Methods and compositions for producing primordial germ cell-like cells

Abstract

Provided herein are methods and compositions for differentiating induced pluripotent stem cells into primordial germ cell-like cells by overexpressing transcription factors such as DLX5, HHEX and / or FIGLA.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 326,656, filed April 1, 2022, which is incorporated by reference herein in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (H049870759WO00-SEQ-KVC.xml, size: 4,453 bytes and creation date: March 27, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] background Primordial germ cells are germline stem cells that give rise to gametes in vertebrates. Primordial germ cells migrate to the developing gonad where they differentiate into sperm or eggs. Primordial germ cell dysfunction underlies many forms of human female infertility, but efficient methods for generating primordial germ cells in vitro remain elusive. Summary of the Invention [Means for solving the problem]

[0004] overview The present disclosure relates, at least in part, to methods and compositions for generating primordial germ cell-like cells (PGCLCs) in vitro from pluripotent stem cells (PSCs). The present disclosure unexpectedly demonstrates that overexpression of certain transcription factors, such as DLX5, HHEX, and FIGLA, can induce the development of PGCLCs (e.g., NANOS3) from PSCs in as little as four days. + , SOX17 + , TFAP2C + , PRDM1 + , OCT4 + , CD38 + ,EPCAM + , ITGA6 + and / or SOX2 -We provide experimental data demonstrating that the α-terminal β-terminal cytoplasmic endothelial cell wall is sufficient to generate PGCLC, which is a cytoplasmic endothelial cell wall wall.

[0005] Some embodiments of the present disclosure provide a PSC comprising an engineered polynucleotide comprising an open reading frame encoding a protein selected from DLX5, HHEX, and FIGLA.

[0006] In some embodiments, the PSC comprises an engineered polynucleotide comprising an open reading frame encoding DLX5.

[0007] In some embodiments, the PSC comprises an engineered polynucleotide comprising an open reading frame encoding HHEX.

[0008] In some embodiments, the PSC comprises an engineered polynucleotide comprising an open reading frame encoding FIGLA.

[0009] In some embodiments, the PSCs express or overexpress DLX5, HHEX, FIGLA, DLX5 and HHEX, DLX5 and FIGLA, HHEX and FIGLA, or DLX5, HHEX and FIGLA.

[0010] In some embodiments, the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0011] In some embodiments, the heterologous promoter is an inducible promoter.

[0012] Another aspect of the present disclosure provides a PSC comprising a protein selected from DLX5, HHEX and FIGLA, wherein the protein is overexpressed.

[0013] In some embodiments, the PSCs express or overexpress DLX5, HHEX, FIGLA, DLX5 and HHEX, DLX5 and FIGLA, HHEX and FIGLA, or DLX5, HHEX and FIGLA.

[0014] In some embodiments, the PSCs are human PSCs.

[0015] In some embodiments, the PSCs are induced PSCs (iPSCs).

[0016] In some embodiments, the PSC comprises 1 to 20, optionally 8 to 10 copies of an engineered polynucleotide comprising an open reading frame encoding a protein selected from DLX5, HHEX and FIGLA.

[0017] Yet another aspect of the disclosure provides a composition comprising a population of PSCs as described in any one of the above paragraphs or elsewhere herein.

[0018] In some embodiments, the population is at least 2500 cells / cm 2 Includes PSCs.

[0019] Some aspects of the 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 DLX5, HHEX and FIGLA in the expanded population of PSCs to produce PGCLCs.

[0020] In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide that includes an open reading frame encoding DLX5.

[0021] In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide that comprises an open reading frame encoding HHEX.

[0022] In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide that includes an open reading frame encoding FIGLA.

[0023] In some embodiments, the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0024] In some embodiments, the heterologous promoter is an inducible promoter.

[0025] In some embodiments, the population is 1×10 2 ~1×10 7 Includes PSCs.

[0026] In some embodiments, the population of PSCs is cultured for about 3-5 days.

[0027] In some embodiments, the population of PSCs is cultured for about 4 days.

[0028] In some embodiments, the PGCLC is NANOS3 + , SOX17 + , TFAP2C + , PRDM1 + , OCT4 + , CD38 + ,EPCAM + , ITGA6 + and / or SOX2 - PGCLC.

[0029] Some aspects of the present disclosure include: (a) delivering to pluripotent stem cells (PSCs) an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from DLX5, HHEX, and FIGLA; (b) culturing the PSCs in a feeder-free, serum-free culture medium to generate an expanded population of PSCs; (c) Expanded populations of PSCs were cultured in a series of induction media containing inducers to induce NANOS3 + , SOX17 + , TFAP2C + , PRDM1 + , OCT4 + , CD38 + ,EPCAM + , ITGA6 + and / or SOX2 - Producing PGCLCs The present invention provides a method comprising:

[0030] In some embodiments, the engineered polynucleotide is a transposon and delivering further comprises delivering a transposase to the PSC.

[0031] In some embodiments, the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

[0032] In some embodiments, the feeder-free, serum-free culture medium of (b) comprises a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

[0033] In some embodiments, the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

[0034] In some embodiments, the ECM protein is selected from laminin, collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

[0035] In some embodiments, the feeder-free, serum-free culture medium of (b) comprises a growth factor selected from recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor beta (rh TGFβ).

[0036] In some embodiments, the culturing in (b) is for about 6 to about 24 hours.

[0037] In some embodiments, the PSCs of the expanded population of (c) are about 2,000 cells / cm. 2 ~Approx. 3,000 cells / cm 2 The cells are cultured at a density of 100 μg / ml.

[0038] In some embodiments, the culturing of (c) comprises culturing the PSCs in a first induction medium, culturing the PSCs in a second induction medium, culturing the PSCs in a third induction medium, and culturing the PSCs in a fourth induction medium.

[0039] 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.

[0040] 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).

[0041] 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).

[0042] 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.

[0043] Another aspect of the present disclosure provides a PGCLC produced by a method according to any one of the above claims.

[0044] The details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will become apparent from the following drawings and detailed description of some embodiments, as well as from the appended claims.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component illustrated in various figures is represented by the same numeral. For clarity, not every component is depicted in every drawing. [Brief description of the drawings]

