Methods and compositions for producing meiotic cells

Abstract

Provided herein are methods and compositions for differentiating induced pluripotent stem cells into meiotic cells by overexpressing BCL2 and one or more of BOLL, HOXB5 and MEIOC.

Description

[0001] METHODS AND COMPOSITIONS FOR PRODUCING MEIOTIC CELLS

[0002] RELATED APPLICATION

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63 / 651,636, filed May 24, 2024, which is incorporated by reference herein in its entirety.

[0004] GOVERNMENT LICENSE RIGHTS

[0005] This invention was made with government support under HD 108898 awarded by National Institutes of Health. The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The content of the electronic sequence listing (H049870818WO00-SEQ-HJD.xml; Size: 7,308 bytes; and Date of Creation: May 21, 2025) is herein incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] Meiosis is a critical step in the formation of gametes (eggs and sperm); thus, activating meiosis is required to produce human gametes in vitro. Additionally, chromosomal recombination during meiosis can be useful for making engineered cell lines. Methods for artificially activate meiosis in human cells are needed.

[0010] SUMMARY

[0011] All sexually reproducing species rely on meiosis to produce haploid gametes from diploid germ cells. To date, the most detailed studies of meiosis have taken place in nonhuman organisms, due to the lack of a reliable in vitro model of human meiosis, as well as technical and ethical barriers to obtaining meiotic cells from humans. Therefore, a method of inducing meiosis in cultured human cells could greatly advance the study of this crucial reproductive process and could also lead to new therapies for people with infertility.

[0012] Research on animals such as mice has revealed important characteristics of mammalian meiosis, including requirements of erasing DNA methylation, as well as retinoic acid and BMP signaling from gonadal somatic cells. Recent studies have demonstrated the initiation of meiosis in mouse cells in vitro, even producing viable offspring from the resulting gametes. Nonetheless, studies attempting to initiate meiosis in human cells have been less successful. These studies based their main conclusions on the production of haploid (IN 1C) cells as assessed by flow cytometry for DNA content. However, this approach has two flaws. First, the IN 1C state is non-physiological in eggs because meiosis is not completed until after fertilization. Second, dead and dying cells with fragmented nuclei can have reduced DNA content, leading to false positives in this assay. Some studies also examined the expression of the meiotic markers SYCP3 and yH2AX (11, 12, 14, 15), but did not convincingly show the expected localization of these proteins during the stages of meiosis, and attempts to reproduce these protocols were unsuccessful (data not shown).

[0013] The technology herein, in some aspects, relates to an in vitro model of meiosis from human induced pluripotent stem cells (hiPSCs). By identifying conditions for activating the expression of meiotic genes, the data shows that, in some embodiments, DNMT1 inhibition, retinoic acid receptor activation, and overexpression of anti-apoptosis and pro-meiosis factors can rapidly initiate meiosis in male and female hiPSCs. The data herein shows a method that, in some embodiments, generates cells corresponding to the leptotene, zygotene, and pachytene stages of meiosis, and that these cells have gene expression similar to meiotic germ cells in vivo. Overall, the methods of the disclosure can be a useful tool for researchers studying human meiosis, and can allow the production of human gametes in vitro.

[0014] Some aspects of the disclosure relate to a method for initiating meiosis in stem cells, the method including transfecting stem cells with a polynucleotide encoding BCL2 and a polynucleotide encoding a protein selected from BOLL, H0XB5, and MEIOC, and culturing the stem cells to initiate meiosis.

[0015] In some embodiments, stem cells comprise human stem cells. In some embodiments, stem cells comprise pluripotent stem cells (PSCs). In some embodiments, PSCs comprise induced PSCs (iPSCs).

[0016] In some embodiments, a method comprises transfecting the stem cells with: (a) a polynucleotide encoding BCL2 and a polynucleotide encoding BOLL; (b) a polynucleotide encoding BCL2 and a polynucleotide encoding H0XB5; (c) a polynucleotide encoding BCL2 and a polynucleotide encoding MEIOC; (d) a polynucleotide encoding BCL2, a polynucleotide encoding BOLL, and a polynucleotide encoding H0XB5; (e) a polynucleotide encoding BCL2, a polynucleotide encoding BOLL, and a polynucleotide encoding MEIOC; (f) a polynucleotide encoding BCL2, a polynucleotide encoding H0XB5, and a polynucleotide encoding MEIOC; or (g) a polynucleotide encoding BCL2, a polynucleotide encoding BOLL, a polynucleotide encoding H0XB5, and a polynucleotide encoding MEIOC. In some embodiments, a polynucleotide encoding BCL2 comprises an inducible promoter operably linked to an open reading frame encoding BCL2, and a polynucleotide encoding a protein selected from BOLL, H0XB5, and MEIOC comprises an inducible promoter operably linked to an open reading frame encoding a protein selected from BOLL, H0XB5, and MEIOC.

[0017] In some embodiments, a polynucleotide encoding BCL2 and a polynucleotide encoding a protein selected from BOLL, H0XB5, and MEIOC are integrated into the respective genomes of the stem cells using a transposase-based integration system (e.g., comprising a PiggyBac™ transposase).

[0018] In some embodiments, a method further comprises seeding the stem cells in a substrate comprising an extracellular matrix protein. In some embodiments, about 25,000 cells / cm2 to about 100,000 cells / cm2, optionally 40,000 cells / cm2 to about 60,000 cells / cm2, preferably about 50,000 cells / cm2, are seeded on the substrate.

[0019] In some embodiments, stem cells are cultured in a first defined, serum-free culture medium formulated for maintenance and expansion of human stem cells. In some embodiments, a first defined, serum-free culture medium comprises one or more agent selected from a DNA methyltransferase 1 inhibitor, a Rho-associated kinase (ROCK) inhibitor, a retinoic acid receptor (RAR) agonist, and an inducing agent.

[0020] In some embodiments, stem cells are cultured in the first defined, serum-free culture medium for about one day (Day 1), and the method further comprises replacing the first defined, serum-free culture medium with a second defined, serum-free culture medium formulated for differentiation of human stem cells into multiple cell lineages.

[0021] In some embodiments, a second defined, serum-free culture medium comprises one or more agent selected from a DNA methyltransferase 1 inhibitor and an inducing agent.

[0022] In some embodiments, a method comprises replacing about 50% of the second defined, serum-free culture medium with fresh second defined, serum-free culture medium comprising a DNA methyltransferase 1 inhibitor every 2 days for about 12 to about 14 days.

[0023] In some embodiments, a DNA methyltransferase 1 inhibitor is withdrawn from the fresh second defined, serum-free culture medium on about Day 5 to about Day 9, optionally Day 7.

[0024] In some embodiments, initiation of meiosis is complete by about Day 12 to about Day 14.

[0025] In some embodiments, culturing the stem cells is performed at a temperature below 37 °C, preferably at a temperature of about 34 °C. In some embodiments, meiotic cells express one or more meiotic biomarker(s) selected from H0RMAD1, SYCP3, TEX12, and gamma-H2AX.

[0026] Other aspects relate to a method for initiating meiosis in stem cells, the method including transfecting stem cells with a BCL2 protein, and a protein selected from BOLL, H0XB5, and MEIOC, and culturing the stem cells to initiate meiosis.

[0027] Yet other aspects relate to a stem cell including an engineered polynucleotide encoding a BCL2 protein and an engineered polynucleotide encoding a protein selected from BOLL, H0XB5, and MEIOC.

[0028] Still other aspects relate to an induced pluripotent stem cell including a BCL2 protein and a protein selected from BOLL, H0XB5, and MEIOC.

[0029] Also provided herein are meiotic cells produced by any one or more of the methods described herein.

