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 (e.g., DLX5, HHEX, and / or FIGLA).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 326,656, 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 (H049870759WO00-SEQ-KVC.xml; size: 4,453 bytes; creation date: March 27, 2023) are incorporated herein by reference in their entirety.

[0005] background

[0006] Primordial germ cells are gamete-producing germline stem cells in vertebrates. Primordial germ cells migrate to the developing gonads, where they differentiate into sperm or eggs. Primordial germ cell dysfunction underlies multiple forms of human female infertility, but effective methods for generating primordial germ cells in vitro remain unclear.

[0007] Overview

[0008] 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 provides experimental data that unexpectedly demonstrates that overexpression of certain transcription factors (e.g., DLX5, HHEX, and FIGLA) is sufficient to generate PGCLCs (e.g., NANOS3) from PSCs in as little as four days. + 、SOX17 + TFAP2C + PRDM1 + 、OCT4 + 、CD38 + ,EPCAM + 、ITGA6 + and / or SOX2 - of PGCLC).

[0009] Certain aspects of the present disclosure provide a PSC comprising: an engineered polynucleotide comprising an open reading frame encoding a protein selected from the group consisting of DLX5, HHEX, and FIGLA.

[0010] In some embodiments, the PSC comprises an engineered polynucleotide comprising an open reading frame encoding DLX5.

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

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

[0013] In some embodiments, the PSC expresses or overexpresses: DLX5; HHEX; FIGLA; DLX5 and HHEX; DLX5 and FIGLA; HHEX and FIGLA; or DLX5, HHEX, and FIGLA.

[0014] In some embodiments, the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0015] In some embodiments, the heterologous promoter is an inducible promoter.

[0016] Other aspects of the present disclosure provide a PSC comprising: a protein selected from the group consisting of DLX5, HHEX and FIGLA, wherein the protein is overexpressed.

[0017] In some embodiments, the PSC expresses or overexpresses: DLX5; HHEX; FIGLA; DLX5 and HHEX; DLX5 and FIGLA; HHEX and FIGLA; or DLX5, HHEX, and FIGLA.

[0018] In some embodiments, the PSC is a human PSC.

[0019] In some embodiments, the PSC is an induced PSC (iPSC).

[0020] In some embodiments, 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 DLX5, HHEX, and FIGLA.

[0021] Other aspects of the present disclosure provide a composition comprising: a population of PSCs as described in any of the preceding paragraphs or elsewhere herein.

[0022] In some embodiments, the population comprises at least 2500 cells / cm 2 PSC.

[0023] Some 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 DLX5, HHEX, and FIGLA in the PSCs of the expanded population to produce PGCLCs.

[0024] In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding DLX5.

[0025] In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding HHEX.

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

[0027] In some embodiments, the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0028] In some embodiments, the heterologous promoter is an inducible promoter.

[0029] In some embodiments, the population comprises 1x10 2 -1x10 7 PSCs.

[0030] In some embodiments, the population of PSCs is cultured for about 3-5 days.

[0031] In some embodiments, the population of PSCs is cultured for about 4 days.

[0032] In some embodiments, the PGCLC is NANOS3 + 、SOX17 + TFAP2C + PRDM1 + 、OCT4 + 、CD38 + ,EPCAM + 、ITGA6 + and / or SOX2 - PGCLC.

[0033] Some aspects of the present disclosure provide a method comprising:

[0034] (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 DLX5, HHEX and FIGLA to pluripotent stem cells (PSCs);

[0035] (b) culturing the PSCs in a feeder-free, serum-free medium to produce an expanded population of PSCs; and

[0036] (c) culturing the expanded population of PSCs in a series of induction media containing an induction agent to produce NANOS3 + 、SOX17+ TFAP2C + PRDM1 + 、OCT4 + 、CD38 + ,EPCAM + 、ITGA6 + and / or SOX2 - PGCLC.

[0037] In some embodiments, the engineered polynucleotide is a transposon, and the delivering further comprises delivering a transposase to the PSC.