[0046] [Figure 1A] We show the identification of overexpressed transcription factors (TFs) that drive enhanced NANOS3+ ​​primordial germ cell yield. We show that TFs are integrated into distinct lineages of NANOS3-mVenus PSCs and overexpressed via doxycycline induction during monolayer primordial germ cell formation. hPGCLC yields were compared with plus dox vs. minus dox and with no TF control conditions. [Figure 1B] Figure 1 shows identification of overexpressed transcription factors (TFs) driving enhanced NANOS3+ ​​primordial germ cell yield. TFs DLX5, HHEX and FIGLA enhance NANOS3+ ​​hPGCLC yield via the same protocol as discussed in Figure 1A. [Figure 2A] Figure 1 shows transcriptome and proteome analysis demonstrating that TFs drive on-target primordial germ cell formation. Transcriptome characterization of hPGCLCs is shown. Results show upregulation of key hPGCLC genes and downregulation of PSCs, consistent with known positive controls. NANOS3+ ​​hPGCLCs were isolated via FACS after TF-based or control induction and subjected to RNA sequencing. [Figure 2B]Figure 1 shows transcriptome and proteome analyses demonstrating that TFs drive on-target primordial germ cell formation. Analysis of key hallmarks of hPGCLC protein expression. Immunofluorescence was performed on TF-induced hPGCLCs for integrin (ITGA6), OCT4, and SOX17 expression. [Figure 3A] Characterization of TF dynamics via dose, time course, and cytokine removal. hPGCLC yield assessed after TF induction in control conditions without cytokines and without hBMP4. Yield change compared to baseline conditions is plotted, showing that DLX5 retains 40% of its activity without hBMP4. [Figure 3B] Characterization of TF dynamics across dose, time course, and cytokine withdrawal. Shown is hPGCLC yield assessed under doxycycline dilution, demonstrating that increasing doxycycline generally increases hPGCLC yield following TF induction. [Figure 3C] Characterization of TF dynamics via dosage, time course and cytokine withdrawal. hPGCLC yield assessed after doxycycline addition at various time points is shown, demonstrating that TFs are generally beneficial when expressed throughout the differentiation process. [Figure 4] FIG. 1 shows a schematic diagram of the TF-assisted hPGCLC formation method. [Diagram 5] FIG. 1 shows that TF induces increased hPGCLC yield across different markers. [Figure 6] Figure 2 shows that TF induces increased hPGCLC yield across differentiation platforms. [Figure 7] TF combination test for hPGCLC formation. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Detailed Description Primordial germ cells (PGCs) are the origin of gametogenesis and serve as the progenitor cell type for both oocyte and spermatocyte development (McLaren et al. 2003). Recently, numerous techniques have been developed to differentiate human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (PSCs) (Mitsunage et al. 2017). Recently, a new monolayer hPGCLC differentiation method has been developed that induces hPGCLC formation without embryoid bodies, allowing for ease of use and scalability. Although many hPGCLC induction methods exist, they all suffer from a large heterogeneity problem in hPGCLC yields depending on the cell line utilized, and many cell lines are no longer used for germ cell formation. Because primordial germ cells are utilized as input cell types for in vitro ovarian and testicular reconstruction, a method for high-yield primordial germ cell formation is needed. Aspects of the present disclosure relate to methods of using direct transcription factor overexpression to induce stem cell differentiation into NANOS3+ ​​(Nanos C2HC-type zinc finger 3), SOX17+ (SRY box transcription factor 17), TFAP2C+ (transcription factor AP-2 gamma), PRDM1+ (PR / SET domain 1), OCT4+ (octamer-binding transcription factor 4) and / or SOX2- (SRY box transcription factor 2) primordial germ cells. It is understood that the term "PGCLC" encompasses cells that express primordial germ cell specific markers, such as NANOS3, SOX17, TFAP2C, PRDM1, OCT4, CD38, EPCAM and / or ITGA6, and / or cells that do not express SOX2, as well as cells that exhibit other characteristics of naturally occurring primordial germ cells.

[0048] Primordial germ cell-like cells Some aspects of the present disclosure provide primordial germ cell-like cells (PGCLCs) and methods for producing such cells. Primordial germ cells (PGCs) are embryonic precursors of gametes (sperm and eggs) and give rise to new organisms capable of producing endless new generations through germ cells. PGCs represent the founder cells of the germ lineage. PGCs are embodied during early mammalian post-implantation development and are uniquely programmed for the transmission of genetic and epigenetic information to successive generations. Primordial germ cells are single cells that can form colonies of cells morphologically similar to undifferentiated embryonic stem cells under specific culture conditions.

[0049] Primordial germ cells and PGCLCs express several different biomarkers that can be used to distinguish PGCLCs from other cell types. For example, PGCLC cells are typically positive for Nanos C2HC-type zinc finger 3 (NANOS3), SRY box transcription factor 17 (SOX17), transcription factor AP-2 gamma (TFAP2C), PR / SET domain 1 (PRDM1), octamer-binding transcription factor 4 (OCT4), and / or negative for SRY box transcription factor 2 (SOX2).

[0050] PGCLCs have other characteristics that distinguish them from non-PGCLCs, including, but not limited to, their overall decreased 5-methyl-cytosine and H3K9me2 levels compared to stem cells, as well as their CXCL12 / SDF1-guided chemotactic motility, cytoplasmic granules.

[0051] pluripotent stem cells The PGCLCs provided herein are differentiated from pluripotent stem cells, which are cells that have the ability to self-renew by division and differentiate into the three major germ layers of the early embryo (e.g., ectoderm, endoderm and mesoderm), and thus have the ability to differentiate into all cells of the adult body, but not into extraembryonic tissues such as the placenta (Shi et al. 2017).

[0052] Non-limiting examples of pluripotent stem cells include induced pluripotent cells (iPSCs), "true" embryonic stem cells (ESCs) derived from embryos, embryonic stem cells generated by somatic cell nuclear transfer (ntESCs) and embryonic stem cells from unfertilized eggs (parthenogenetic embryonic stem cells or pESCs). In some embodiments, the pluripotent cells are human pluripotent cells.

[0053] In some embodiments, the pluripotent stem cells are embryonic stem cells, such as human embryonic stem cells. "Embryonic stem cells" is a general term that refers to pluripotent stem cells that are generated using embryos or eggs, rather than genetically reprogrammed cells from the body. As used herein, "ESCs" encompass true ESCs, ntESCs, and pESCs.

[0054] In other embodiments, the pluripotent stem cells are induced pluripotent stem cells, such as human induced pluripotent stem cells. iPSCs can be derived from skin or blood cells that have been reprogrammed back to an embryonic-like pluripotent state, allowing for the generation of an unlimited source of cells that can become any human cell type.

[0055] Some aspects of the disclosure provide PSCs comprising a protein selected from DLX5, HHEX and FIGLA, wherein the protein is expressed or overexpressed. In some embodiments, the protein is expressed at a level of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, or at least 100% above a control level. In some embodiments, the control level is an endogenous level of the protein, such as in a spontaneously occurring pluripotent stem cell. In some embodiments, the PSC comprises DLX5. In some embodiments, the PSC expresses or overexpresses DLX5. In some embodiments, the PSC comprises HHEX. In some embodiments, the PSC expresses or overexpresses HHEX. In some embodiments, the PSC comprises FIGLA. In some embodiments, the PSC expresses or overexpresses FIGLA.

[0056] The data provided herein show that expression of only one of DLX5, HHEX or FIGLA is superior to combinatorial expression of all three. For example, combinatorial expression of DLX5, HHEX and FIGLA in PSCs significantly increased expression of NANOS3 compared to no TF controls. + , SOX17 + , TFAP2C + , PRDM1 + , OCT4 + , CD38 + ,EPCAM + , ITGA6 + and / or SOX2 - It increases the efficiency of PGCLC production by 2-fold. However, unexpectedly, combinatorial expression of DLX5, HHEX and FIGLA in PSCs reduces the efficiency by 5-45-fold compared to PSC controls expressing only one of DLX5, HHEX or FIGLA. Thus, in some embodiments, PSCs contain DLX5, HHEX or FIGLA, but not necessarily all three together.

[0057] In other embodiments, the PSC comprises DLX5 and HHEX. In some embodiments, the PSC expresses or overexpresses DLX5 and HHEX. In some embodiments, the PSC comprises DLX5 and FIGLA. In some embodiments, the PSC expresses or overexpresses DLX5 and FIGLA. In some embodiments, the PSC comprises HHEX and FIGLA. In some embodiments, the PSC expresses or overexpresses HHEX and FIGLA.

[0058] Transcription factors The PGCLCs provided herein, in some embodiments, are differentiated from pluripotent stem cells by expressing one or more (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) transcription factors (i.e., proteins that control the transcription rate). Differentiation is the process by which undifferentiated or partially differentiated cells differentiate into specialized cell fates. Aspects of the present disclosure relate to the differentiation of undifferentiated pluripotent stem cells into PGCLC fates.

