrathecal, parenchymal, or intraventricular injection.

[0165] In some embodiments, the subject is a human subject. The subject may suffer from infertility, such as Turner syndrome. Infertility includes diseases in which the development of reproductive cells in a female fetus is severely affected. Analysis of blood samples can be used to diagnose infertility. In some embodiments, infertility may include oogonia damage.

[0166] The composition can be administered to a subject in a therapeutically effective amount. The term "therapeutically effective amount" refers to the amount of oogonia-like cells required to produce a therapeutic effect on a subject, alone or in combination with at least one other active agent. As will be appreciated by those skilled in the art, the effective amount varies depending on the route of administration, excipient use, and co-use with other active agents. The amount to be administered depends on the subject to be treated, including, for example, the strength of the individual's immune system or genetic predisposition. A suitable dosage range is readily determined by those skilled in the art, and it may be micrograms of the polypeptide of the present disclosure. The dosage of the formulations disclosed herein may depend on the route of administration and vary according to the size of the subject.

[0167] It is believed that those skilled in the art can make full use of the present invention according to the above description. Therefore, the following specific embodiments should be interpreted as illustrative only, and do not limit the remainder of the present disclosure in any way. All publications cited in this application are incorporated by reference for the purpose or theme cited in the present disclosure.

[0168] Additional Implementation

[0169] The present disclosure also relates to additional embodiments listed in the following numbered paragraphs:

[0170] 1. A pluripotent stem cell (PSC), comprising: an engineered polynucleotide containing 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).

[0171] 2. The PSC of paragraph 1, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding ZNF281.

[0172] 3. The PSC of paragraph 1 or 2, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding LHX8.

[0173] 4. The PSC of any of the preceding paragraphs, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding SOHLH1.

[0174] 5. The PSC of any of the preceding paragraphs, wherein the PSC expresses or overexpresses: ZNF281; LHX8; SOHLH1; ZNF281 and LHX8; ZNF281 and SOHLH1; LHX8 and SOHLH1; or ZNF281, LHX8 and SOHLH1.

[0175] 6. The PSC of any of the preceding paragraphs, wherein the PSC further comprises an engineered polynucleotide comprising an open reading frame encoding a folliculogenesis-specific BHLH transcription factor (FIGLA) protein, optionally wherein the PSC expresses or overexpresses FIGLA.

[0176] 7. The PSC of paragraph 6, wherein the PSC further comprises: an engineered polynucleotide comprising an open reading frame encoding a distal less homeobox 5 (DLX5) protein; and an engineered polynucleotide comprising an open reading frame encoding a hematopoietic expression homeobox (HHEX) protein, optionally wherein the PSC expresses or overexpresses the DLX5 protein and the HHEX protein.

[0177] 8. The PSC of paragraph 7, wherein the PSC further comprises: an engineered polynucleotide containing an open reading frame encoding a DDX4 protein; an engineered polynucleotide containing an open reading frame encoding a DAZL protein; and an engineered polynucleotide containing an open reading frame encoding a BOLL protein, optionally wherein the PSC expresses or overexpresses DDX4 protein, DAZL protein and BOLL protein.

[0178] 9. The PSC of any of the preceding paragraphs, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0179] 10. The PSC of paragraph 9, wherein the heterologous promoter is an inducible promoter.

[0180] 11. A pluripotent stem cell (PSC), comprising: a protein selected from the group consisting of ZNF281, LHX8 and SOHLH1, wherein the protein is overexpressed.

[0181] 12. The PSC of paragraph 11, wherein the PSC expresses or overexpresses: ZNF281; LHX8; SOHLH1; ZNF281 and LHX8; ZNF281 and SOHLH1; LHX8 and SOHLH1; or ZNF281, LHX8 and SOHLH1.

[0182] 13. The PSC of paragraph 12, wherein the PSC further comprises FIGLA protein, optionally wherein the PSC expresses or overexpresses FIGLA.

[0183] 14. The PSC of paragraph 13, wherein the PSC further comprises: DLX5 protein and HHEX protein, optionally wherein the PSC expresses or overexpresses DLX5 protein and HHEX protein.