[0038] In some embodiments, the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

[0039] 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.

[0040] In some embodiments, the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

[0041] In some embodiments, the ECM protein is selected from laminin, collagen IV, heparin sulfate proteoglycans, and entactin / nidogen.

[0042] 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).

[0043] In some embodiments, the culturing of (b) continues for about 6 to about 24 hours.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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).

[0048] 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).

[0049] 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.

[0050] Other aspects of the disclosure provide PGCLCs produced by the method of any one of the preceding claims.

[0051] 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.

[0052] BRIEF DESCRIPTION OF THE DRAWINGS

[0053] 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:

[0054] Figure 1A-1B Identification of overexpressed transcription factors (TFs) driving increased NANOS3+ ​​primordial germ cell yield is shown. Figure 1A TF was shown to be integrated into each line of NANOS3-mVenus PSCs and overexpressed by doxycycline induction during monolayer primordial germ cell formation. hPGCLC yields were compared under dox plus versus dox minus conditions and compared to no TF control conditions. Figure 1B Display TF DLX5, HHEX and FIGLA through the Figure 1A The same protocol discussed in enhanced NANOS3+ ​​hPGCLC yield.

[0055] Figure 2A-2BTranscriptomic and proteomic analyses are shown, indicating that TFs drive on-target primordial germ cell formation. Figure 2A Transcriptomic 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 by FACS after TF-based or control induction and subjected to RNA sequencing. Figure 2B Analysis of key features of hPGCLC protein expression is shown. Immunofluorescence was performed on TF-induced hPGCLCs to detect integrin (ITGA6), OCT4, and SOX17 expression.

[0056] Figures 3A-3C Characterization of TF dynamics by dose, time series, and cytokine withdrawal is shown. Figure 3A The hPGCLC yields assessed after TF induction under control conditions, in the absence of cytokines and hBMP4 are shown. The change in yield compared to normal conditions is plotted, showing that DLX5 retains 40% of its activity in the absence of hBMP4. Figure 3B Shown are hPGCLC yields assessed at doxycycline dilutions, indicating that increased doxycycline generally increases hPGCLC yields following TF induction. Figure 3C Shown are hPGCLC yields assessed after addition of doxycycline at various time points, indicating that TF is generally beneficial when expressed throughout the differentiation process.

[0057] Figure 4 A schematic diagram of the TF-assisted hPGCLC formation method is shown.

[0058] Figure 5 TF induced an increase in the yield of hPGCLCs of different markers.

[0059] Figure 6 The increase in hPGCLC yield in the TF-induced differentiation platform was shown.

[0060] Figure 7 Shown are the TF combination tests for hPGCLC formation.

[0061] Detailed Description

[0062] Primordial germ cells (PGCs) are the origin of gametogenesis and serve as the ancestral cell type for the development of oocytes and spermatocytes (McLaren et al., 2003). Recently, a large number of 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, new monolayer hPGCLC differentiation methods have been developed that can induce hPGCLC formation without embryoid bodies, making them easy to use and scalable. Although there are many hPGCLC induction methods, there is a great heterogeneity in the hPGCLC yield of all methods, depending on the cell line used, and many cell lines cannot be used for germ cell formation. Since primordial germ cells are used as input cell types for in vitro ovarian and testicular reconstruction, methods for high-yield primordial germ cell formation are needed. Aspects of the present disclosure relate to methods of inducing stem cell differentiation into NANOS3+ ​​(Nanos C2HC-type zinc finger 3), SOX17+ (SRY-Box transcription factor 17), TFAP2C+ (transcription factor AP-2γ), PRDM1+ (PR / SET domain 1), OCT4+ (octamer binding transcription factor 4) and / or SOX2- (SRY-Box transcription factor 2) primordial germ cells using direct transcription factor overexpression. It should be understood that the term "PGCLC" encompasses cells that express primordial germ cell-specific markers (e.g., NANOS3, SOX17, TFAP2C, PRDM1, OCT4, CD38, EPCAM and / or ITGA6) and / or do not express SOX2 and exhibit other characteristics of naturally occurring primordial germ cells.