[0059] In some embodiments, the transcription factor is selected from DLX5, HHEX, and FIGLA. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are engineered to express or overexpress DLX5. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are engineered to express or overexpress HHEX. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are engineered to express or overexpress FIGLA. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are engineered to express or overexpress DLX5 and HHEX. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are engineered to express or overexpress DLX5 and FIGLA. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are engineered to express or overexpress HHEX and FIGLA. In some embodiments, pluripotent stem cells, such as hPSCs or hiPSCs, are engineered to express or overexpress DLX5, HHEX and FIGLA.

[0060] A cell "expresses" a particular protein if it is possible to detect levels of the protein in the cell (e.g., using a known protein assay). A cell "overexpresses" a particular protein if the levels of the protein are higher (e.g., at least 5%, at least 10%, or at least 20% higher) than the levels of protein expressed from an endogenous, naturally occurring polynucleotide encoding the protein (e.g., an engineered polynucleotide encoding the protein).

[0061] Engineered Polynucleotides and Polypeptides The pluripotent stem cells of the present disclosure, in some embodiments, comprise engineered polynucleotides. Engineered polynucleotides are non-naturally occurring nucleic acids (e.g., at least two nucleotides covalently linked together, in some cases containing phosphodiester bonds, referred to as a phosphodiester backbone). Engineered polynucleotides include recombinant and synthetic nucleic acids. Recombinant nucleic acids are molecules constructed by linking nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids, or combinations thereof) from two different organisms (e.g., human and mouse). Synthetic nucleic acids are molecules that have been amplified or synthesized chemically or by other means. Synthetic nucleic acids include those that have been chemically modified or otherwise modified, provided that they are capable of base pairing (binding) with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids include molecules resulting from any of the above replications.

[0062] An engineered polynucleotide can be DNA (e.g., genomic DNA, cDNA, or a combination of genomic DNA and cDNA), RNA, or a hybrid molecule, e.g., the nucleic acid can contain any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural) and any combination of two or more bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine.

[0063] In some embodiments, the polynucleotide is complementary DNA (cDNA), which is synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.

[0064] The engineered polynucleotides of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the nucleic acids are produced using GIBSON ASSEMBLY® cloning (see, e.g., Gibson, DG et al. Nature Methods, 343-345, 2009 and Gibson, DG et al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). In GIBSON ASSEMBLY®, typically three enzyme activities are used in a single tube reaction: a 5' exonuclease activity, a 3' extension activity of a DNA polymerase, and a DNA ligase activity. The 5' exonuclease activity chews back the 5' end sequence to expose complementary sequences for annealing. Gaps on the annealed domains are then filled by the polymerase activity. The nicks are then repaired by DNA ligase to covalently link the DNA fragments together. The overlapping sequences of the adjoining fragments are much longer than those used in Golden Gate assembly, resulting in a higher percentage of correct assembly. Other methods for producing engineered polynucleotides can be used in accordance with the present disclosure.

[0065] In some embodiments, the engineered polynucleotide comprises a promoter operably linked to the open reading frame. A promoter is a nucleotide sequence (e.g., ATG) to which RNA polymerase binds in coordination with initial transcription. A promoter is typically located directly upstream (5' end) of the transcription start site. In some embodiments, the promoter is a heterologous promoter. A heterologous promoter is not naturally associated with the open reading frame to which it is operably linked.

[0066] In some embodiments, the promoter is an inducible promoter. An inducible promoter can be regulated in vivo, for example, by a chemical agent, temperature, or light. An inducible promoter allows, for example, temporal and / or spatial control of gene expression. Inducible promoters for use according to the present disclosure include any inducible promoter described herein or known to one of skill in the art. Examples of inducible promoters include, but are not limited to, chemically / biochemically regulated promoters and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems including the tetracycline repressor protein (tetR), the tetracycline operator sequence (tetO) and the tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters from the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptor and the steroid / retinoid / thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (a protein that binds and sequesters metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters) and light-regulated promoters (e.g., light-responsive promoters from plant cells). In some embodiments, the inducible promoter is a tetracycline-inducible promoter. In some embodiments, the inducible promoter is a doxycycline-inducible promoter. In other embodiments, the promoter is a constitutive promoter (in vivo active, unregulated).

[0067] An open reading frame is a contiguous stretch of codons that begins with a start codon (e.g., ATG), ends with a stop codon (e.g., TAA, TAG, or TGA), and encodes a polypeptide, such as a protein. An open reading frame is operably linked to a promoter if that promoter controls transcription of the open reading frame.

[0068] Vectors used for delivery of engineered polynucleotides include minicircles, plasmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes. Transposon-based systems such as the piggyBac™ system (e.g., Chen et al. Nature Communications. 2020; 11(1): 3446) are also contemplated herein.

[0069] The pluripotent stem cells, in some embodiments, comprise an engineered polynucleotide that comprises an open reading frame encoding a protein selected from DLX5, HHEX, and 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. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding FIGLA.

[0070] In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide comprising an open reading frame encoding DLX5 and an engineered polynucleotide comprising an open reading frame encoding HHEX. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide comprising an open reading frame encoding DLX5 and an engineered polynucleotide comprising an open reading frame encoding FIGLA. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide comprising an open reading frame encoding HHEX and an engineered polynucleotide comprising an open reading frame encoding FIGLA. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide comprising an open reading frame encoding DLX5, an engineered polynucleotide comprising an open reading frame encoding HHEX and an engineered polynucleotide comprising an open reading frame encoding FIGLA.

[0071] An engineered polynucleotide coding for an open reading frame encoding a follicle formation specific BHLH transcription factor (FIGLA) (e.g., UniprotKB Accession No. Q6QHK4), in some embodiments, [ka] It encodes a protein comprising the sequence:

[0072] An engineered polynucleotide coding for Distal-Less Homeobox 5 (DLX5) (e.g., UniprotKB Accession No. P56178) can, in some embodiments, be [ka] It encodes a protein comprising the sequence:

[0073] An engineered polynucleotide coding for an open reading frame encoding a hematopoietic expressed homeobox (HHEX) (e.g., UniprotKB Accession No. Q03014) can, in some embodiments, be [ka] It encodes a protein comprising the sequence:

[0074] The number of copies of the engineered polynucleotide delivered to the PSC can vary. In some embodiments, the PSC comprises 1-20 copies of the engineered polynucleotide. For example, the PSC can comprise 1-15, 1-10, 2-10, 2-15, 2-10, 5-20, 5-15, or even 5-10 copies of the engineered polynucleotide. In some embodiments, the PSC comprises 8-10 copies of the engineered polynucleotide. More than 20 copies are also contemplated herein.

[0075] Methods for Producing PGCLCs The methods of producing PGCLCs provided herein, in some aspects, include culturing a population of pluripotent stem cells (PSCs) in a culture medium to produce an expanded population of PSCs, and expressing a protein selected from DLX5, HHEX, and FIGLA in the expanded population of PSCs to produce PGCLCs.

[0076] In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide that comprises an open reading frame encoding DLX5. In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide that comprises an open reading frame encoding HHEX. In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide that comprises an open reading frame encoding FIGLA.

[0077] In some embodiments, the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0078] In some embodiments, the heterologous promoter is an inducible promoter, non-limiting examples of which are provided elsewhere herein.