[0030] The entire content of the following publication and its Supplemental Materials is incorporated herein by reference:

[0031] Pierson Smela et al. bioRxiv 2024 doi.org / 10.1101 / 2024.05.31.596483.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0034] FIG. 1A is a graph showing induction efficiency of meiosis-promoting factors (three iPSC lines, two replicates per line). FIG. IB shows co-expression of pro-meiosis factors MEIOC, BOLL, or HOXB5 with the anti-apoptotic factor BCL2 during an exemplary meiotic induction protocol. Cells were fixed after 15 days and stained for the meiotic markers HORMAD1, SYCP3, gamma-H2AX, and Actin.

[0035] FIG. 2 is a graph showing the effects of inducing meiosis at 34 °C and 37 °C (two female iPSC lines with two replicates per line, and one male iPSC line with four replicates per line).

[0036] FIG. 3A shows immunofluorescence microscopy images of marker expression from days 0 to 15 of meiosis induction. Three stages of meiosis (leptonema, labeled a; zygonema, labeled b; pachynema, labeled c) are visible. A gamma-H2AX positive sex body is labeled with an arrow. FIG. 3B is a graph showing change in cell number over time (three iPSC lines). FIG. 3C shows representative images showing marker protein expression over time. FIG. 3D is a graph showing quantification of marker protein expression at each day from 0 to 15 (three iPSC lines, two replicates per line).

[0037] FIG. 4A is a graph from a time course scRNAseq analysis of meiosis induction showing proportions of common (>5% abundance) cell types over time. FIG. 4B is a graph from a time course scRNAseq analysis of meiosis induction showing proportions of rare (<5% abundance) cell types over time.

[0038] FIG. 5 shows violin plots showing expression of marker genes from days 0 to 15 of meiosis induction. Units for the color scale are log2(CPM+l). Categories include exogenous transgenes, pluripotency markers, primordial germ cell markers, oogonia markers, meiosis markers, synaptonemal complex components, recombination markers, oocyte markers, and sperm markers.

[0039] DETAILED DESCRIPTION

[0040] Meiosis is a specialized type of cell division that is crucial for sexual reproduction in eukaryotes. Unlike mitosis, which results in two genetically identical daughter cells, meiosis produces four daughter cells, each with half the number of chromosomes of the parent cell. This reduction in chromosome number is essential for maintaining genetic stability across generations, ensuring that when gametes (sperm and egg cells in animals, pollen and ovules in plants) fuse during fertilization, the resulting offspring has the appropriate number of chromosomes.

[0041] Gametes are specialized cells involved in sexual reproduction, carrying half the genetic information of an organism to combine with another gamete during fertilization, resulting in offspring with a full set of chromosomes. There are several types of gametes, classified based on the organisms that produce them and their roles in reproduction. Spermatozoa (also referred to as sperm cells are the male gametes in many sexually reproducing organisms, including animals, plants (in the form of pollen grains), and some fungi. Sperm cells are typically motile, equipped with flagella or other structures to facilitate movement towards the female gamete. Ova (also referred to as egg cells are the female gametes found in animals, including humans, and many other organisms. They are usually larger than sperm cells and contain nutrients to support the early stages of development of the embryo after fertilization. Unlike sperm, egg cells are typically non-motile.

[0042] The process of meiosis is divided into two successive phases: Meiosis I and Meiosis II. Meiosis I reduces the chromosome number by half through the separation of homologous chromosomes, while Meiosis II, similar to mitosis, separates the sister chromatids. This results in the formation of four genetically distinct haploid gametes. Through fertilization, these gametes have the potential to combine and form a diploid zygote, thereby initiating the development of a new organism.

[0043] Chromosomal recombination, also known as genetic recombination, is a process that occurs during meiosis in sexually reproducing organisms. It involves the exchange of genetic material between homologous chromosomes, leading to the production of new combinations of alleles. This process enhances genetic diversity within a population, which is crucial for evolution and adaptation. Chromosomal recombination has significant implications for genetics and evolution. It is one of the key mechanisms by which genetic variation is introduced into populations, providing the raw material for natural selection to act upon. This genetic diversity is critical for a population's ability to adapt to changing environments and to evolve over time. Recombination also plays an important role in the repair of DNA damages and in ensuring the correct distribution of chromosomes to gametes during meiosis, thus contributing to genetic health and stability.

[0044] Provided herein are methods to initiate meiosis in stem cells, such as human stem cells. In some embodiments, the stem cells comprise pluripotent stem cells (e.g., human induced pluripotent stem cells (hiPSCs)). In some embodiments, methods of the disclosure include transfecting stem cells with a polynucleotide encoding BCL2 and a polynucleotide encoding a protein selected from BOLL, H0XB5, and MEIOC. In some embodiments, methods of the disclosure include transfecting stem cells with a polynucleotide encoding BCL2 and a polynucleotide encoding BOLL. In some embodiments, methods of the disclosure include transfecting stem cells with a polynucleotide encoding BCL2 and a polynucleotide encoding H0XB5. In some embodiments, methods of the disclosure include transfecting stem cells with a polynucleotide encoding BCL2 and a polynucleotide encoding MEIOC. In some embodiments, methods of the disclosure include transfecting stem cells with a polynucleotide encoding BCL2, a polynucleotide encoding BOLL, and a polynucleotide encoding H0XB5. In some embodiments, methods of the disclosure include transfecting stem cells with a polynucleotide encoding BCL2, a polynucleotide encoding BOLL, and a polynucleotide encoding MEIOC. In some embodiments, methods of the disclosure include transfecting stem cells with a polynucleotide encoding BCL2, a polynucleotide encoding H0XB5, and a polynucleotide encoding MEIOC. In some embodiments, methods of the disclosure include transfecting stem cells with a polynucleotide encoding BCL2, a polynucleotide encoding BOLL, a polynucleotide encoding H0XB5, and a polynucleotide encoding MEIOC. Meiotic Cells

[0045] The methods of the disclosure can be used to produce meiotic cells (e.g., activate meiosis in stem cells). A meiotic cell includes a cell that undergoes meiosis, a specialized type of cell division responsible for producing gametes (sex cells) in sexually reproducing organisms. Meiosis reduces the chromosome number by half, resulting in cells that are haploid (n), meaning they contain one set of chromosomes. This contrasts with the body's somatic cells, which are diploid (2n) and contain two sets of chromosomes, one from each parent. Meiotic cells undergo two consecutive rounds of cell division, termed Meiosis I and Meiosis II, but only one round of DNA replication. This process ensures that each gamete receives a unique combination of genes, contributing to genetic diversity among offspring.

[0046] The first division is a reductional division where homologous chromosomes (each consisting of two sister chromatids) pair up, exchange genetic material through crossing over, and then segregate into two daughter cells. This results in each cell containing one chromosome from each homologous pair, but each chromosome still consists of two sister chromatids.

[0047] The second division is similar to mitosis, where the sister chromatids of each chromosome are separated and distributed into two new daughter cells. This division results in four haploid cells, each with a unique set of chromosomes due to the recombination and independent assortment of chromosomes during Meiosis I.

[0048] The transition of a cell through meiosis is critical for sexual reproduction, as it produces gametes that can fuse during fertilization to form a new organism with a complete set of chromosomes. The genetic diversity generated through meiotic recombination and the reduction of chromosome number are fundamental to the evolution and survival of species.