[0063] Primordial germ cell-like cells

[0064] 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) that produce new organisms that can generate countless new generations through germ cells. PGCs represent the founder cells of the germ line. PGCs are specialized during early mammalian post-implantation development and are uniquely programmed to pass genetic and epigenetic information to offspring. Primordial germ cells are single cells that can form cell colonies with morphology similar to undifferentiated embryonic stem cells under certain culture conditions.

[0065] Primordial germ cells and PGCLC express many different biomarkers that can be used to distinguish PGCLC from other cell types. For example, PGCLC cells are often positive for Nanos C2HC-type zinc finger 3 (NANOS3), SRY-Box transcription factor 17 (SOX17), transcription factor AP-2γ (TFAP2C), PR / SET domain 1 (PRDM1), octamer binding transcription factor 4 (OCT4), and / or negative for SRY-Box transcription factor 2 (SOX2).

[0066] PGCLCs also have other features that are different from non-PGCLCs, including but not limited to their overall decreased 5-methylcytosine and H3K9me2 levels compared with stem cells, as well as their CXCL12 / SDF1-directed chemotactic motility and cytoplasmic granules.

[0067] Pluripotent stem cells

[0068] The PGCLCs provided herein are differentiated from pluripotent stem cells. Pluripotent stem cells are cells that have the ability to self-renew by division and are able 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 adult body, excluding extraembryonic tissues such as the placenta (Shi et al., 2017).

[0069] 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.

[0070] 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.

[0071] 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.

[0072] Some aspects of the present disclosure provide a PSC comprising: a protein selected from DLX5, HHEX and FIGLA, 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 naturally occurring pluripotent stem cells. 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.

[0073] The data presented here demonstrate that expression of only one of DLX5, HHEX, or FIGLA outperforms the combined expression of all three. For example, combined expression of DLX5, HHEX, and FIGLA in PSCs resulted in NANOS3 expression compared to no TF controls. + 、SOX17 + TFAP2C + PRDM1 + 、OCT4 + 、CD38 + ,EPCAM + 、ITGA6 + and / or SOX2 - The efficiency of PGCLC generation increased by 2-fold. However, unexpectedly, the combined expression of DLX5, HHEX, and FIGLA in PSCs resulted in a 5- to 45-fold decrease in efficiency compared to PSC controls expressing only one of DLX5, HHEX, or FIGLA. Thus, in some embodiments, the PSCs comprise DLX5, HHEX, or FIGLA, but not all three together.

[0074] 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.

[0075] Transcription Factors

[0076] In some embodiments, the PGCLCs 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 transcription rates). Differentiation is the process by which an uncommitted cell or partially committed cell is directed to a specialized cell fate. Aspects of the present disclosure relate to the differentiation of uncommitted pluripotent stem cells to a PGCLC fate.

[0077] In some embodiments, the transcription factor is selected from DLX5, HHEX and FIGLA. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress DLX5. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress HHEX. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress FIGLA. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress DLX5 and HHEX. In some embodiments, pluripotent stem cells, such as hPSC or hiPSC, are engineered to express or overexpress DLX5 and FIGLA. In some embodiments, pluripotent stem cells (such as hPSC or hiPSC) are engineered to express or overexpress HHEX and FIGLA. In some embodiments, pluripotent stem cells (such as hPSC or hiPSC) are engineered to express or overexpress DLX5, HHEX and FIGLA.

[0078] 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.

[0079] Engineered polynucleotides and peptides

[0080] 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.

[0081] 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).

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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).

[0086] 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.

[0087] 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).

[0088] In some embodiments, the pluripotent stem cell comprises an engineered polynucleotide comprising 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.