[0184] 15. The PSC of paragraph 14, wherein the PSC further comprises: DDX4 protein, DAZL protein and BOLL protein, optionally wherein the PSC expresses or overexpresses DDX4 protein, DAZL protein and BOLL protein.

[0185] 16. The PSC of any of the preceding paragraphs, wherein the PSC is a human PSC.

[0186] 17. The PSC of any of the preceding paragraphs, wherein the PSC is an induced PSC (iPSC).

[0187] 18. The PSC of any of the preceding paragraphs, wherein the PSC comprises 1-20, optionally 8-10 copies of an engineered polynucleotide comprising an open reading frame encoding a protein selected from the group consisting of ZNF281, LHX8 and SOHLH1.

[0188] 19. A composition comprising: a population of PSCs as described in any of the preceding paragraphs or elsewhere herein.

[0189] 20. The composition of paragraph 18, wherein the population comprises at least 2500 / cm 2 PSC.

[0190] 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 PSCs of the expanded population to produce oogonia-like cells.

[0191] 22. The method of paragraph 21, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding ZNF281.

[0192] 23. The method of paragraph 21 or 22, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding LHX8.

[0193] 24. The method of any of paragraphs 21-23, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding SOHLH1.

[0194] 25. The method of any of paragraphs 21-24, wherein the PSCs of the expanded population further comprise an engineered polynucleotide comprising an open reading frame encoding a FIGLA protein.

[0195] 26. The method of paragraph 25, wherein the PSCs of the expanded population further comprise: an engineered polynucleotide comprising an open reading frame encoding a DLX5 protein; and an engineered polynucleotide comprising an open reading frame encoding a HHEX protein.

[0196] 27. The method of paragraph 26, wherein the PSCs of the expanded population further comprise: an engineered polynucleotide comprising an open reading frame encoding a DDX4 protein; an engineered polynucleotide comprising an open reading frame encoding a DAZL protein; and an engineered polynucleotide comprising an open reading frame encoding a BOLL protein.

[0197] 28. The method of any of the preceding paragraphs, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0198] 29. The method of any of the preceding paragraphs, wherein the heterologous promoter is an inducible promoter.

[0199] 30. The method of any of the preceding paragraphs, wherein the population comprises 1x10 2 -1x10 7 PSCs.

[0200] 31. The method of any of the preceding paragraphs, wherein the population of PSCs is cultured for about 3-5 days, optionally about 4 days.

[0201] 32. The method of paragraph 31, wherein the population of PSCs is cultured for about days.

[0202] 33. The method of any of the preceding paragraphs, wherein the oogonia-like cells are DDX4 + .

[0203] 34. The method of any of the preceding paragraphs, wherein the oogonia-like cells independently have a diameter of about 20 microns to 180 microns.

[0204] 35. A method comprising:

[0205] (a) delivering an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from the group consisting of ZNF281, LHX8 and SOHLH1 to pluripotent stem cells (PSCs);

[0206] (b) culturing the PSCs in a feeder-free, serum-free medium to produce an expanded population of PSCs; and

[0207] (c) culturing the expanded population of PSCs in a series of induction media containing induction agents to produce DDX4 + Oogonia-like cells.

[0208] 36. The method of paragraph 35, wherein the engineered polynucleotide is a transposon, and the delivering further comprises delivering a transposase to the PSC.

[0209] 37. The method of paragraph 35 or 36, wherein the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

[0210] 38. The method of any of paragraphs 35-37, wherein the feeder-free, serum-free medium of (b) comprises a solubilized basement membrane preparation extracted from an Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

[0211] 39. The method of paragraph 38, wherein the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

[0212] 40. The method of paragraph 39, wherein the ECM protein is selected from the group consisting of laminin, collagen IV, heparin sulfate proteoglycans, and paranectin / nidogen.

[0213] 41. The method of any of paragraphs 35-40, wherein the feeder-free, serum-free medium of (b) comprises a growth factor selected from recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ).

[0214] 42. The method of any of paragraphs 35-41, wherein the culturing of (b) continues for about 6 to about 24 hours.

[0215] 43. The method of any of paragraphs 35-42, wherein the PSCs of the expanded population of (c) are approximately 2,000 cells / cm 2 About 3,000 cells / cm 2 Density culture.