[0049] Meiotic cells of the disclosure, in some embodiments, express one or more (e.g., two, three, or four) meiotic biomarker(s) selected from H0RMAD1 (HORMA Domain- Containing Protein 1), SYCP3 (Synaptonemal Complex Protein 3), TEX12 (Testis Expressed 12), and gamma-H2AX(Phosphorylated H2AX). In some embodiments, the meiotic cells express H0RMAD1. In some embodiments, the meiotic cells express SYCP3. In some embodiments, the meiotic cells express TEX12. In some embodiments, the meiotic cells express gamma-H2AX. Other non-limiting examples of biomarkers for meiosis include, SYCP1 (Synaptonemal Complex Protein 1), REC8 (Rec8 Cohesin Complex Component), MLH1 (MutL Homolog 1), DMC1 (Disrupted Meiotic cDNA 1), RAD51, STRA8 (Stimulated by Retinoic Acid Gene 8), and PRDM9 (PR Domain Zinc Finger Protein 9). Stem Cells

[0050] The methods of the disclosure can be used induce meiosis in stem cells. Stem cells can by sourced from several locations, each with unique properties and applications. Embryonic stem cells, typically derived from the inner cell mass of blastocysts in early-stage embryos, possess the ability to differentiate into any cell type. Adult stem cells, also known as somatic or tissue-specific stem cells, can be found in various tissues such as bone marrow, brain, liver, and skin. Induced pluripotent stem cells (iPSCs) can be generated by reprogramming adult cells, such as skin or blood cells, to revert to a pluripotent state, similar to that of embryonic stem cells. Additionally, perinatal stem cells, sourced from umbilical cord blood, placental tissue, and amniotic fluid, present a middle ground with a higher differentiation capability than adult stem cells and a less contentious origin than embryonic stem cells.

[0051] In some embodiments, methods of the disclosure are used to induce meiosis in pluripotent stem cells (PSCs). Pluripotent stem cells include cells that have the capacity to self-renew by dividing, and to develop into the three primary germ cell layers of the early embryo (e.g., ectoderm, endoderm, and mesoderm), and therefore into all cells of the adult body, but not extra-embryonic tissues such as the placenta. Non-limiting examples of PSCs include iPSCs, “true” embryonic stem cell (ESCs) derived from embryos, embryonic stem cells made by somatic cell nuclear transfer (ntESCs), and embryonic stem cells from unfertilized eggs (parthenogenesis embryonic stem cells, or pESCs). In some embodiments, a PSC is a human PSC. In some embodiments, a PSC is an iPSC. In some embodiments, a PSC is a human iPSC.

[0052] In some embodiments, a PSC is an embryonic stem cell (ESC), such as a human ESC. “Embryonic stem cell” is a general term for PSCs that are made using embryos or eggs, rather than for cells genetically reprogrammed from the body. As used herein, “ESCs” encompass true ESCs, ntESCs, and pESCs.

[0053] In some embodiments, a PSC is an induced pluripotent stem cell, such as a human iPSC.

[0054] Protein for Initiation of Meiosis

[0055] 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., engineered polynucleotide encoding the protein) if the level of the protein is higher than (e.g., at least 5%, at least 10%, or at least 20% higher than) the level of the protein expressed from an endogenous (i.e., naturally occurring) polynucleotide encoding the protein.

[0056] Some aspects of the present disclosure provide a stem cell comprising (e.g., expressing or overexpressing): BCL2 and a protein selected from BOLL, HOXB5, and MEIOC, wherein the protein is expressed or overexpressed. In some embodiments, the protein is expressed at a level that 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 a control level. In some embodiments, a control level is an endogenous level of the protein, for example in a naturally occurring stem cell (e.g., PSC). Overexpression refers to an expression level above an endogenous control level. In some embodiments, a stem cell (e.g., PSC) expresses or overexpresses BCL2. In some embodiments, a stem cell (e.g., PSC) expresses or overexpresses BOLL. In some embodiments, a stem cell (e.g., PSC) expresses or overexpresses HOXB5. In some embodiments, a stem cell (e.g., PSC) expresses or overexpresses MEIOC). In some embodiments, a PSC comprises BOLL. In some embodiments, a PSC expresses or overexpresses BOLL. In some embodiments, a stem cell (e.g., PSC) expresses or overexpresses BCL2 and BOLL. In some embodiments, a stem cell (e.g., PSC) expresses or overexpresses BCL2 and HOXB5. In some embodiments, a stem cell (e.g., PSC) expresses or overexpresses BCL2 and MEIOC. In some embodiments, a stem cell (e.g., PSC) expresses or overexpresses BCL2, BOLL, and HOXB5. In some embodiments, a stem cell (e.g., PSC) expresses or overexpresses BCL2, BOLL, and MEIOC. In some embodiments, a stem cell (e.g., PSC) expresses or overexpresses BCL2, HOXB5, and MEIOC. In some embodiments, a stem cell (e.g., PSC) expresses or overexpresses BCL2, BOLL, HOXB5, and MEIOC.

[0057] BCL2

[0058] BCL2 Apoptosis Regulator (BCL2) is a protein encoded by the BCL2 gene and plays a crucial role in regulating apoptosis. BCL2 is an anti-apoptotic protein localized to the outer membrane of mitochondria, where it plays a role in promoting cellular survival. There are two isoforms of BCL2, which differ in their ability to bind to pro-apoptotic proteins (e.g., BCL2 Associated Agonist of Cell Death, BAD, and BCL2 Homologous Antagonist Killer, BAK). An engineered polynucleotide comprising an open reading frame encoding BCL2 Apoptosis Regulator (BCL2) (e.g., UniprotKB Accession No. P10415), in some embodiments, encodes a protein comprising the sequence of: MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPAAS RDPVARTSPLQTPAAPGAAAGPALSPVPPWHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPF TARGRFATWEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLH TWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHK ( SEQ ID NO : 1 ) .

[0059] An engineered polynucleotide encoding an open reading frame encoding BCL2 Apoptosis Regulator (BCL2) (e.g., UniprotKB Accession No. A0A1L4AQQ4), in some embodiments, encodes a protein comprising the sequence of: MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPAAS RDPVARTSPLQTPAAPGAAAGPALSPVPPWHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPF TARGRFATWEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLH TWIQDNGGW ( SEQ ID NO : 2 ) .

[0060] HOXB5

[0061] Homeobox B5 (HOXB5) is a protein encoded by the HOXB5 gene that comprises a homeobox DNA-binding domain. HOXB5 is a member of the Hox gene cluster, which plays a role in the patterning of the body plan during embryogenesis and is involved in specifying the identity of cells along the anterior-posterior axis during development. An engineered polynucleotide comprising an open reading frame encoding HomeoboxB5 (HOXB5) (e.g., UniprotKB Accession No. P09067), in some embodiments, encodes a protein comprising the sequence of: MSSYFVNSFSGRYPNGPDYQLLNYGSGSSLSGSYRDPAAMHTGSYGYNYNGMDLSVNRSSAS SSHFGAVGESSRAFPAPAQEPRFRQAASSCSLSSPESLPCTNGDSHGAKPSASSPSDQATSA SSSANFTEIDEASASSEPEEAASQLSSPSLARAQPEPMATSTAAPEGQTPQIFPWMRKLHIS HDMTGPDGKRARTAYTRYQTLELEKEFHFNRYLTRRRRIEIAHALCLSERQIKIWFQNRRMK WKKDNKLKSMSLATAGSAFQP ( SEQ ID NO : 3 ) .

[0062] BOLL

[0063] Boule Homolog, RNA Binding Protein (BOLL) is a protein encoded by the BOLL gene expressed specifically in germ cells. An engineered polynucleotide comprising an open reading frame encoding Boule Homolog, RNA Binding Protein (BOLL) (e.g., UniprotKB Accession No. Q8N9W6), in some embodiments, encodes a protein comprising the sequence of: MQTDSLSPSPNPVSPVPLNNPTSAPRYGTVIPNRIFVGGIDFKTNESDLRKFFSQYGSVKEV KIVNDRAGVSKGYGFVTFETQEDAQKILQEAEKLNYKDKKLNIGPAIRKQQVGIPRSSIMPA AGTMYLTTSTGYPYTYHNGVAYFHTPEVTSVPPPWPSRSVCSSPVMVAQPIYQQPAYHYQAT TQYLPGQWQWSVPQPSASSAPFLYLQPSEVIYQPVEIAQDGGCVPPPLSLMETSVPEPYSDH GVQATYHQVYAPSAITMPAPVMQPEPIKTVWSIHY ( SEQ ID NO : 4 ) .