[0089] 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 DLX5 and an engineered polynucleotide containing an open reading frame encoding FIGLA. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding HHEX and an engineered polynucleotide containing an open reading frame encoding FIGLA. In some embodiments, the pluripotent stem cells comprise an engineered polynucleotide containing an open reading frame encoding DLX5, an engineered polynucleotide containing an open reading frame encoding HHEX, and an engineered polynucleotide containing an open reading frame encoding FIGLA.

[0090] 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:

[0091] MDPAPGVLDPRAAPPALLGTPQAEVLEDVLREQFGPLPQLAAVCRLKRLPSGGYSSTENLQLVLERRRVANAKERERIKNLNRGFARLKALVPFLPQSRKPSKVDILKGAT EYIQVLSDLLEGAKDSKKQDPDEQSYSNNSSESHTSSARQLSRNITQHISCAFGLKNEEEGPWADGGSGEPAHACRHSVMSTTEIISPTRSLDRFPEVELLSHRLPQV(SEQ ID NO:1)

[0092] 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:

[0093] MTGVFDRRRVPSIRSGDFQAPFQTSAAMHHPSQESPTLPESSATDSDYYSPTGGAPHGYCSPTSASYGKALNPYQYQYHGVNGSAGSYPAKAYADYSYASSYHQYGGAYNRVPSATNQPEKEVTEPEVRMVNGKPKKVRKPRTIYSS FQLAALQRRFQKTQYLALPERAELAASLGLTQTQVKIWFQNKRSKIKKIMKNGEMPPEHSPSSSDPMACNSPQSPAVWEPQGSSRSLSHHPHAHPPTSNQSPASSYLENSASWYTSAASSINSHLPPPGSLQHPLALASGTLY(SEQ IDNO: 2)

[0094] 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:

[0095] MQYPHPGPAAGAVGVPLYAPTPLLQPAHPTPFYIEDILGRGPAAPTPAPTLPSPNSSFTSLVSPYRTPVYEPTPIHPAFSHHSAAALAAAYGPGGFGGPLYPFPRTVNDYTHALLRHDPLGKPLLWSPFLQRPLHKR KGGQVRFSNDQTIELEKKFETQKYLSPPERKRLAKMLQLSERQVKTWFQNRRAKWRRLKQENPQSNKKEELESLDSSCDQRQDLPSEQNKGASLDSSQCSPSPASQEDLESEISEDSDQEVDIEGDKSYFNAG(SEQ ID NO:3)

[0096] 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 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.

[0097] Methods for Producing PGCLCs

[0098] In some aspects, the method of producing PGCLCs provided herein comprises 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.

[0099] In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide containing an open reading frame encoding DLX5. In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide containing an open reading frame encoding HHEX. In some embodiments, the PSCs of the expanded population comprise an engineered polynucleotide containing an open reading frame encoding FIGLA.

[0100] In some embodiments, the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0101] In some embodiments, the heterologous promoter is an inducible promoter, non-limiting examples of which are provided elsewhere herein.

[0102] 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 1x10 1 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.

[0103] 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.

[0104] 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 DLX5, HHEX, and FIGLA 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 NANOS3. + 、SOX17 + TFAP2C + PRDM1 + 、OCT4 + 、CD38 + ,EPCAM + 、ITGA6 + and / or SOX2 -PGCLC. 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.

[0105] 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.

[0106] 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.

[0107] 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 2In 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 2 PSC, 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 / 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 / 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

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] Transfection method

[0114] 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.

[0115] 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.

[0116] 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).

[0117] 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.

[0118] Thus, in some embodiments, the method further comprises delivering a transposon comprising the engineered polynucleotide to the PSC, and also delivering a transposase.

[0119] 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.

[0120] 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 integrated engineered polynucleotides can be obtained. In some embodiments, antibiotic drug selection is used to select a cell population containing integrated engineered polynucleotides. 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.

[0121] Culture medium

[0122] 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.

[0123] 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).

[0124] 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.

[0125] 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).

[0126] 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.

[0127] 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.

[0128] 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).