[0216] 44. The method of any one of paragraphs 35-43, wherein 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.

[0217] 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.

[0218] 46. ​​The method of paragraph 44 or 45, wherein 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).

[0219] 47. The method of any of paragraphs 44-46, wherein 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).

[0220] 48. The method of any of paragraphs 44-47, wherein 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.

[0221] 49. An oogonia-like cell produced by the method of any of the preceding paragraphs. Example

[0222] Example 1. Induction of DDX4+ oogonia-like cells from hiPSCs by a mixture of three transcription factors

[0223] The current method of generating DDX4+ human oogonium-like cells from human induced pluripotent stem cells (hiPSC) is laborious, expensive and time-consuming. For example, one of the methods uses the co-culture of induced human primordial germ cell-like cells (hPGCLC) and mouse E12.5 fetal gonadal cells (mFGC) to form xenogeneic reconstructed ovaries (xrOvaries), which induce a small group of human DDX4+ oogonium-like cells when grown for more than about 120 days (Yamashiro et al., Science. 2018 Oct 19; 362 (6412): 356-360). Although this method can generate oogonium-like cells, it requires a lot of rodent handling expertise, expensive culture techniques and a long differentiation period. This embodiment describes an efficient and reliable method for generating DDX4+ oogonium-like cells from hiPSC within four days using direct transcription factor (TF) overexpression combined with growth factor culture.

[0224] The present disclosure relates to the identification of three transcription factors (ZNF281, LHX8, SOHLH1) that, when overexpressed alone or in combination, drive the formation of DDX4+ oogonia-like cells from hiPSCs.

[0225] To identify these three transcription factors (ZNF281, LHX8, and SOHLH1), a computational algorithm was developed to predict transcription factors involved in human oocyte differentiation (Kramme et al. Cell Reports Methods, 100082, 2021). Using the predicted set of 53 transcription factors, 53 individual hiPSC lines, each carrying one TF, and a pool of TF combinations spanning 42 million unique combinations were generated. Through TF induction followed by bulk RNA sequencing (RNA-seq) and single-cell RNA-Seq, a subset of TFs that drive gene expression signatures of oogenesis were identified ( Figure 1 Next, we utilized the DDX4-tdTomato hiPSC reporter line, which uses cytokines combined with individual TF overexpression to induce the formation of human germ cells (hPGCLCs). We identified three TFs (ZNF281, LHX8, and SOHLH1) that, when individually overexpressed in hiPSCs, generated a small population of high DDX4-expressing cells ( Figure 2A-2B Next, a combinatorial hiPSC line carrying integrated expression vectors for all three TFs was constructed, termed DDX4-3 (D3), which, when induced during hPGCLC formation, drove higher yields of DDX4+ cells than individual TF expression ( Figure 2C ). Bulk RNA-Seq was used to characterize this high DDX4+ population driven by three TFs and showed that combinatorial overexpression of the three TFs drives upregulation of key oocyte regulatory genes ( Figure 3A Transcriptome comparison with the human ovarian atlas reference showed that the D3 combination drives a robust oocyte-like transcriptome signature, as observed in a statistically significant TROM classification when compared to secondary and antral oocytes ( Figure 3B ). Taken together, these results indicate that directly overexpressing these three TFs alone and in combination with hPGCLC differentiation is sufficient to generate DDX4+ oogonia-like cells from hiPSCs within four days.

[0226] This TF-based differentiation protocol for high DDX4-expressing cells is highly scalable and cost-effective ( Figure 4 ). Briefly, TFs were integrated into female hiPSCs individually or in combination as doxycycline-inducible gene expression cassettes using the piggyBac transposase system. Antibiotic drug selection was used to obtain a pure population of cells containing the integrated expression cassettes. The hiPSCs containing the TFs were then cultured in serum-free mTeSR TM Culture medium No feeder layer is grown on the substrate.