[0064] MEIOC

[0065] Meiosis Specific with Coiled-Coil Domain (MEIOC) is a protein encoded by the MEIOC gene that is required for meiosis completion in both female and male germ cells. MEIOC confers stability to meiotic mRNAs, allowing proper initiation and progression into prophase 1. An engineered polynucleotide comprising an open reading frame encoding Meiosis Specific with Coiled-Coil Domain (MEIOC) (e.g., UniprotKB Accession No. A2RUB1), in some embodiments, encodes a protein comprising the sequence of: MEVRRGDTCPRPHPSGLREEGLEPKVAFPGGANRCWNLGADAGSRLTDVFGSVMLTGSAS FYDCYTSQSEDNVDLRQTYTPFSSTEYSSSVDSSLFCAPWSTYGDDIKQPSNSQISIKNR IQTERNDYGSETDLYGLVSNILEEQDKSQPYFAEGTCSSNLKSVWPMNTSRFADHHDLLT ETKRPIDTVISQQAFYSDESVSAMEKQYLRNSNLTPQQKIDELHHGFTGLDLEEQWMYPS RSDHSNCHNIQTNDTAKTTFQEYPLIKNCFTPQTGLSDIMKESGVDIYHYGRDRICTKGL EAPLQQKRAEMFLSQFNRYNENVDYCRYPEYVHPNKAKLNKCSNFSVQDSKKLANGTPET PTVEADTYTKLFQVKPANQKKMEETIPDQQNFTFPKTTPHLTEKQFAKEAVFTADFGLTS EYGLKPHTACPANDFANVTEKQQFAKPDPPHSEYFKSVNLLSNSATSSGGINLNRPTWMN VQTKNNTPIPYRNQGNLMKLNSHLSAASKGSNHSSDFPQLSSTNLTPNSNLFQKYCQENP SAFSSFDFSYSGAERIQSVNHIEGLTKPGEENLFKLVTDKKIKQPNGFCDNYSAQKYGII ENVNKHNFQAKPQSGHYDPEEGPKHLDGLSQNTYQDLLESQGHSNSHRTRGGDNSRVNRT QVSCFSNNYMMGDLRHNQCFQQLGSNGFPLRSTHPFGHSWPLLDSYDLLSYDDLSHLYP YFNMMYGDNSFSGLMPTFGFQRPIKTRSGPASELHIRLEECCEQWRALEKERKKTELALA KNYPGKKVSSTNNTPVPRLTSNPSRVDRLIVDELRELARWTLLGKMERLRSSLLHASIS TALDRHLESIHIVQSRRKDEIVNASNRQRQGVPRCQDDRDVFALASAIKEMCVATRKTRT ALWCALQMTLPKTASTADWKPLQDTVNCEDKVHESINSSNPMNQRGETNKH ( SEQ ID NO : 5 ) .

[0066] Engineered Polynucleotides and Polypeptides The stem cells (e.g., PSCs) of the present disclosure, in some embodiments, comprise engineered polynucleotides. An engineered polynucleotide includes a nucleic acid (e.g., at least two nucleotides covalently linked together, and in some instances, containing phosphodiester bonds, referred to as a phosphodiester backbone) that does not occur in nature. Engineered polynucleotides include recombinant nucleic acids and synthetic nucleic acids. A recombinant nucleic acid includes a molecule that is constructed by joining nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids or a combination thereof) from two different organisms (e.g., human and mouse). A synthetic nucleic acid includes a molecule that is amplified or chemically, or by other means, synthesized. A synthetic nucleic acid includes those that are chemically modified, or otherwise modified, but can base pair with (bind to) naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.

[0067] An engineered polynucleotide can comprise DNA (e.g., genomic DNA, cDNA or a combination of genomic DNA and cDNA), RNA or a hybrid molecule, for example, where the nucleic acid contains 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.

[0068] In some embodiments, a polynucleotide is a complementary DNA (cDNA). cDNA is synthesized from a single- stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.

[0069] Engineered polynucleotides of the present disclosure can be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, nucleic acids are produced using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009; and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated by reference herein). Other methods of producing engineered polynucleotides can be used in accordance with the present disclosure.

[0070] In some embodiments, an engineered polynucleotide comprises a promoter operably linked to an open reading frame. A promoter includes a nucleotide sequence to which RNA polymerase binds to initial transcription (e.g., ATG). Promoters are typically located directly upstream from (at the 5' end of) a transcription initiation site. In some embodiments, a promoter is a heterologous promoter. A heterologous promoter is not naturally associated with the open reading frame to which is it operably linked. In some embodiments, a promoter is an inducible promoter. An inducible promoter can be regulated in vivo by a chemical agent, temperature, or light, for example. Inducible promoters enable, for example, temporal and / or spatial control of gene expression. Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, 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 a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein (tTA)), steroid- regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid 25 receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester 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, a promoter is a constitutive promoter (active in vivo, unregulated).

[0071] An open reading frame includes a continuous 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, for example, a protein. An open reading frame is operably linked to a promoter if that promoter regulates transcription of the open reading frame.

[0072] Vectors used for delivery of an engineered polynucleotide 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), is also contemplated herein.

[0073] A stem cell (e.g., PSC), in some embodiments, comprises an engineered polynucleotide comprising an open reading frame encoding a protein selected from BCL2, BOLL, HOXB5, and MEIOC. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding BCL2. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding HOXB5. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding BOLL. In some embodiments, the engineered polynucleotide comprises an open reading frame encoding MEIOC.

[0074] In some embodiments, a stem cell (e.g., PSC) comprises an engineered polynucleotide comprising an open reading frame encoding BCL2 and an engineered polynucleotide comprising an open reading frame encoding BOLL. In some embodiments, a stem cell (e.g., PSC) comprises an engineered polynucleotide comprising an open reading frame encoding BCL2 and an engineered polynucleotide comprising an open reading frame encoding H0XB5. In some embodiments, a stem cell (e.g., PSC) comprises an engineered polynucleotide comprising an open reading frame encoding BCL2 and an engineered polynucleotide comprising an open reading frame encoding MEIOC.

[0075] In some embodiments, a stem cell (e.g., PSC) comprises an engineered polynucleotide comprising an open reading frame encoding BCL2, an engineered polynucleotide comprising an open reading frame encoding BOLL, and an engineered polynucleotide comprising an open reading frame encoding H0XB5. In some embodiments, a stem cell (e.g., PSC) comprises an engineered polynucleotide comprising an open reading frame encoding BCL2, an engineered polynucleotide comprising an open reading frame encoding BOLL, and an engineered polynucleotide comprising an open reading frame encoding MEIOC. In some embodiments, a stem cell (e.g., PSC) comprises an engineered polynucleotide comprising an open reading frame encoding BCL2, an engineered polynucleotide comprising an open reading frame encoding H0XB5, and an engineered polynucleotide comprising an open reading frame encoding MEIOC.

[0076] In some embodiments, a stem cell (e.g., PSC) comprises an engineered polynucleotide comprising an open reading frame encoding BCL2, an engineered polynucleotide comprising an open reading frame encoding BOLL, an engineered polynucleotide comprising an open reading frame encoding H0XB5, and an engineered polynucleotide comprising an open reading frame encoding MEIOC.