[0129] GlutaMAX TM The supplement contains L-alanyl-L-glutamine, which is a dipeptide substitute for L-glutamine.

[0130] Activin-A is a dimeric glycoprotein that belongs to the transforming growth factor-β (TGF-β) family.

[0131] 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.

[0132] 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).

[0133] 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.

[0134] Therapeutic compositions and methods of use

[0135] In some embodiments, the present disclosure provides a therapeutic composition comprising the PGCLC produced herein. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition is cryopreserved.

[0136] 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.

[0137] In some embodiments, the subject is a human subject. Subjects who may benefit from such compositions include patients struggling with male or female factor infertility, wherein such patients are unable to produce viable gametes.

[0138] The composition can be administered to a subject in a therapeutically effective amount. The term "therapeutically effective amount" refers to the amount of PGCLC required to produce a therapeutic effect on a subject alone or in combination with at least one other active agent. As recognized 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 easily determined by those skilled in the art, and it can be a polypeptide of the present disclosure in micrograms. The dosage of the formulation disclosed herein may depend on the route of administration and vary according to the size of the subject.

[0139] 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.

[0140] Additional Implementation

[0141] The present disclosure also relates to additional embodiments listed in the following numbered paragraphs:

[0142] 1. A pluripotent stem cell (PSC), comprising: an engineered polynucleotide containing an open reading frame encoding a protein selected from the group consisting of DLX5, HHEX and FIGLA.

[0143] 2. The PSC of paragraph 1, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding DLX5.

[0144] 3. The PSC of paragraph 1 or 2, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding HHEX.

[0145] 4. The PSC of any of the preceding paragraphs, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding FIGLA.

[0146] 5. The PSC of any of the preceding paragraphs, wherein the PSC expresses or overexpresses: DLX5; HHEX; FIGLA; DLX5 and HHEX; DLX5 and FIGLA; HHEX and FIGLA; or DLX5, HHEX, and FIGLA.

[0147] 6. The PSC of any of the preceding paragraphs, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0148] 7. The PSC of paragraph 6, wherein the heterologous promoter is an inducible promoter.

[0149] 8. A pluripotent stem cell (PSC), comprising: a protein selected from the group consisting of DLX5, HHEX and FIGLA, wherein the protein is overexpressed.

[0150] 9. The PSC of paragraph 8, wherein the PSC expresses or overexpresses: DLX5; HHEX; FIGLA; DLX5 and HHEX; DLX5 and FIGLA; HHEX and FIGLA; or DLX5, HHEX and FIGLA.

[0151] 10. The PSC of any of the preceding paragraphs, wherein the PSC is a human PSC.

[0152] 11. The PSC of any of the preceding paragraphs, wherein the PSC is an induced PSC (iPSC).

[0153] 12. 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 DLX5, HHEX and FIGLA.

[0154] 13. A composition comprising: a population of PSCs as described in any of the preceding paragraphs or elsewhere herein.

[0155] 14. The composition of paragraph 13, wherein the population comprises at least 2500 cells / cm 2 PSC.

[0156] 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 the group consisting of DLX5, HHEX and FIGLA in the PSCs of the expanded population to produce PGCLCs.

[0157] 16. The method of paragraph 15, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding DLX5.

[0158] 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.

[0159] 18. The method of any of paragraphs 15-17, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding FIGLA.

[0160] 19. The method of any of the preceding paragraphs, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

[0161] 20. The method of any of the preceding paragraphs, wherein the heterologous promoter is an inducible promoter.

[0162] 21. The method of any of the preceding paragraphs, wherein the population comprises 1x10 2 -1x10 7 PSCs.

[0163] 22. The method of any of the preceding paragraphs, wherein the population of PSCs is cultured for about 3-5 days.

[0164] 23. The method of paragraph 22, wherein the population of PSCs is cultured for about 4 days.

[0165] 24. The method of any of the preceding paragraphs, wherein the PGCLC is NANOS3 + 、SOX17 + TFAP2C + PRDM1 + 、OCT4 + 、CD38 + ,EPCAM + 、ITGA6 + and / or SOX2 - PGCLC.