[0227] To induce oogonia formation, hiPSCs were plated at 2,500 cells / cm2 The cells were seeded at a density of 100 μM in mTeSR medium supplemented with approximately 8-10 μM Y-27623 and 0.5-3 μg / mL doxycycline. TM In the culture medium Matrix-coated plates. After approximately six hours, the culture medium was removed and the plates were briefly washed with DMEM / F12. The culture medium was then replaced with the mesoderm induction medium (culture medium #1) listed in Table 1. After approximately 18 hours of culture in culture medium #1, the culture medium was removed, the plates were briefly washed again with DMEM / F12, and hPGCLC induction medium (culture medium #2 listed in Table 1) was added and cultured for 24 hours. After 24 hours, the culture medium was replaced with culture medium #3 and incubated again for 24 hours. Finally, culture medium #3 was replaced with culture 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.

[0228] Figure 5 Tests of TF combinations for oogonia formation are shown. 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 showed the production of DDX4+ cells. All three combined overexpression showed a 100-1000-fold increase in the efficiency of DDX4+ oogonia production. The combination of the two, especially the combination with LHX8, showed a 10-50-fold increase compared to overexpression alone. In addition, adding FIGLA to the D3 combo increased the yield by 10 times compared to D3 alone, and also induced NPM2+ oocyte-like formation. Similarly, it was found that adding DLX5, HHEX and FIGLA to the D3 combo showed an increased DDX4+ yield compared to D3 alone, and the combination is referred to herein as D3N3. Finally, additional overexpression of DDX4, DAZL and BOLL in combination with D3N3 showed increased DDX4+ yield compared to D3 alone, this combination is 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.

[0229] Methods and Materials

[0230] iPSC culture

[0231] iPSCs were coated with hESC-qualified iPSCs were cultured in mTESR1 medium (Stemcell Technologies) on standard polystyrene plates with 1% succinimum iodine. The medium was changed daily. Passaging was performed with 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; all cells tested negative.

[0232] TF plasmid construction

[0233] TF cDNAs were synthesized as full-length transcripts or obtained as Gateway entry clones from ORFeome (The ORFeome Colobration, Nat Methods. 13, 191-192 (2016)). They were cloned into doxycycline-inducible PiggyBac expression plasmids (Addgene #175503) using MegaGate (Kramme et al., STAR Protoc. 2, 100907 (2021)). The final expression constructs were verified by Sanger sequencing, which was also used to determine the barcode sequence of each TF.

[0234] Integration of TF plasmids into hiPSCs

[0235] An expression plasmid containing TF cDNA under the control of a doxycycline inducible promoter was integrated into iPSCs using PiggyBac transposase. For integration, approximately 50-100 fmol of TF cDNA plasmid, approximately 150-250 ng of PiggyBac transposase expression plasmid, and approximately 100,000-200,000 iPSCs were mixed in Lonza P3 buffer and electroporated using a Lonza Nucleofector 4D. After electroporation, cells were plated in mTeSR TM Plus medium + about 8-12μM Y-27632 in a 24-well plate. Selection with an appropriate reagent (usually puromycin) begins 48 hours after electroporation and lasts for about 3-5 days. The cells are then passaged for about 3 days without the use of drug selectins to allow the loss of unintegrated plasmids. Finally, the cells are passaged again under drug selection to produce a pure, selected integron pool. The presence and approximate copy number of the integrated TF plasmid are confirmed by qPCR of genomic DNA. For oogonia, hiPSC pools are used instead of clones selected from single cells. The average copy number is 8-10.

[0236] Protocol for inducing primordial germ cells by TF overexpression

[0237] hiPSCs containing integrated TF expression plasmids were mTeSR on substrate TM For induction in monolayer format, use StemPro TM Accutase TM Cell dissociation reagent dissociates hiPSCs into single cells and 2,500-3,000 cells / cm on matrix or vitronectin XF coated plates 2 The density of mTeSR TM Inoculate in medium + about 8-10 μM Y-27632 and about 0.5-3 μg / ml doxycycline for about 6 hours. Then remove the medium and wash with dPBS or DMEM / F12 and replace with aRB27 medium #1. After about 12-18 hours of induction, remove medium #1 and wash with dPBS or DMEM / F12, then replace with medium #2. About 24 hours later, remove medium #2 and replace it with medium #3. About 24 hours later, replace medium #3 with medium #4. After about 24 hours of culture in medium #4 or after additional culture in medium #4 for about two more days (day 6 of the protocol), harvest the oogonia for use. Isolate oogonia-like cells using the DDX4 reporter factor. In addition, oogonia can also be generated through embryoid body formation with similar efficiency as the monolayer protocol by the methods established by 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.