[0077] The number of copies of an engineered polynucleotide delivered to a stem cell (e.g., PSC) may vary. In some embodiments, a stem cell (e.g., PSC) comprises 1-20 copies of an engineered polynucleotide. Lor example, a stem cell (e.g., PSC) may comprise 1-15, 1-10, 2- 10, 2-15, 2-10, 5-20, 5-15, or 5-10 copies of an engineered polynucleotide. In some embodiments, a stem cell (e.g., PSC) comprises 8-10 copies of an engineered polynucleotide. Greater than 20 copies are also contemplated herein. Methods of Inducing Meiosis in Pluripotent Stem Cells

[0078] Methods for initiating (also referred to as inducing) meiosis, in some embodiments, include transfecting stem cells with a polynucleotide encoding BCL2 and a polynucleotide encoding a protein selected from BOLL , H0XB5, and MEIOC, and culturing the stem cells to initiate meiosis, thereby producing meiotic cells.

[0079] In some embodiments, methods for initiating meiosis comprise culturing, in culture media, a population of stem cells (e.g., PSCs) to produce an expanded population of stem cells; and expressing in PSCs of the expanded population a BCL2 protein and a (one, two, or three) protein selected from BOLL, H0XB5, and MEIOC to induce meiosis (produce meiotic cells).

[0080] In some embodiments, the stem cells (e.g. PSC) of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding BCL2. In some embodiments, the stem cells (e.g. PSC) of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding BOLL. In some embodiments, the stem cells (e.g. PSC) of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding H0XB5. In some embodiments, the stem cells (e.g. PSC) of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding MEIOC. In some embodiments, the stem cells (e.g. PSC) of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding BCL2 and an engineered polynucleotide comprising an open reading frame encoding BOLL. In some embodiments, the stem cells (e.g. PSC) of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding BCL2 and an engineered polynucleotide comprising an open reading frame encoding H0XB5. In some embodiments, the stem cells (e.g. PSC) of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding BCL2 and an engineered polynucleotide comprising an open reading frame encoding MEIOC. In some embodiments, the stem cells (e.g. PSC) of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding BCL2, an engineered polynucleotide comprising an open reading frame encoding BOLL, and an engineered polynucleotide comprising an open reading frame encoding H0XB5. In some embodiments, the stem cells (e.g. PSC) of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding BCL2, an engineered polynucleotide comprising an open reading frame encoding BOLL, and an engineered polynucleotide comprising an open reading frame encoding MEIOC. In some embodiments, the stem cells (e.g. PSC) of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding BCL2, an engineered polynucleotide comprising an open reading frame encoding H0XB5, and an engineered polynucleotide comprising an open reading frame encoding MEIOC. In some embodiments, the stem cells (e.g. PSC) of the expanded population comprise an engineered polynucleotide comprising an open reading frame encoding BCL2, an engineered polynucleotide comprising an open reading frame encoding BOLL, an engineered polynucleotide comprising an open reading frame encoding H0XB5, and an engineered polynucleotide comprising an open reading frame encoding MEIOC.

[0081] In some embodiments, an open reading frame of the engineered polynucleotide is operably linked to a heterologous promoter (one that is not operably linked to the open reading frame in nature). In some embodiments, a heterologous promoter is an inducible promoter, non-limiting examples of which are provided elsewhere herein.

[0082] Transfection Methods

[0083] The engineered polynucleotide of the present disclosure can be delivered to a PSC using any one or more transfection method, including chemical transfection methods, viral transduction methods, and electroporation.

[0084] In some embodiments, an engineered polynucleotide is delivered on a vector. A vector is any vehicle, for example, a virus or a plasmid, that is used to transfer a desired polynucleotide into a host cell, such as a PSC. In some embodiments, the vector is a viral vector. In some embodiments, a viral vector is not a naturally occurring viral vector. The viral vector can be from adeno-associated virus (AAV), adenovirus, herpes simplex virus, lentiviral, retrovirus, varicella, variola virus, hepatitis B, cytomegalovirus, JC polyomavirus, BK polyomavirus, monkeypox virus, Herpes Zoster, Epstein-Barr virus, human herpes virus 7, Kaposi's sarcoma-associated herpesvirus, or human parvovirus B 19. Other viral vectors are encompassed by the present disclosure.

[0085] In some embodiments, a viral vector is an AAV vector. AAV is a small, nonenveloped virus that packages a single- stranded linear DNA genome that is approximately 5 kb long and has been adapted for use as a gene transfer vehicle. The coding regions of AAV are flanked by inverted terminal repeats (ITRs), which act as the origins for DNA replication and serve as the primary packaging signal. Thus, an AAV vector typically includes ITR sequences. Both positive and negative strands are packaged into virions equally well and capable of infection. In addition, a small deletion in one of the two ITRs allows packaging of self-complementary vectors, in which the genome self-anneals after viral uncoating. This results in more efficient transduction of cells but reduces the coding capacity by half. In some embodiments, a polynucleotide encoding BCL2 and a (one or more) polynucleotide encoding a protein selected from BOLL, H0XB5, and MEIOC are integrated into the genome of a stem cells using a transposase-based integration system (e.g., comprising a piggyBac™ transposase). In some embodiments, a polynucleotide is delivered to a cell using a transposon / transposase system. For example, the piggyBac™ transposon system can be used. A piggyBac™ transposon is a mobile genetic element that efficiently transposes between vectors and chromosomes via a “cut and paste” mechanism (Woodard et al. 2015). During transposition, the piggyBac™ transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the contents from the original sites and integrates them into TTAA chromosomal sites. The piggyBac™ transposon system facilitates efficient integration of a polynucleotide into a cell genome.

[0086] Thus, in some embodiments, the method further comprises delivering to a PSC a transposon comprising an engineered polynucleotide and delivering a transposase.

[0087] In some embodiments, an engineered polynucleotide is delivered to a cell using electroporation. Electroporation is a physical transfection method that uses an electrical pulse to create temporary pores in cell membranes through which the engineered polynucleotide can pass into cells. See, e.g., Chicaybam L et al. Front. Bioeng. Biotechnol., 23 January 2017.

[0088] Following transfection, the engineered polynucleotides can be integrated into the genome of a PSC. In some embodiments, an engineered polynucleotide may further comprise an antibiotic resistance gene to confer resistance to an antibiotic used in an antibiotic drug selection process. In this way, a ‘pure’ population of cells comprising an integrated engineered polynucleotide can be obtained. In some embodiments, a population of cells comprising an integrated engineered polynucleotide are selected using antibiotic drug selection. Antibiotic drug selection is the process of treating a population of cells with an antibiotic so that only cells that are capable of surviving in the presence of said 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, zeocin, and actinomycin.

[0089] Culture Conditions

[0090] In some embodiments, a method comprises seeding (e.g., depositing) stem cells in / on a substrate comprising extracellular matrix (ECM) protein(s). Non-limiting examples of ECM proteins used in cell culture include Collagen Type I and IV, Fibronectin, Vitronectin, Matrigel, Gelatin, and Elastin. Matrigel is the preferred ECM for stem cell culture and differentiation. Matrigel is a gelatinous protein mixture derived from the Engelbreth-Holm- Swarm (EHS) mouse sarcoma, rich in ECM proteins such as laminin, collagen IV, entactin, and heparan sulfate proteoglycans. In some embodiments, the ECM proteins comprise Matrigel.

[0091] A substrate can be a cell culture substrate designed to enhance cellular adhesion, growth, and differentiation across multiple cell types. These substrates include, for example, surfaces coated with ECM proteins such as Collagen Type I and IV, Fibronectin, Laminin, Vitronectin, and Gelatin. Additional non-limiting examples of substrates include synthetic polymer coatings like poly-L- lysine and poly-D-lysine; elastin-coated substrates; hyaluronic acid coatings; synthetic polymer coatings, such as polylactic acid (PLA) and polyethylene glycol (PEG); nanofiber scaffolds made from materials like polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA); and decellularized extracellular matrix substrates derived from natural tissues.