[0166] 25. A method comprising:

[0167] (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 DLX5, HHEX and FIGLA to pluripotent stem cells (PSCs);

[0168] (b) culturing the PSCs in a feeder-free, serum-free medium to produce an expanded population of PSCs; and

[0169] (c) culturing the expanded population of PSCs in a series of induction media containing an induction agent to produce NANOS3 + 、SOX17 + TFAP2C + PRDM1 + 、OCT4 + 、CD38 + ,EPCAM + 、ITGA6 + and / or SOX2 - PGCLC.

[0170] 26. The method of paragraph 25, wherein the engineered polynucleotide is a transposon, and the delivering further comprises delivering a transposase to the PSC.

[0171] 27. The method of paragraph 25 or 26, wherein the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

[0172] 28. The method of any of paragraphs 25-27, wherein the feeder-free, serum-free medium of (b) comprises a solubilized basement membrane preparation extracted from an Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

[0173] 29. The method of paragraph 28, wherein the solubilized basement membrane preparation comprises extracellular matrix (ECM) proteins and growth factors.

[0174] 30. The method of paragraph 29, wherein the ECM protein is selected from the group consisting of laminin, collagen IV, heparin sulfate proteoglycans, and paranectin / nidogen.

[0175] 31. The method of any of paragraphs 25-30, 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).

[0176] 32. The method of any of paragraphs 25-31, wherein the culturing of (b) continues for about 6 to about 24 hours.

[0177] 33. The method of any of paragraphs 25-32, wherein the PSCs of the expanded population of (c) are approximately 2,000 cells / cm 2 About 3,000 cells / cm 2 Density culture.

[0178] 34. The method of any one of paragraphs 25-33, 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.

[0179] 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.

[0180] 36. The method of paragraph 34 or 35, 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).

[0181] 37. The method of any of paragraphs 34-36, 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).

[0182] 38. The method of any of paragraphs 34-37, 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.

[0183] 39. A primordial germ cell-like cell produced by the method of any of the preceding paragraphs. Example

[0184] Example 1. Generation of Primordial Germ Cells

[0185] Primordial germ cells (PGCs) are the origin of gametogenesis and serve as the ancestral cell type for the development of oocytes and spermatocytes. Recently, a large number of technologies have been developed to differentiate human primordial germ cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs) (Mitsunage et al., 2017). Recently, new monolayer hPGCLC differentiation methods have been developed that can induce hPGCLC formation without the need for embryoid bodies, making them easy to use and scalable. Although there are many methods for hPGCLC induction, there is a great heterogeneity in the hPGCLC yield of all methods, depending on the cell line used, and many cell lines cannot be used for germ cell formation. Since primordial germ cells are used as the input cell type for in vitro ovarian and testicular reconstruction, methods for high-yield primordial germ cell formation are needed. Partly to meet these needs, a technology based on transcription factor (TF) overexpression was developed that significantly improves primordial germ cell yield.

[0186] 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 are three TFs identified in the screen. Overexpression of these three TFs induces a robust increase in NANOS3+ ​​germ cell yield during cytokine-based germ cell induction. Importantly, none of these three TFs have been previously described in the process of primordial germ cell formation, and to date there has been no protocol using overexpression of these three TFs to increase primordial germ cell yield.

[0187] Overexpression of the three TFs alone showed a 35- to 80-fold increase in primordial germ cell yield, as evidenced by the percentage of NANOS3 (germ cell marker) cells ( Figure 1B). It is noteworthy that the germ cell lines used had almost no germ cell competence, showing an average of 0.2 to 3% natural germ cell yield. After the addition of these TFs alone under doxycycline induction, the hPGCLC yield increased to 10-35%. Based on their gene expression and protein expression, it was determined that the TF-induced germ cells are indeed true germ cells. It can be seen that TF-induced germ cells show characteristic expression of SOX17, TFAP2C and PRDM1, as well as upregulation of germ cell genes (such as NANOS3) and downregulation of hiPSC genes (such as SOX2) ( Figure 2A ). In addition, TF-induced germ cells exhibited characteristic OCT4 and SOX17 double-positive protein expression, as can be seen by immunofluorescence imaging ( Figure 2B ).