[0079] The starting population was approximately 1 × 10 2 ~1×10 10 , about 1×10 2 ~1×10 9 , about 1×10 2 ~1×10 8 Or about 1 x 10 2 ~1×10 7 In some embodiments, the population comprises about 1 x 10 PSCs. 3 ~1×10 8 Or about 1 x 10 3 ~1×10 7 In some embodiments, the population comprises about 1 x 10 PSCs. 4 ~1×10 7 Or about 1 x 10 5 ~1×10 6 In some embodiments, the population comprises about 1 x 10 PSCs. 1 PSCs, approximately 1 x 10 2 PSCs, approximately 1 x 10 3 PSCs, approximately 1 x 10 4 PSCs, approximately 1 x 10 5 PSCs, approximately 1 x 10 6 PSCs, approximately 1 x 10 7 PSCs, approximately 1 x 10 8 PSCs, approximately 1 x 10 9 PSCs or approximately 1 x 10 10 Includes PSCs.

[0080] 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.

[0081] Some methods of the disclosure include (a) delivering to PSCs an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from DLX5, HHEX, and FIGLA; (b) culturing the PSCs in a feeder-free, serum-free culture medium to produce an expanded population of PSCs; and (c) culturing the expanded population of PSCs in a series of induction media comprising an inducer to produce NANOS3. + , SOX17 + , TFAP2C + , PRDM1 + , OCT4 + , CD38 + ,EPCAM + , ITGA6 + and / or SOX2 - and producing a PGCLC. In some embodiments, the series of induction mediums comprises a first, a second, a third and a fourth induction medium.

[0082] In some embodiments, the PSCs are cultured in a feeder-free, serum-free culture medium for about 6 to about 24 hours. For example, the PSCs may be cultured in a feeder-free, serum-free culture medium for about 6 to about 12 hours. In some embodiments, the PSCs are cultured in a feeder-free, serum-free culture medium for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours.

[0083] In some embodiments, the expanded population of PSCs comprises at least 5×10 3 For example, an expanded population (e.g., upon induction) may contain at least 1 x 10 PSCs. 4 , at least 1 × 10 5 , at least 1 × 10 6 Or at least 1 × 10 7 In some embodiments, the expanded population of PSCs may comprise about 5×10 3 PSCs ~ approx. 1 × 10 7Includes PSCs.

[0084] In some embodiments, the expanded population of PSCs comprises about 2,000 cells / cm. 2 ~Approx. 3,000 cells / cm 2 In some embodiments, the PSCs of the expanded population are cultured at a density of about 500 cells / cm. 2 ~10000 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 1000 cells / cm. 2 ~9500 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 1500 cells / cm. 2 ~9000 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 2000 cells / cm. 2 ~8500 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 2500 cells / cm. 2 ~8000 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 3000 cells / cm. 2 ~7500 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 3500 cells / cm. 2 ~7000 pieces / cm 2 In some embodiments, the population is cultured at a density of PSCs of 4000 cells / cm. 2 ~6500 pieces / cm 2 In some embodiments, the expanded population of PSCs comprises about 4500 PSCs / cm. 2 ~6000 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of about 5000 cells / cm. 2 ~5500 pieces / cm 2 In some embodiments, the expanded population of PSCs are cultured at a density of at least 500 cells / cm. 2 PSCs, at least 1000 pieces / cm 2PSC, at least 1500 pieces / cm 2 PSCs, at least 2000 pieces / cm 2 PSCs, at least 2500 pieces / cm 2 PSCs, at least 3000 pieces / cm 2 PSC, at least 3500 particles / cm 2 PSCs, at least 4000 pieces / cm 2 PSCs, at least 4500 particles / cm 2 PSCs, at least 5000 pieces / cm 2 PSC, at least 5500 particles / cm 2 PSCs, at least 6000 pieces / cm 2 PSCs, at least 6500 particles / cm 2 PSCs, at least 7000 particles / cm 2 PSCs, at least 7500 particles / cm 2 PSCs, at least 8000 pieces / cm 2 PSCs, at least 8500 particles / cm 2 PSCs, at least 9000 particles / cm 2 PSC, at least 9500 particles / cm 2 PSC or at least 10,000 particles / cm 2 PSCs are cultured at a density of 100x.

[0085] In some embodiments, the PSCs of the expanded population are cultured for 8 days or less, 7 days or less, 6 days or less, 5 days or less, or 4 days or less. For example, the PSCs of the expanded population can be cultured for about 2 to about 8 days, about 2 to about 7 days, about 2 to about 6 days, about 2 to about 5 days, about 2 to about 4 days, about 3 to about 8 days, about 3 to about 7 days, about 3 to about 6 days, about 3 to about 5 days, or about 3 to about 4 days. In some embodiments, the PSCs of the expanded population are cultured for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, or about 8 days.

[0086] In some embodiments, the PSCs of the expanded population are cultured in the first induction medium for about 6 to about 36 hours. For example, the PSCs can be cultured in the first induction medium for about 6 to about 24 hours, about 6 to about 18 hours, about 6 to about 12 hours, 12 to about 36 hours, about 12 to about 24 hours, about 12 to about 18 hours, 18 to about 36 hours, or about 18 to about 24 hours. In some embodiments, the PSCs are cultured in the first induction medium for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours.

[0087] In some embodiments, the PSCs of the expanded population are cultured in the second induction medium for about 6 to about 36 hours. For example, the PSCs can be cultured in the second induction medium for about 6 to about 24 hours, about 6 to about 18 hours, about 6 to about 12 hours, 12 to about 36 hours, about 12 to about 24 hours, about 12 to about 18 hours, 18 to about 36 hours, or about 18 to about 24 hours. In some embodiments, the PSCs are cultured in the second induction medium for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours.

[0088] In some embodiments, the PSCs of the expanded population are cultured in the third induction medium for about 6 to about 36 hours. For example, the PSCs can be cultured in the third induction medium for about 6 to about 24 hours, about 6 to about 18 hours, about 6 to about 12 hours, 12 to about 36 hours, about 12 to about 24 hours, about 12 to about 18 hours, 18 to about 36 hours, or about 18 to about 24 hours. In some embodiments, the PSCs are cultured in the third induction medium for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours.

[0089] In some embodiments, the PSCs of the expanded population are cultured in the fourth induction medium for about 6 to about 36 hours. For example, the PSCs can be cultured in the fourth induction medium for about 6 to about 24 hours, about 6 to about 18 hours, about 6 to about 12 hours, 12 to about 36 hours, about 12 to about 24 hours, about 12 to about 18 hours, 18 to about 36 hours, or about 18 to about 24 hours. In some embodiments, the PSCs are cultured in the fourth induction medium for about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, or about 30 hours.

[0090] Transfection methods The engineered polynucleotides of the present disclosure may be delivered to the PSCs using any one or more transfection methods, including chemical transfection methods, viral transduction methods, and electroporation methods.

[0091] In some embodiments, the engineered polynucleotide is delivered in a vector. A vector is any vehicle, e.g., a virus or a plasmid, used to introduce a desired polynucleotide into a host cell, such as a PSC. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is not a naturally occurring viral vector. The viral vector can be derived from adeno-associated virus (AAV), adenovirus, herpes simplex virus, lentivirus, retrovirus, varicella virus, smallpox virus, hepatitis B virus, cytomegalovirus, JC polyomavirus, BK polyomavirus, monkeypox virus, varicella zoster virus, Epstein-Barr virus, human herpesvirus type 7, Kaposi's sarcoma associated herpes virus, or human parvovirus B19. Other viral vectors are encompassed by the present disclosure.