[0092] In some embodiments, about 25,000 cells / cm2to about 100,000 cells / cm2, for example, 40,000 cells / cm2to about 60,000 cells / cm2are seeded on a substrate. In some embodiments, about 25,000 cells / cm2, about 30,000 cells / cm2, about 35,000 cells / cm2, about 40,000 cells / cm2, about 45,000 cells / cm2, about 50,000 cells / cm2, about 50,000 cells / cm2, about 60,000 cells / cm2, about 65,000 cells / cm2, about 70,000 cells / cm2, about 75,000 cells / cm2, about 80,000 cells / cm2, about 85,000 cells / cm2, about 90,000 cells / cm2, about 95,000 cells / cm2, or about 100,000 cells / cm2. In some embodiments, about 40,000 cells / cm2are seeded on a substrate. In some embodiments, about 50,000 cells / cm2are seeded on a substrate. In some embodiments, about 60,000 cells / cm2are seeded on a substrate.

[0093] In some embodiments, stem cells are cultured for about 2 days to about 20 days. In other embodiments, stem cells are cultured for about 2 days to about 15 days. In some embodiments, stem cells are cultured for no more than 15 days, no more than 14 days, nor more than 13 days, or no more than 12 days. In some embodiments, meiosis has been initiated (commences) at about Day 12 to about Day 14 of any one of the methods described herein. In some embodiments, meiosis has been initiated at about Day 13 of any one of the methods described herein.

[0094] In some embodiments, stem cells are cultured in a first defined, serum-free culture medium formulated for maintenance and expansion of human stem cells. In some embodiments, the first culture medium comprises mTeSRl (STEMCELL TECHNOLOGIES™). mTeSRl is a defined, serum-free medium specifically formulated for the maintenance and expansion of stem cells under feeder- free conditions. mTeSRl contains essential nutrients, growth factors, and signaling molecules that create an optimal environment for stem cell culture, promoting robust cell growth and maintaining the cells' pluripotent state. This medium may be used in conjunction with specific substrates, such as Matrigel or vitronectin, which provide the necessary extracellular matrix support for stem cells.

[0095] In some embodiments, the first defined, serum-free culture medium comprises (or further comprises) one or more agent selected from a DNA methyltransferase 1 (DNMT1) inhibitor (e.g., GSK3484862), a Rho-associated kinase (ROCK) inhibitor (e.g., Y-27632), a retinoic acid receptor (RAR) agonist (e.g., AM580), and an inducing agent (e.g., doxycycline).

[0096] GSK3484862 is a non-covalent inhibitor of DNMT1. It is designed to selectively inhibit DNMT1 without affecting other DNMT family members like DNMT3A / 3L or DNMT3B / 3L. This compound induces global DNA demethylation and has been shown to target DNMT1 for degradation in both cancer cell lines and murine embryonic stem cells (mESCs). GSK3484862 achieves this by promoting proteasome-dependent degradation of DNMT1, leading to significant reductions in DNA methylation with minimal non-specific toxicity. Other non-limiting examples of DNMT1 inhibitors include 5-azacytidine, decitabine, RG108, and zebularine, MG98, SGI-1027, and MC3343.

[0097] In some embodiments, a DNMT1 inhibitor (e.g., GSK3484862) is used at a concentration of about 1 pM to about 20 pM, e.g., 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, or 20 pM. In some embodiments, a DNMT1 inhibitor is used at a concentration of about 2 pM. In some embodiments, a DNMT1 inhibitor is used at a concentration of about 3 pM. In some embodiments, a DNMT1 inhibitor is used at a concentration of about 4 pM. In some embodiments, a DNMT1 inhibitor is used at a concentration of about 5 pM. In some embodiments, a DNMT1 inhibitor is used at a concentration of about 6 pM. In some embodiments, a DNMT1 inhibitor is used at a concentration of about 7 pM. In some embodiments, an DNMT1 agonist is used at a concentration of about 8 pM. In some embodiments, a DNMT1 inhibitor (e.g., GSK3484862) is used at a concentration of about 2 pM to about 8 pM.

[0098] Y-27632 is a selective inhibitor of ROCK, which plays a key role in the regulation of the cytoskeleton and cell motility. By inhibiting ROCK, Y-27632 prevents the phosphorylation of downstream targets involved in actin filament organization and cell contraction. This inhibition promotes cell survival, particularly in human embryonic stem cells and human induced pluripotent stem cells, by reducing apoptosis and improving cell attachment and proliferation during cell culture. Other non-limiting examples of ROCK inhibitors include fasudil, ripasudil, netarsudil, H-1152, GSK429286A, SR3677, and AS 189280.

[0099] In some embodiments, a ROCK inhibitor (e.g., Y-27632) is used at a concentration of about 1 pM to about 20 pM, e.g., 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, or 20 pM. In some embodiments, a ROCK inhibitor is used at a concentration of about 8 pM. In some embodiments, a ROCK inhibitor is used at a concentration of about 9 pM. In some embodiments, a ROCK inhibitor is used at a concentration of about 10 pM. In some embodiments, a ROCK inhibitor is used at a concentration of about 11 pM. In some embodiments, a ROCK inhibitor is used at a concentration of about 12 pM. In some embodiments, a ROCK inhibitor (e.g., Y-27632) is used at a concentration of about 9 pM to about 12 pM.

[0100] AM580 is a synthetic retinoid and a potent agonist of the retinoic acid receptor (RAR) a (alpha). It is structurally related to all-trans retinoic acid (ATRA) and has been used to study the roles of retinoic acid signaling in development and disease, given its selectivity and potency for RARa. Other non-limiting examples of RARa agonists include all-trans retinoic acid (ATRA), bexarotene, tamibarotene, tazarotene, TTNPB (Ro 13-7410), and Ro 40-6055.

[0101] In some embodiments, an RAR agonist (e.g., AM580) is used at a concentration of about 0.5 pM to about 5 pM. For example, an RAR agonist can be used at a concentration of about 0.5 pM, about 1 pM, about 1.5 pM, about 2 pM, about 2.5 pM, about 3 pM, about 3.5 pM, about 4 pM, about 5.5 pM, or about 5 pM. In some embodiments, an RAR agonist is used at a concentration of about 0.5 pM. In some embodiments, an RAR agonist is used at a concentration of about 1 pM. In some embodiments, an RAR agonist is used at a concentration of about 1.5 pM. In some embodiments, an RAR agonist is used at a concentration of about 2 pM. In some embodiments, an RAR agonist (e.g., AM580) is used at a concentration of about 0.5 pM to about 2 pM.

[0102] Non-limiting examples of inducing agents include doxycycline, tetracycline, and tamoxifen (and others).

[0103] In some embodiments, an inducing agent (e.g., doxycycline) is used at a concentration of about 0.5 pg / mL to about 5 pg / mL. For example, an inducing agent can be used at a concentration of about 0.5 pg / mL, about 1 pg / mL, about 1.5 pg / mL, about 2 pg / mL, about 2.5 pg / mL, about 3 pg / mL, about 3.5 pg / mL, about 4 pg / mL, about 5.5 pg / mL, or about 5 g / mL. In some embodiments, an inducing agent is used at a concentration of about 0.5 |jg / mL. In some embodiments, an inducing agent is used at a concentration of about 1 |jg / mL. In some embodiments, an inducing agent is used at a concentration of about 1.5 |jg / mL. In some embodiments, an inducing agent is used at a concentration of about 2 |jg / mL. In some embodiments, an inducing agent (e.g., doxycycline) is used at a concentration of about 0.5 |jg / mL to about 2 |jg / mL.

[0104] In some embodiments, stem cells are cultured in a first defined, serum-free culture medium for about 12 hours to about 48 hours. In some embodiments, stem cells are cultured in a first defined, serum-free culture medium for about 24 hours / one day (Day 1). In some embodiments, a method further comprises replacing the first defined, serum-free culture medium with a second defined, serum-free culture medium formulated for differentiation of human stem cells into multiple cell lineages. In some embodiments, a second defined, serum- free culture medium formulated for differentiation of human stem cells into multiple cell lineages comprises APEL2. APEL2 is a chemically defined, serum-free medium specifically designed for the feeder- free culture of human pluripotent stem cells, including both human embryonic stem cells and human induced pluripotent stem cells. It supports the maintenance of pluripotency and the growth of hPSCs without the need for additional growth factors or serum.