[0238] Protocol for expansion of oogonia-like cells after isolation

[0239] Expansion of sorted oogonia-like cells was performed by FACS isolation of DDX4+ cells in step 6 on approximately day 4 or day 6. Isolated oogonia were inoculated into S-CM medium with Y-27632 and doxycycline as described by Kobayashi et al. (2022). Half of the medium is replaced every 2-3 days and cells can be expanded, passaged, and repurified as needed.

[0240] Table 1. Composition of the culture medium used in Example 1

[0241]

[0242] *Optional antibiotics

[0243] Table 2. Cytokines used in Example 1

[0244]

[0245] Table 3. Inhibitors used in Example 1

[0246] Inhibitors supplier batch number Solvents CHIR99021 (hydrochloride) Axon Medchem 12 66% H2O, 33% DMSO PD173074 Selleck S126404 DMSO XAV939 Tocris 5B / 251235 DMSO Y-27632 Ambeed Various H2O

[0247] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which they are cited, which in some cases may be the entire document.

[0248] The indefinite articles "a" and "an" as used in this specification and claims are to be understood as meaning "at least one", unless expressly stated otherwise.

[0249] It should also be understood that in any method claimed herein that includes multiple steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described unless explicitly stated otherwise.

[0250] In the claims and the foregoing description, all transitional phrases, such as "comprises," "comprising," "carrying," "having," "containing," "involving," "holding," "comprising," etc., shall be understood to be open-ended, i.e., meaning including but not limited to. 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 U.S. Patent Office Manual of Patent Examining Procedures.

[0251] The terms "about" and "substantially" preceding a numerical value mean ±10% of the numerical value.

[0252] Where a range of values ​​is provided, every value between and including the upper and lower limits of that range is specifically contemplated and described herein.

Claims

1. A pluripotent stem cell (PSC), comprising: an engineered polynucleotide containing 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 of claim 1, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding ZNF281.

3. The PSC of claim 1, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding LHX8.

4. The PSC of claim 1, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding SOHLH1.

5. The PSC of claim 1, wherein the PSC expresses or overexpresses: ZNF281; LHX8; SOHLH1; ZNF281 and LHX8; ZNF281 and SOHLH1; LHX8 and SOHLH1; or ZNF281, LHX8, and SOHLH1.

6. The PSC of claim 1, wherein the PSC further comprises an engineered polynucleotide comprising an open reading frame encoding a folliculogenesis-specific BHLH transcription factor (FIGLA) protein, optionally wherein the PSC expresses or overexpresses FIGLA.

7. The PSC of claim 6, wherein the PSC further comprises: an engineered polynucleotide containing an open reading frame encoding a distal less homeobox 5 (DLX5) protein; and an engineered polynucleotide containing an open reading frame encoding a hematopoietic expression homeobox (HHEX) protein, optionally wherein the PSC expresses or overexpresses the DLX5 protein and the HHEX protein.

8. The PSC of claim 7, wherein the PSC further comprises: an engineered polynucleotide containing an open reading frame encoding a DDX4 protein; an engineered polynucleotide containing an open reading frame encoding a DAZL protein; and an engineered polynucleotide containing an open reading frame encoding a BOLL protein, optionally wherein the PSC expresses or overexpresses DDX4 protein, DAZL protein and BOLL protein.

9. The PSC of claim 1, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

10. The PSC of claim 9, wherein the heterologous promoter is an inducible promoter.

11. A pluripotent stem cell (PSC), comprising: a protein selected from the group consisting of ZNF281, LHX8 and SOHLH1, wherein the protein is overexpressed.

12. The PSC of claim 11, wherein the PSC expresses or overexpresses: ZNF281; LHX8; SOHLH1; ZNF281 and LHX8; ZNF281 and SOHLH1; LHX8 and SOHLH1; or ZNF281, LHX8, and SOHLH1.