[0188] TFs were further characterized to determine whether they exhibited dose dependence, induction time point specificity, and induction efficiency in the absence of cytokines. In general, it was determined that germ cell yield increased with increasing TF dose, reaching a peak of maximal protein expression at approximately 400 ng-600 ng doxycycline ( Figure 3A ). In addition, it would be informative to determine whether TF overexpression is generally responsible throughout germ cell formation, with HHEX being useful during early mesoderm steps ( Figure 3B ). It was determined that DLX5 overexpression induced a 50% rescue rate of germ cell formation compared to wild type in the absence of BMP4, suggesting that it could be used to help reduce or eliminate cytokine dependence during differentiation ( Figure 3C Individual independent expression of FIGLA, DLX5, or HHEX was beneficial, whereas combinations of two or all three showed minimal additive or even deleterious effects ( Figure 5 and 7 ).

[0189] A method for generating high-yield germ cells from stem cells in monolayer culture conditions was designed and described below and Figure 4 The composition of the culture medium is described in more detail in , and the composition of the culture medium is shown in Table 1 . TFs DLX5, HHEX, or FIGLA, or all three together, were integrated into iPSCs by piggyBac or lentivirus, and the pools were purified by adding antibiotic selection. To induce germ cells, 2,500 TF-containing iPSCs per cm2 were plated in mTeSR on substrate TMThe cells were then grown in 4% paraformaldehyde for 6 hours in the culture medium. After approximately 6 hours, the culture medium was removed and washed and replaced with culture medium #1, the composition of which is listed in Table 1. After approximately 12-18 hours, culture medium #1 was removed and the cells were washed again, and then culture medium #2 was added. The cells were then grown for approximately 24 hours, and then culture medium #2 was replaced with culture medium #3. The cells were grown again for approximately 24 hours, and then culture medium #3 was replaced with culture medium #4. Finally, the cells were grown again for approximately 24 hours, at which time the primordial germ cells were harvested and isolated by FACS.

[0190] This process is carried out entirely in monolayers, without the need for embryoid body formation, and is completed in approximately 5-6 days. However, these TFs show induction potential in both embryoid bodies (EBs) and monolayers ( Figure 6 ), making it broadly applicable to many paradigms of germ cell formation. The process is highly scalable, where large numbers of germ cells can be easily obtained by performing the same protocol in larger plates. Without being bound by a particular theory, the process is also highly amenable to high-throughput screening and can be easily parallelized to test culture conditions, drug interactions, or other developmental processes.

[0191] Methods and Materials

[0192] iPSC culture

[0193] 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.

[0194] TF plasmid construction

[0195] 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.

[0196] Integration of TF plasmids into hiPSCs

[0197] 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.

[0198] Protocol for inducing primordial germ cells by TF overexpression

[0199] hiPSCs with integrated TF expression plasmids were cultured in mTeSR1 medium on Matrigel. For induction as a monolayer, hiPSCs were dissociated into single cells using Accutase and plated at 2,500-3,000 cells / cm on Matrigel or vitronectin XF-coated plates. 2 The density of mTeSR TMInoculate 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, and replace with aRB27 medium #1 (see the list of ingredients in the detailed scheme). After induction for about 12-18 hours, remove medium #1 and wash with dPBS, and then replace with medium #2. After about 24 hours, remove medium #2 and replace it with medium #3. After about 24 hours, replace medium #3 with medium #4. After about 24 hours of cultivation in medium #4 or additionally in medium #4 for about two days (the 6th day of the scheme), harvest hPGCLC for use. Isolate hPGCLC by NANOS3 reporter expression, CD38 cell surface expression, a combination of the two, or EPCAM / ITGA6 double positive cell surface markers. In addition, hPGCLCs can also be generated through embryoid body formation with similar efficiency compared to 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.