[0105] In some embodiments, a second defined, serum-free culture medium comprises (or further comprises) one or more agent selected from a DNMT1 inhibitor and an inducing agent. Exemplary concentrations that can be used for the DNMT1 inhibitor and the inducing agent are described above.

[0106] In some embodiments, a method comprises replacing about 50% (e.g., about 40% to about 60%) of the second defined, serum-free culture medium with fresh second defined, serum-free culture medium comprising a DNA methyltransferase 1 inhibitor. The medium can be replaced every day, every 2 days, or every 3 days, for example, for about 10 days to about 20 days, for example, about 12 to about 14 days (e.g., for about 12 days, about 13 days, or about 14 days).

[0107] In some embodiments, the DNMT1 inhibitor is withdrawn from the fresh second defined, serum-free culture medium (i.e., is not added to the cells) on about Day 5 to about Day 10, about Day 5 to about Day 9, or about Day 5 to about Day 8. In some embodiments, the DNMT1 inhibitor is withdrawn from the fresh second defined, serum- free culture medium (i.e., is not added to the cells) on Day 7. In some embodiments, initiation of meiosis is complete by about Day 12 to about Day 14. In some embodiments, initiation of meiosis is complete by about Day 12. In some embodiments, initiation of meiosis is complete by about Day 13, In some embodiments, initiation of meiosis is complete by about Day 14.

[0108] In some embodiments, stem cells are cultured at or below a temperature below 37 °C. For example, stem cells can be cultured at a temperature of about 33 °C, about 34 °C, about 35 °C, or about 36 °C. In preferred embodiments, stem cells are cultured at about 34 °C (see Examples below).

[0109] Compositions and Method of Use

[0110] The present disclosure provides, in some embodiments, compositions comprising the meiotic cells produced using the methods herein. In some embodiments, the compositions further comprise a pharmaceutically acceptable excipient. The compositions, in some embodiments, are cryopreserved.

[0111] Such compositions can be used to produce gametes for use in in vitro fertilization or other reproductive therapies. In some embodiments, the gametes are eggs. In some embodiments, the gametes are spermatids or sperm. In some embodiments, the meiotic cells are incorporated into an ovarian organoid for the production of eggs. In some embodiments, the meiotic cells are incorporated into a testicular organoid for the production of sperm.

[0112] 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.

[0113] In some embodiments, a subject is a human subject. A subject may have an infertility disorder, such as Turner syndrome. Infertility disorders include disorders in which germ cell development is severely affected in a female fetus. Blood sample analysis can be used to diagnose an infertility disorder. In some embodiments, the infertility disorder may include damage to meiotic cells.

[0114] The compositions can be administered to a subject in a therapeutically effective amount. The term “therapeutically effective amount” refers to the amount of meiotic cells required to confer therapeutic effect on a subject, either alone or in combination with at least one other active agent. Effective amounts vary, as recognized by those skilled in the art, depending on the route of administration, excipient usage, and co-usage with other active agents. The quantity to be administered depends on the subject to be treated, including, for example, the strength of an individual’s immune system or genetic predispositions. Suitable dosage ranges are readily determinable by one skilled in the art and can be on the order of micrograms of the polypeptide of this disclosure. The dosage of the preparations disclosed herein may depend on the route of administration and varies according to the size of the subject.

[0115] It is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited in the present application are incorporated by reference for the purposes or subject matter referenced in this disclosure.

[0116] EXAMPLES

[0117] An in vitro model of human meiosis can accelerate research into this important reproductive process and development of therapies for infertility. The technology developed herein relates to a method to induce meiosis starting from male or female human pluripotent stem cells. The data demonstrates that DNMT1 inhibition, retinoid signaling activation, and overexpression of regulatory factors (anti-apoptotic BCL2, and pro-meiotic H0XB5, BOLL, or MEIOC) rapidly activates meiosis, with leptonema beginning at 6 days, zygonema at 9 days, and pachynema at 12 days. Immunofluorescence microscopy shows key aspects of meiosis, including chromosome synapsis and sex body formation. The meiotic cells express genes similar to meiotic oogonia in vivo, including synaptonemal complex components and machinery for meiotic recombination. These findings establish an accessible system for inducing human meiosis in vitro.

[0118] Example 1. Meiosis-promoting Factors

[0119] A constitutive EFla-driven BCL2 expression plasmid was integrated into F2 DDX4- tdTomato / DAZL-GFP, F3 DDX4-tdTomato / TFAP2C-GFP, and PGP1 DDX4- tdTomato / REC8-GFP reporter hiPSCs under hygromycin selection. Then, all eight possible combinations of H0XB5, BOLL, and MEIOC expression vectors were integrated under puromycin selection. Cells were fixed and stained for H0RMAD1, SYCP3, gamma-H2AX, and TEX12 after 13 days of differentiation, with the first 3 days at 37 °C and the remainder at 34 °C (FIG. 1A and FIG. IB). Example 2. Timing and Temperature

[0120] To evaluate the effects of lower temperature on male meiosis, PGP1 D4TR8G reporter hiPSCs containing integrated expression vectors for BCL2, H0XB5, BOLL, and MEIOC were seeded in 8-well dishes at 50,000 cells / cm2in mTeSRl with a DNA methyltransferase 1 inhibitor, doxycycline, and a Rho-associated kinase inhibitor. After 24 hours, the media was replaced with APEL2 containing DNA methyltransferase 1 inhibitor and doxycycline. A 50% media change was performed every 2 days, and the DNA methyltransferase 1 inhibitor was withdrawn starting on about day 7. Initially, all cells were cultured at 37 °C. One plate was moved to a 34 °C incubator after the first day. Cells were fixed on day 13, stained for H0RMAD1, SYCP3, and gamma-H2AX, and imaged.

[0121] As a confirmatory experiment, F2 D4TDZG, F3 D4TS3G, and PGP1 D4TR8G reporter hiPSCs containing integrated expression vectors for BCL2, HOXB5, BOLL, and MEIOC were differentiated according to the same protocol. The following conditions were tested:

[0122] 1. 34 °C starting on day 3, fixation at day 13

[0123] 2. 34 °C starting on day 1, fixation at day 14

[0124] 3. 34 °C starting on day 3, fixation at day 15

[0125] 4. 34 °C starting on day 3, fixation at day 16

[0126] 5. Continuous 37 °C, fixation at day 16

[0127] 6. 34 °C starting on day 1, fixation at day 17

[0128] 7. 34 °C starting on day 3, fixation at day 19

[0129] 8. 34 °C starting on day 3, fixation at day 21

[0130] After fixation, cells were stained for HORMAD1, SYCP3, gamma-H2AX, and actin, and imaged (FIG. 2).

[0131] Example 3. Meiosis Induction Protocol

[0132] A constitutive or doxycycline-inducible expression vector for the anti- apop to tic factor BCL2, as well as doxycycline-inducible expression vectors for meiosis-promoting factors (HOXB5, BOLL, and / or MEIOC), were integrated into human iPSCs using PiggyBac™ transposase on Day 0. The iPSCs were seeded at 50,000 cells / cm2on Matrigel® coated plates in mTeSRl supplemented with a DNA methyltransferase 1 inhibitor (e.g., about 2 pM to about pM 10), doxycycline (e.g., about 0.5 pg to about 5 pg), a Rho-associated kinase inhibitor (e.g., about 5 pM to about 15 |aM), and in some instances, a retinoic acid receptor agonist (e.g., about 0.5 pM to about 5 pM). On Day 1, the media was replaced with STEMdiff™ APEL™2 media (STEMCELL TECHNOLOGIES™) containing a DNA methyltransferase 1 inhibitor e.g., about 2 pM to about pM 10) and doxycycline (e.g., about 0.5 pg to about 5 pg). A 50% media change was performed every 2 days, and the DNA methyltransferase 1 inhibitor was withdrawn starting on Day 7. Initiation of meiosis was complete by about Day 13.