[0092] In some embodiments, the viral vector is an AAV vector. AAV is a small non-enveloped virus that packages a single-stranded linear DNA genome of approximately 5 kb length, adapted for use as a gene transfer vehicle (Samulski, RJ et al., Annu Rev Virol. 2014; 1(1):427-51). The coding region of AAV is flanked by inverted terminal repeats (ITRs), which act as DNA replication origins and serve as primary packaging signals (McLaughlin, SK et al. Virol. 1988; 62(6): 1963-73;Hauswirth, WW et al. 1977; 78(2):488-99). For this reason, AAV vectors typically contain ITR sequences. Both the positive and negative strands are packaged equally well into virions and are infectious (Zhong, L et al. Mol Ther. 2008; 16(2):290-5; Zhou, X et al. Mol Ther. 2008; 16(3):494-9; Samulski, RJ et al. Virol. 1987; 61(10):3096-101). In addition, a small deletion in one of the two ITRs allows packaging of a self-complementary vector, and the genome self-anneals after viral uncoating. This results in more efficient transduction of cells, but reduces the coding capacity by half (McCarty, DM et al. Mol Ther. 2008; 16(10): 1648-56; McCarty, DM et al. Gene Ther. 2001; 8(16): 1248-54).

[0093] In some embodiments, polynucleotides are delivered to cells using a transposon / transposase system. For example, the piggyBac™ transposon system can be used. The piggyBac™ transposon is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism (Woodard et al. 2015). Upon transposition, the piggyBac™ transposase recognizes transposon-specific inverted terminal repeats (ITRs) located at both ends of the transposon vector, efficiently displacing the contents from the original site and integrating it into the TTAA chromosomal site. The piggyBac™ transposon system facilitates efficient integration of polynucleotides into the cell genome.

[0094] Thus, in some embodiments, the method further comprises delivering a transposon comprising the engineered polynucleotide to the PSC and also delivering a transposase.

[0095] In some embodiments, engineered polynucleotides are delivered to cells using electroporation. Electroporation is a physical transfection method that uses an electric pulse to create a temporary hole in the cell membrane through which engineered polynucleotides can enter cells.See, for example, Chicaybam L et al. Front. Bioeng. Biotechnol., 23 January 2017.

[0096] After transfection, the engineered polynucleotide may be integrated into the genome of the PSC. In some embodiments, the engineered polynucleotide may further comprise an antibiotic resistance gene that confers resistance to an antibiotic used in the antibiotic drug selection process. In this way, a "pure" cell population containing the integrated engineered polynucleotide may be obtained. In some embodiments, the cell population containing the integrated engineered polynucleotide is selected using antibiotic drug selection. Antibiotic drug selection is a process in which a cell population is treated with an antibiotic such that only cells capable of surviving in the presence of the antibiotic remain in the population. Non-limiting examples of antibiotics that may be used for antibiotic drug selection include puromycin, blasticidin, geneticin, hygromycin, mycophenolic acid, zeocin, carbenicillin, kanamycin, ampicillin, and actinomycin.

[0097] Culture medium The methods provided herein, in some embodiments, include culturing PSCs in a feeder-free, serum-free culture medium. The culture medium can include, for example, a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., Corning® Matrigel® Matrix) (75-150 μl / cm 2 (coated with a lot-based diluted suspension of). In some embodiments, the solubilized basement membrane preparation comprises one or more extracellular matrix (ECM) proteins and one or more growth factors. For example, the ECM proteins can be selected from laminin, collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

[0098] In some embodiments, the culture medium further comprises one or more growth factors selected from, for example, recombinant human basic fibroblast growth factor (rh bFGF) (e.g., 80 ng / ml to 120 ng / ml) and recombinant human transforming growth factor beta (rh TGFβ) (e.g., 20 to 25 pM). In some embodiments, the culture medium further comprises rh bFGF and rh TGFβ. In some embodiments, the culture medium comprises mTeSR™ medium (STEMCELL Technologies).

[0099] In some embodiments, the first induction medium comprises one or more (e.g., 2, 3, 4 or more) of B-27 supplement (e.g., 90x to 110x), L-alanyl-L-glutamine (e.g., 1.8 mM to 2.2 mM), an inducer (e.g., doxycycline (e.g., 50 ng / ml to 2000 ng / ml)), activin A (e.g., 50 ng / ml to 150 ng / ml), a glycogen synthase kinase (GSK) 3 inhibitor (e.g., 2.8 μM to 3.2 μM), a selective FGFR1 and FGFR3 inhibitor (e.g., 90 nM to 110 nM), and a small molecule ROCK inhibitor (e.g., 8 μM to 12 μM). In some embodiments, the first induction medium comprises B-27, L-alanyl-L-glutamine, an inducer (e.g., doxycycline), activin A, a glycogen synthase kinase (GSK) 3 inhibitor, and a selective FGFR1 and FGFR3 inhibitor. For example, the first induction medium can comprise aRB27 medium, doxycycline, activin A, CHIR99021, and PD173074.

[0100] 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-110x), an inducer (e.g., doxycycline (e.g., 50ng / ml-2000ng / ml)), a small molecule inhibitor of tankyrase (TNKS) (e.g., 0.9μM-1.1μM), and human bone morphogenetic protein 4 (hBMP4) (e.g., 20ng / ml-250ng / ml). In some embodiments, the second induction medium comprises B-27, an inducer (e.g., doxycycline), a small molecule inhibitor of tankyrase (TNKS), and human bone morphogenetic protein 4 (hBMP4). For example, the second induction medium can comprise aRB27 medium, doxycycline, XAV939, and human bone morphogenetic protein 4 (hBMP4).

[0101] In some embodiments, the third induction medium comprises one or more (e.g., 2, 3, 4 or more) of B-27, an inducer (e.g., doxycycline), a small molecule inhibitor of tankyrase (e.g., 0.9 μM to 1.1 μM), stem cell factor (SCF) (e.g., 25 ng / ml to 200 ng / ml), and epidermal growth factor (EGF) (e.g., 25 ng / ml to 100 ng / ml). In some embodiments, the third induction medium comprises B-27 supplement (e.g., 90× to 110×), an inducer (e.g., doxycycline (e.g., 50 ng / ml to 2000 ng / ml)), a small molecule inhibitor of tankyrase (e.g., 0.9 μM to 1.1 μM), stem cell factor (SCF) (e.g., 25 ng / ml to 200 ng / ml), and epidermal growth factor (EGF) (e.g., 25 ng / ml to 100 ng / ml). For example, the third induction medium can include aRB27 medium, doxycycline, XAV939, SCF and EGF.

[0102] In some embodiments, the fourth induction medium comprises one or more (e.g., 2, 3, 4 or more) of B-27 supplement (90-110x), an inducer (e.g., doxycycline (e.g., 50ng / ml-2000ng / ml)), a small molecule inhibitor of tankyrase (e.g., 0.9μM-1.1μM), hBMP4 (e.g., 20ng / ml-250ng / ml), SCF (e.g., 25ng / ml-200ng / ml), and EGF (e.g., 25ng / ml-100ng / ml). In some embodiments, the fourth induction medium comprises B-27, an inducer (e.g., doxycycline), a small molecule inhibitor of tankyrase, hBMP4, SCF, and EGF. For example, the fourth induction medium may comprise aRB27 medium, doxycycline, XAV939, hBMP4, SCF, and EGF.

[0103] As used herein, "aRB27 medium" includes modified RPMI, B-27™ supplement, minus or plus Vitamin A (Thermo Fisher), GlutaMAX™ supplement (Thermo Fisher), non-essential amino acids (NEAA), Primocin® (broad-spectrum antibiotic), and Y-27632 (a small molecule ROCK inhibitor).

[0104] GlutaMAX™ supplement contains L-alanyl-L-glutamine, an alternative dipeptide to L-glutamine.