13. The PSC of claim 12, wherein the PSC further comprises FIGLA protein, optionally wherein the PSC expresses or overexpresses FIGLA.

14. The PSC of claim 13, wherein the PSC further comprises: a DLX5 protein and a HHEX protein, optionally wherein the PSC expresses or overexpresses the DLX5 protein and the HHEX protein.

15. The PSC of claim 14, wherein the PSC further comprises: DDX4 protein, DAZL protein and BOLL protein, optionally wherein the PSC expresses or overexpresses DDX4 protein, DAZL protein and BOLL protein.

16. The PSC of claim 1, wherein the PSC is a human PSC.

17. The PSC of claim 1, wherein the PSC is an induced PSC (iPSC).

18. The PSC of claim 1, wherein the PSC comprises 1-20, optionally 8-10 copies of an engineered polynucleotide comprising an open reading frame encoding a protein selected from the group consisting of ZNF281, LHX8, and SOHLH1.

19. A composition comprising: a population of the PSCs of claim 1.

20. The composition of claim 18, wherein the population comprises at least 2500 cells / cm 2 PSC.

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 PSCs of the expanded population to generate oogonia-like cells.

22. The method of claim 21, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding ZNF281.

23. The method of claim 21, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding LHX8.

24. The method of claim 21, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding SOHLH1.

25. The method of claim 21, wherein the PSCs of the expanded population further comprise an engineered polynucleotide comprising an open reading frame encoding a FIGLA protein.

26. The method of claim 25, wherein the expanded population of PSCs further comprises: an engineered polynucleotide comprising an open reading frame encoding a DLX5 protein; and an engineered polynucleotide comprising an open reading frame encoding a HHEX protein.

27. The method of claim 26, wherein the PSCs of the expanded population further comprise: an engineered polynucleotide comprising an open reading frame encoding a DDX4 protein; an engineered polynucleotide comprising an open reading frame encoding a DAZL protein; and an engineered polynucleotide comprising an open reading frame encoding a BOLL protein.

28. The method of claim 21, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

29. The method of claim 21, wherein the heterologous promoter is an inducible promoter.

30. The method of claim 21, wherein the population comprises 1 x 10 2 -1x10 7 PSCs.

31. The method of claim 21, wherein the population of PSCs is cultured for about 3-5 days, optionally about 4 days.

32. The method of claim 31, wherein the population of PSCs is cultured for about days.

33. The method of claim 21, wherein the oogonia-like cell is DDX4 + .

34. The method of claim 21, wherein the oogonia-like cells independently have a diameter of about 20 microns to 180 microns.

35. A method comprising: (a) delivering an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from the group consisting of ZNF281, LHX8 and SOHLH1 to pluripotent stem cells (PSCs); (b) culturing the PSCs in a feeder-free, serum-free medium to produce an expanded population of PSCs; and (c) culturing the expanded population of PSCs in a series of induction media containing induction agents to produce DDX4 + Oogonia-like cells.

36. The method of claim 35, wherein the engineered polynucleotide is a transposon and the delivering further comprises delivering a transposase to the PSC.

37. The method of claim 35, wherein the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

38. The method of claim 35, wherein the feeder-free, serum-free medium of (b) comprises a solubilized basement membrane preparation extracted from an Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

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

40. The method of claim 39, wherein the ECM protein is selected from the group consisting of laminin, collagen IV, heparin sulfate proteoglycans, and paranectin / nidogen.

41. The method of claim 35, wherein the feeder-free, serum-free medium of (b) comprises a growth factor selected from the group consisting of recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ).

42. The method of claim 35, wherein the culturing of (b) continues for about 6 to about 24 hours.

43. The method of claim 35, wherein the PSCs of the expanded population of (c) are approximately 2,000 cells / cm 2 About 3,000 cells / cm 2 Density culture.

44. The method of claim 35, wherein the culturing of (c) comprises 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.

45. The method of claim 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.

46. ​​The method of claim 44, wherein 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).

47. The method of claim 44, wherein 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).

48. The method of claim 44, wherein 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.

49. An oogonia-like cell produced by the method of claim 35.

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