[0200] Table 1. Composition of the culture medium used in Example 1 *Optional antibiotics

[0201] Table 2. Cytokines used in Example 1

[0202]

[0203] Table 3. Inhibitors used in Example 1

[0204] 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

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

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

[0210] 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 the group consisting of DLX5, HHEX and FIGLA.

2. The PSC of claim 1, wherein the PSC comprises an engineered polynucleotide comprising an open reading frame encoding DLX5.

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

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

5. The PSC of claim 1, wherein the PSC expresses or overexpresses: DLX5; HHEX; FIGLA; DLX5 and HHEX; DLX5 and FIGLA; HHEX and FIGLA; or DLX5, HHEX, and FIGLA.

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

7. The PSC of claim 6, wherein the heterologous promoter is an inducible promoter.

8. A pluripotent stem cell (PSC), comprising: a protein selected from the group consisting of DLX5, HHEX and FIGLA, wherein the protein is overexpressed.

9. The PSC of claim 8, wherein the PSC expresses or overexpresses: DLX5; HHEX; FIGLA; DLX5 and HHEX; DLX5 and FIGLA; HHEX and FIGLA; or DLX5, HHEX, and FIGLA.

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

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

12. 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 DLX5, HHEX and FIGLA.

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

14. The composition of claim 13, wherein the population comprises at least 2500 cells / cm 2 PSC.

15. A method comprising: A population of pluripotent stem cells (PSCs) is cultured in a culture medium to produce an expanded population of PSCs; and a protein selected from the group consisting of DLX5, HHEX and FIGLA is expressed in the PSCs of the expanded population to produce PGCLCs.

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

17. The method of claim 15, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding HHEX.

18. The method of claim 15, wherein the PSCs of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding FIGLA.

19. The method of claim 15, wherein the open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter.

20. The method of claim 15, wherein the heterologous promoter is an inducible promoter.

21. The method of claim 15, wherein the population comprises 1 x 10 2 -1x10 7 PSCs.

22. The method of claim 15, wherein the population of PSCs is cultured for about 3-5 days.

23. The method of claim 22, wherein the population of PSCs is cultured for about 4 days.

24. The method of claim 15, wherein the PGCLC is NANOS3 + 、SOX17 + TFAP2C + PRDM1 + 、OCT4 + 、CD38 + ,EPCAM + 、ITGA6 + and / or SOX2 - PGCLC.

25. 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 DLX5, HHEX and FIGLA 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 an induction agent to produce NANOS3 + 、SOX17 + TFAP2C + PRDM1 + 、OCT4 + 、CD38 + ,EPCAM + 、ITGA6 + and / or SOX2 - PGCLC.

26. The method of claim 25, wherein the engineered polynucleotide is a transposon and the delivering further comprises delivering a transposase to the PSC.

27. The method of claim 25, wherein the inducible promoter is a chemically inducible promoter, optionally a doxycycline inducible promoter.

28. The method of claim 25, wherein the feeder-free, serum-free medium of (b) comprises a solubilized basement membrane preparation extracted from an Engelbreth-Holm-Swarm (EHS) mouse sarcoma.

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

30. The method of claim 29, wherein the ECM protein is selected from the group consisting of laminin, collagen IV, heparin sulfate proteoglycans, and paranectin / nidogen.

31. The method of claim 25, 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).

32. The method of claim 25, wherein the culturing of (b) continues for about 6 to about 24 hours.

33. The method of claim 25, wherein the PSCs of the expanded population of (c) are approximately 2,000 cells / cm 2 About 3,000 cells / cm 2 Density culture.

34. The method of claim 25, 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.

35. The method of claim 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.

36. The method of claim 34, 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).

37. The method of claim 34, 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).

38. The method of claim 34, 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.

39. A primordial germ cell-like cell produced by the method of claim 25.

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