[0105] Activin-A is a dimeric glycoprotein that belongs to the transforming growth factor-β (TGF-β) family.

[0106] CHIR99021 inhibits GSK3β (IC 50 = 6.7 nM) and GSK3α (IC 50 CHIR99021 is an aminopyrimidine derivative that is a highly potent glycogen synthase kinase (GSK) 3 inhibitor that inhibits both vasopressin and vasopressin (vasopressin-1, vasopressin-2, and vasopressin-3) at elevated levels (T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, T12, T14, T16, T18, T18, T20, T16, T18, T21, T18 ...

[0107] PD173074 is a selective FGFR1 and FGFR3 inhibitor (IC50 values ​​of approximately 5 nM, approximately 21.5 nM, approximately 100 nM, approximately 17600 nM and approximately 19800 nM for FGFR3, FGFR1, VEGFR2, PDGFR and c-Src, respectively, and >50000 nM for EGFR, InsR, MEK and PKC).

[0108] XAV939 is a potent small molecule inhibitor of tankyrase (TNKS) 1 and 2 (IC 50 = 11 and 4 nM) (Huang et al.). By inhibiting TNKS activity, XAV939 increases the protein levels of the axin-GSK3β complex and promotes the degradation of β-catenin in SW480 cells, thereby inhibiting downstream effects of the WNT pathway (Huang et al.).

[0109] Therapeutic Compositions and Methods of Use The present disclosure provides, in some embodiments, a therapeutic composition comprising the PGCLC produced herein. In some embodiments, the composition further comprises a pharma- ceutically acceptable excipient. In some embodiments, the composition is cryopreserved.

[0110] Such compositions may be administered to a subject, such as a human subject, using any suitable route of administration. Suitable routes of administration include parenteral routes, such as intravenous, intrathecal, intraparenchymal, intraventricular routes, etc. Suitable routes of administration include parenteral routes, such as intravenous, intrathecal, intraparenchymal, intraventricular injection, etc.

[0111] In some embodiments, the subject is a human subject. Subjects who may benefit from such compositions include patients suffering from male or female factor infertility, the inability to produce viable gametes.

[0112] The composition may be administered to a subject in a therapeutically effective amount. The term "therapeutically effective amount" refers to the amount of PGCLC required to provide a therapeutic effect to a subject, either alone or in combination with at least one other active agent. The effective amount varies depending on the route of administration, excipient use, and co-use of other active agents, as will be appreciated by those skilled in the art. The dosage depends on the subject being treated, including, for example, the strength of the individual's immune system or genetic predisposition. Suitable dosage ranges can be easily determined by those skilled in the art, and may be on the order of micrograms for the polypeptides of the present disclosure. The dosage of the preparations disclosed herein may depend on the route of administration and vary depending on the size of the subject.

[0113] It is believed that one skilled in the art can utilize the present invention to its fullest extent based on the above description. The following specific embodiments are therefore to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited in this application are incorporated by reference for the purposes or subject matter mentioned in this disclosure.

[0114] Additional Embodiments The present disclosure also relates to additional embodiments set forth in the following numbered paragraphs.

[0115] 1. A pluripotent stem cell (PSC) comprising an engineered polynucleotide comprising an open reading frame encoding a protein selected from DLX5, HHEX and FIGLA.

[0116] 2. The PSC of paragraph 1, comprising an engineered polynucleotide comprising an open reading frame encoding DLX5.

[0117] 3. The PSC of paragraph 1 or 2, comprising an engineered polynucleotide comprising an open reading frame encoding HHEX.

[0118] 4. A PSC according to any one of the above paragraphs, comprising an engineered polynucleotide comprising an open reading frame encoding FIGLA.

[0119] 5. A PSC according to any one of the above paragraphs which expresses or overexpresses DLX5, HHEX, FIGLA, DLX5 and HHEX, DLX5 and FIGLA, HHEX and FIGLA, or DLX5, HHEX and FIGLA.

[0120] 6. The PSC of any one of the above paragraphs, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0121] 7. The PSC of paragraph 6, wherein the heterologous promoter is an inducible promoter.

[0122] 8. A pluripotent stem cell (PSC) comprising a protein selected from DLX5, HHEX and FIGLA, wherein the protein is overexpressed.

[0123] 9. The PSC of paragraph 8, which expresses or overexpresses DLX5, HHEX, FIGLA, DLX5 and HHEX, DLX5 and FIGLA, HHEX and FIGLA, or DLX5, HHEX and FIGLA.

[0124] 10. The PSC of any one of the above paragraphs, which is a human PSC.

[0125] 11. The PSC of any one of the above paragraphs, which is an induced PSC (iPSC).

[0126] 12. A PSC according to any one of the above paragraphs, comprising 1 to 20, optionally 8 to 10 copies of an engineered polynucleotide comprising an open reading frame encoding a protein selected from DLX5, HHEX and FIGLA.

[0127] 13. A composition comprising a population of PSCs as described in any one of the above paragraphs or elsewhere herein.

[0128] 14. The population should be at least 2500 cells / cm2 14. The composition of paragraph 13, comprising a PSC of

[0129] 15. A method comprising culturing a population of pluripotent stem cells (PSCs) in a culture medium to produce an expanded population of PSCs, and expressing a protein selected from DLX5, HHEX and FIGLA in the expanded population of PSCs to produce PGCLCs.

[0130] 16. The method of paragraph 15, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding DLX5.

[0131] 17. The method of paragraph 15 or 16, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding HHEX.

[0132] 18. The method of any one of paragraphs 15-17, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding FIGLA.

[0133] 19. The method of any one of the above paragraphs, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0134] 20. The method of any one of the above paragraphs, wherein the heterologous promoter is an inducible promoter.

[0135] 21. The population is 1 x 10 2 ~1×10 7 The method of any one of the above paragraphs, comprising a PSC.

[0136] 22. The method of any one of the above paragraphs, wherein the population of PSCs is cultured for about 3 to 5 days.

[0137] 23. The method of paragraph 22, wherein the population of PSCs is cultured for about 4 days.

[0138] 24. PGCLC is NANOS3 + , SOX17 + , TFAP2C + , PRDM1 + , OCT4 + , CD38 + ,EPCAM + , ITGA6 + and / or SOX2 - The method of any one of the above paragraphs, wherein the PGCLC.

[0139] 25. (a) delivering to a pluripotent stem cell (PSC) an engineered polynucleotide comprising an inducible promoter operably linked to an open reading frame encoding a protein selected from DLX5, HHEX, and FIGLA; (b) culturing the PSCs in a feeder-free, serum-free culture medium to generate an expanded population of PSCs; (c) Expanded populations of PSCs were cultured in a series of induction media containing inducers to induce NANOS3 + , SOX17 + , TFAP2C + , PRDM1 + , OCT4 + , CD38 + ,EPCAM + , ITGA6 + and / or SOX2 - Producing PGCLCs The method includes:

[0140] 26. The method of paragraph 25, wherein the engineered polynucleotide is a transposon and the delivery further comprises delivering a transposase to the PSC.

[0141] 27. The method of paragraph 25 or 26, wherein the inducible promoter is a chemically inducible promoter, optionally a doxycycline-inducible promoter.

[0142] 28. The method of any one of paragraphs 25 to 27, wherein the feeder-free, serum-free culture medium of (b) comprises a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

[0143] 29. The method of paragraph 28, wherein the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

[0144] 30. The method of paragraph 29, wherein the ECM protein is selected from laminin, collagen IV, heparan sulfate proteoglycan and entactin / nidogen.

[0145] 31. The method according to any one of paragraphs 25 to 30, wherein the feeder-free, serum-free culture medium of (b) comprises a growth factor selected from recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ).

[0146] 32. The method of any one of paragraphs 25 to 31, wherein the culturing in (b) is for about 6 to about 24 hours.

[0147] 33.(c) The expanded population of PSCs is approximately 2,000 cells / cm 2 ~Approx. 3,000 cells / cm 2 33. The method of any one of paragraphs 25 to 32, wherein the cells are cultured at a density of

[0148] 34. The method of any one of paragraphs 25 to 33, wherein the culturing in (c) includes culturing the PSCs in a first induction medium, culturing the PSCs in a second induction medium, culturing the PSCs in a third induction medium, and culturing the PSCs in a fourth induction medium.

[0149] 35. The method of paragraph 34, 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.

[0150] 36. The method of paragraph 34 or 35, wherein the second induction medium comprises one or more of B-27, an inducer (e.g., doxycycline), a small molecule inhibitor of tankyrase (TNKS), and human bone morphogenetic protein 4 (hBMP4).

[0151] 37. The method of any one of paragraphs 34 to 36, wherein the third induction medium comprises one or more of B-27, an inducer (e.g., doxycycline), a small molecule inhibitor of tankyrase, stem cell factor (SCF), and epidermal growth factor (EGF).

[0152] 38. The method of any one of paragraphs 34 to 37, wherein the fourth induction medium comprises one or more of B-27, an inducer (e.g., doxycycline), a small molecule inhibitor of tankyrase, hBMP4, SCF, and EGF.

[0153] 39. A primordial germ cell-like cell produced by the method according to any one of the above paragraphs. EXAMPLES

[0154] Working Example Example 1. Production of primordial germ cells Primordial germ cells (PGCs) are the origin of gametogenesis and serve as the progenitor cell type for both oocyte and spermatocyte development. Recently, numerous techniques have been developed to differentiate human primordial germ cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs) (Mitsunage et al. 2017). Recently, a new monolayer hPGCLC differentiation method has been developed that induces hPGCLC formation without embryoid bodies, allowing for ease of use and scalability. Although many hPGCLC induction methods exist, all suffer from significant heterogeneity in hPGCLC yields depending on the cell line utilized, and many cell lines are no longer used for germ cell formation. As primordial germ cells are utilized as input cell types for in vitro ovarian and testicular reconstruction, a method for high-yield primordial germ cell formation is needed. To partially address these needs, we developed a transcription factor (TF) overexpression-based technique that significantly enhances primordial germ cell yields.

[0155] Fifty-three TFs were screened for their ability to induce robust germ cell formation from induced pluripotent stem cells (hiPSCs) (Figure 1A). DLX5, HHEX, and FIGLA were three TFs identified in the screen. Overexpression of these three TFs throughout the cytokine-based germ cell induction process induced a robust increase in NANOS3+ ​​germ cell yield. Importantly, none of these three TFs have been previously described in the primordial germ cell formation process, and no protocol has been previously described that uses overexpression of these three TFs to increase primordial germ cell yield.

[0156] Overexpression of the three TFs individually increases primordial germ cell yield, as seen in the percentage of NANOS3 (germ cell marker) cells, by 35-80 fold (Figure 1B). Notably, the utilized germ cell lineages largely lacked germ cell formation potential, showing an average of 0.2-3% native germ cell yield. When these TFs were added individually under doxycycline induction, the hPGCLC yield was boosted to 10-35%. The TF-induced germ cells were indeed determined to be bona fide germ cells based on their gene and protein expression. As can be seen, the TF-induced germ cells show characteristic expression of SOX17, TFAP2C and PRDM1, accompanied by upregulation of germ cell genes such as NANOS3 and downregulation of hiPSC genes such as SOX2 (Figure 2A). In addition, as seen from immunofluorescence imaging, the TF-induced germ cells show characteristic OCT4 and SOX17 double positive protein expression (Figure 2B).

[0157] TFs were further characterized to determine whether they exhibited dose-dependence, time point of induction specificity, and induction efficiency in the absence of cytokines. In general, germ cell yield was determined to increase with TF dose, reaching a peak of maximum protein expression at approximately 400ng-600ng doxycycline (Figure 3A). In addition, overexpression of TFs throughout the germ cell formation process was determined to be generally beneficial, with HHEX being useful at the early mesoderm step (Figure 3B). DLX5 overexpression induced 50% germ cell formation restoration compared to wild type in the absence of BMP4, and thus may be used to help reduce or eliminate cytokine dependency on the differentiation process (Figure 3C). Individual independent expression of FIGLA, DLX5, or HHEX was beneficial, with combinations of two or all three showing minimal additive or even deleterious effects (Figures 5 and 7).

[0158] Using the culture medium composition described in Table 1, we designed a method for high yield germ cell formation from stem cells in monolayer culture conditions, as described in more detail below and in Figure 4. The TFs DLX5, HHEX or FIGLA or all three together were integrated into iPSCs via piggyBac or lentivirus, and purified pools were selected via antibiotic addition. For germ cell induction, cells were cultured in mTeSR™ medium on Corning® Matrigel® matrix, per cm 2 2,500 TF-containing iPSCs were seeded per well for 6 hours. After approximately 6 hours, the medium was removed, washed, and replaced with medium #1, whose ingredients are listed in Table 1. After approximately 12-18 hours, medium #1 was removed, the cells were washed again, and medium #2 was added. The cells were then grown for approximately 24 hours, after which medium #2 was replaced with medium #3. The cells were again grown for approximately 24 hours, after which medium #3 was replaced with medium #4. Finally, the cells were again grown for approximately 24 hours, at which point primordial germ cells were harvested and isolated via FACS.

[0159] This process did not require embryoid body formation, was fully monolayer, and was completed in approximately 5-6 days. However, these TFs showed induction ability in both embryoid bodies (EBs) and monolayers (Figure 6), making it broadly applicable to many paradigms of germ cell formation. This process is fairly scalable, and larger numbers of germ cells can be easily obtained by performing the same protocol in larger plates. Without being bound by a particular theory, this process is also highly amendable to high-throughput screening that allows simple parallelization to examine culture conditions, drug interactions, or other developmental processes.

[0160] Methods and Materials iPSC culture iPSCs were cultured in mTESR1 medium (Stemcell Technologies) on standard polystyrene plates coated with hESC-qualified Corning® Matrigel® matrix. Medium was changed daily. Passages were performed using TRYPLE (Gibco). After each passage, iPSCs were treated with approximately 8-12 μM Y-27632 (Ambeed) for 24 hours. Mycoplasma testing was performed by PCR every 3 months, and all tested cells were negative.

[0161] TF plasmid construction TF cDNAs were synthesized as full-length transcripts or obtained from ORFeome (The ORFeome Colobration, Nat Methods. 13, 191-192 (2016)) as Gateway entry clones. They were cloned into doxycycline-inducible PiggyBac expression plasmids (Addgene #175503) using MegaGate (Kramme et al., STAR Protoc. 2, 100907 (2021)). Final expression constructs were verified by Sanger sequencing, which was also used to determine the barcode sequence for each TF.

[0162] Integration of TF plasmids into hiPSCs An expression plasmid containing TF cDNA under the control of a doxycycline-inducible promoter was integrated into iPSCs using PiggyBac transposase. To perform the integration, approximately 50-100 fmol of TF cDNA plasmid, approximately 150-250 ng PiggyBac transposase expression plasmid, and approximately 100,000-200,000 iPSCs were combined using Lonza P3 buffer and electroporated using a Lonza Nucleofector 4D. After electroporation, cells were seeded in 24-well plates in mTeSR™ Plus medium + approximately 8-12 μM Y-27632. 48 hours after electroporation, selection with the appropriate agent (typically puromycin) was initiated and continued for approximately 3-5 days. Cells were then passaged for approximately 3 days without drug selection to allow for non-integrated plasmid loss. Finally, cells were passaged again under drug selection to generate a pure selected integrant pool. The presence and approximate copy number of integrated TF plasmid was confirmed by qPCR on genomic DNA. In oogonia, hiPSC pools and no single cell selected clones were used. The average copy number was 8-10.

[0163] Protocol for primordial germ cell induction via TF overexpression hiPSCs containing integrated TF expression plasmids were cultured on Matrigel in mTeSR1 medium. For monolayer induction, hiPSCs were dissociated into single cells using Accutase and plated at 2,500–3,000 cells / cm. 2hPGCLCs were seeded on Matrigel or Vitronectin XF coated plates in mTeSR™ medium + about 8-10 μM Y-27632 and about 0.5-3 μg / ml doxycycline at a density of 1000 μg / ml for about 6 hours. The medium was then removed, washed with dPBS, and replaced with aRB27 medium #1 (see ingredients list for detailed protocol). After about 12-18 hours of induction, medium #1 was removed, washed with dPBS, and replaced with medium #2. After about 24 hours, medium #2 was removed and replaced with medium #3. After about 24 hours, medium #3 was replaced with medium #4. After about 24 hours in medium #4 or additionally after about 2 more days of culture in medium #4 (day 6 of the protocol), hPGCLCs were harvested for use. hPGCLCs were isolated via NANOS3 reporter expression, CD38 cell surface expression, a combination of both, or EPCAM / ITGA6 double positive cell surface markers. In addition, hPGCLCs can be generated with similar efficiency by embryoid body formation compared to monolayer protocols via methods established in Yamashiro et al. Science, 362(6412), 356-360, Kobayashi et al., Stem Cell Reports, 9(3), 999-1015, Stem Cell Reports, 9(3), 999-1015, Murase et al., The EMBO Journal, 1-25, 2020 and Mitsunaga et al., Proceedings of the National Academy of Sciences of the United States of America, 114(46), E9913-E9922.

[0164] [Table 1]

[0165] [Table 2]

[0166] [Table 3]

[0167] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may include the entire document.

[0168] The indefinite articles "a" and "an," as used in the specification and claims, should be understood to mean "at least one," unless clearly indicated to the contrary.

[0169] It is also to be understood that in any method claimed herein that includes two or more steps or actions, unless clearly indicated to the contrary, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are described.

[0170] In the claims and the foregoing specification, transitional phrases such as "comprise," "include," "carry," "have," "contain," "involve," "hold," "comprise," and the like, are all to be understood as open-ended, i.e., to mean inclusive, but not exclusive. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the United States Patent Office Manual of Patent Examining Procedure.

[0171] The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value.

[0172] When a range of values ​​is provided, each value therebetween, including the upper and lower limits of the range, is specifically contemplated and described herein.

Claims

1. Pluripotent stem cells (PSCs) containing engineering polynucleotides, including an open reading frame encoding a protein selected from DLX5, HHEX, and FIGLA.

2. The PSC according to claim 1, which expresses or overexpresses DLX5, HHEX, FIGLA, DLX5 and HHEX, DLX5 and FIGLA, HHEX and FIGLA, or DLX5, HHEX and FIGLA.

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

4. A pluripotent stem cell (PSC) comprising a protein selected from DLX5, HHEX, and FIGLA, wherein the protein is overexpressed in the pluripotent stem cell (PSC).

5. The PSC according to claim 4, which expresses or overexpresses DLX5, HHEX, FIGLA, DLX5 and HHEX, DLX5 and FIGLA, HHEX and FIGLA, or DLX5, HHEX and FIGLA.

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

7. The PSC according to claim 1, comprising 1 to 20 or 8 to 10 copies of the engineering polynucleotide, which includes the open reading frame encoding the protein selected from DLX5, HHEX, and FIGLA.

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

9. 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 DLX5, HHEX, and FIGLA in the PSCs of the expanded population to produce PGCLCs.

10. The method according to claim 9, wherein the PSC of the expanded population comprises an engineering polynucleotide including an open reading frame encoding a protein selected from DLX5, HHEX, and FIGLA.

11. The method according to claim 9, wherein the open reading frame of the engineering-operated polynucleotide is operably linked to a heterologous promoter or an inducible promoter.

12. The aforementioned group is 1 × 10 ２ ~1 x 10 ７ The method according to claim 9, comprising a PSC.

13. The method according to claim 9, wherein the group of PSCs is cultured for about 3 to 5 days or about 4 days.

14. The PGCLC is NANOS3 ＋ , SOX17 ＋ , TFAP2C ＋ , PRDM1 ＋ , OCT4 ＋ , CD38 ＋ , EPCAM ＋ , ITGA6 ＋ and / or SOX2 － The method according to claim 9, wherein the PGCLC is as described above.

15. (a) Delivering an engineered polynucleotide to pluripotent stem cells (PSCs) that includes an inducible promoter operably linked to an open reading frame encoding a protein selected from DLX5, HHEX, and FIGLA, (b) Culturing the PSCs in a feeder-free, serum-free culture medium to produce an expanded population of PSCs, (c) Culturing the expanded population of PSCs in a series of induction media containing an inducer to obtain NANOS3 ＋ SOX17 ＋ TFAP2C ＋ PRDM1 ＋ , OCT4 ＋ CD38 ＋ EPCAM ＋ ITGA6 ＋ and / or SOX2 － To produce PGCLCs and A method that includes this.

16. The method according to claim 15, wherein the engineering polynucleotide is a transposon, and the delivery further comprises delivering a transposase to the PSC.

17. The method according to claim 15, wherein the inducible promoter is a chemically inducible promoter or a doxycycline-inducible promoter.

18. (b) The feeder-free serum-free culture medium is Solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma; or Growth factors selected from recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ). The method according to claim 15, including the method described in claim 15.

19. The method according to claim 18, wherein the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

20. The method according to claim 19, wherein the ECM protein is selected from laminin, collagen IV, heparan sulfate proteoglycan, and entactin / nidogen.

21. The method according to claim 15, wherein the culture in (b) lasts for about 6 to about 24 hours.

22. The method according to claim 15, wherein the culture in (c) comprises culturing the PSCs at a density of about 2,000 cells / cm² to about 3,000 cells / cm², or the culture in (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.

23. The first induction medium comprises B-27, L-alanyl-L-glutamine, an inducer, activin A, a glycogen synthase kinase (GSK) 3 inhibitor, and one or more selective FGFR1 and FGFR3 inhibitors. The second induction medium comprises one or more of B-27, an inducer, a low-molecular-weight inhibitor of tankyrase (TNKS), and human bone morphogenetic protein 4 (hBMP4). The third induction medium comprises one or more of B-27, an inducer, a low-molecular-weight inhibitor of tankirase, stem cell factor (SCF), and epidermal growth factor (EGF), and / or The method according to claim 22, wherein the fourth induction medium comprises one or more of B-27, an inducer, a low molecular weight inhibitor of tankirase, hBMP4, SCF, and EGF.

24. The method according to claim 23, wherein the inducer of the first inducement medium, the second inducement medium, the third inducement medium, and / or the fourth inducement medium comprises doxycycline.

25. Primordial germ cell-like cells produced by the method of claim 15.

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