[0133] FIGs. 3A-3D show immunofluorescence microscopy of marker expression from days 0 to 15 of meiosis induction. Three stages of meiosis (leptonema, labeled a; zygonema, labeled b; pachynema, labeled c) are visible (FIG. 3A). A gamma-H2AX positive sex body is labeled with an arrow. Change in cell number over time (three iPSC lines) is shown in FIG. 3B. FIG. 3C shows representative images of marker protein expression over time. FIG. 3D shows a quantification of marker protein expression at each day from 0 to 15 (three iPSC lines, two replicates per line).

[0134] Example 4. Timecourse scRNAseq and Imaging

[0135] Plasmids for constitutive expression of BCL2 and doxycycline-inducible expression of HOXB5, BOLL, and MEIOC were integrated into F2 DDX4-tdTomato / DAZL-GFP, F3 DDX4-tdTomato / TFAP2C-GFP, and PGP1 DDX4-tdTomato / REC8-GFP reporter hiPSCs. Meiosis was initiated following the protocol described in Example 3. At each day from Day 0 (hiPSC) to Day 15, cells were fixed for imaging and harvested for scRNAseq. Stains used for imaging were: rabbit anti-HORMADl, goat anti-SYCP3, mouse anti gamma-H2AX, and rat anti-T2A; and rabbit anti-TEX12, goat anti-SYCP3, mouse anti-RAD51, and rat anti- KI67. Samples for scRNAseq were counted and fixed using the Parse Biosciences fixation kit. Library preparation was performed using the Parse Biosciences WT v3 kit. Sequencing was performed on two lanes of a Novaseq X Plus 25B PE 150 flowcell.

[0136] FIGs. 4A-4B show graphs from a time course scRNAseq analysis of meiosis induction.

[0137] FIG. 5 shows violin plots showing expression of marker genes from Days 0 to 15 of meiosis induction.

[0138] 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 encompass the entirety of the document. The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0139] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0140] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean 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 the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0141] The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value.

[0142] Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.

Claims

What is claimed is:CLAIMS1. A method for initiating meiosis in stem cells, the method comprising: transfecting stem cells with a polynucleotide encoding BCL2 and a polynucleotide encoding a protein selected from BOLL, H0XB5, and MEIOC; and culturing the stem cells to initiate meiosis, thereby producing meiotic cells.

2. The method of claim 1, wherein the stem cells comprise human stem cells.

3. The method of claim 1 or 2, wherein the stem cells comprise pluripotent stem cells(PSCs).

4. The method of claim 3, wherein the PSCs comprise induced PSCs (iPSCs).

5. The method of any of the preceding claims, wherein the method comprises transfecting the stem cells with:(a) a polynucleotide encoding BCL2 and a polynucleotide encoding BOLL;(b) a polynucleotide encoding BCL2 and a polynucleotide encoding H0XB5;(c) a polynucleotide encoding BCL2 and a polynucleotide encoding MEIOC;(d) a polynucleotide encoding BCL2, a polynucleotide encoding BOLL, and a polynucleotide encoding H0XB5;(e) a polynucleotide encoding BCL2, a polynucleotide encoding BOLL, and a polynucleotide encoding MEIOC;(f) a polynucleotide encoding BCL2, a polynucleotide encoding H0XB5, and a polynucleotide encoding MEIOC; or(g) a polynucleotide encoding BCL2, a polynucleotide encoding BOLL, a polynucleotide encoding H0XB5, and a polynucleotide encoding MEIOC.

6. The method of any of the preceding claims, wherein the polynucleotide encoding BCL2 comprises an inducible promoter operably linked to an open reading frame encoding BCL2, and the polynucleotide encoding a protein selected from BOLL, H0XB5, and MEIOCcomprises an inducible promoter operably linked to an open reading frame encoding a protein selected from BOLL, H0XB5, and MEIOC.

7. The method of any of the preceding claims, wherein the polynucleotide encoding BCL2 and the polynucleotide encoding a protein selected from BOLL, H0XB5, and MEIOC are integrated into the respective genomes of the stem cells using a transposase-based integration system (e.g., comprising a PiggyBac™ transposase).

8. The method of any of the preceding claims, wherein the method further comprises seeding the stem cells in a substrate comprising an extracellular matrix protein.

9. The method of claim 8, wherein about 25,000 cells / cm2to about 100,000 cells / cm2, optionally 40,000 cells / cm2to about 60,000 cells / cm2, preferably about 50,000 cells / cm2, are seeded on the substrate.

10. The method of any of the preceding claims, wherein the stem cells are cultured in a first defined, serum-free culture medium formulated for maintenance and expansion of human stem cells.

11. The method of claim 10, wherein the first defined, serum- free culture medium comprises one or more agent selected from a DNA methyltransferase 1 inhibitor, a Rho- associated kinase (ROCK) inhibitor, a retinoic acid receptor (RAR) agonist, and an inducing agent.

12. The method of claim 10 or 11, wherein the stem cells are cultured in the first defined, serum-free culture medium for about one day (Day 1), and the method further comprises replacing the first defined, serum-free culture medium with a second defined, serum-free culture medium formulated for differentiation of human stem cells into multiple cell lineages.

13. The method of claim 12, wherein the second defined, serum- free culture medium comprises one or more agent selected from a DNA methyltransferase 1 inhibitor and an inducing agent.

14. The method of claim 12 or 13, wherein the method comprises replacing about 50% of the second defined, serum-free culture medium with fresh second defined, serum-free culture medium comprising a DNA methyltransferase 1 inhibitor every 2 days for about 12 to about14 days.

15. The method of claim 14, wherein the DNA methyltransferase 1 inhibitor is withdrawn from the fresh second defined, serum-free culture medium on about Day 5 to about Day 9, optionally Day 7.

16. The method of any of the preceding claims, wherein initiation of meiosis is complete by about Day 12 to about Day 14.

17. A method for initiating meiosis in stem cells, the method comprising: transfecting stem cells with a BCL2 protein and a protein selected from BOLL, HOXB5, and MEIOC; and culturing the stem cells to initiate meiosis.

18. The method of claim 17, wherein the stem cells comprise human stem cells.

19. The method of claim 17 or 18, wherein the stem cells comprise pluripotent stem cells(PSCs).

20. The method of claim 19, wherein the PSCs comprise induced PSCs (iPSCs).

21. The method of any of claims 17-20, wherein the method comprises transfecting the stem cells with:(a) a polynucleotide encoding BCL2 and a polynucleotide encoding BOLL;(b) a polynucleotide encoding BCL2 and a polynucleotide encoding HOXB5;(c) a polynucleotide encoding BCL2 and a polynucleotide encoding MEIOC;(d) a polynucleotide encoding BCL2, a polynucleotide encoding BOLL, and a polynucleotide encoding HOXB5;(e) a polynucleotide encoding BCL2, a polynucleotide encoding BOLL, and a polynucleotide encoding MEIOC;(f) a polynucleotide encoding BCL2, a polynucleotide encoding HOXB5, and a polynucleotide encoding MEIOC; or(g) a polynucleotide encoding BCL2, a polynucleotide encoding BOLL, a polynucleotide encoding HOXB5, and a polynucleotide encoding MEIOC.

22. The method of any of the preceding claims, wherein culturing the stem cells is performed at a temperature below 37 °C, preferably at a temperature of about 34 °C.

23. The method of any of the preceding claims, wherein the meiotic cells express one or more meiotic biomarker(s) selected from HORMAD1, SYCP3, TEX12, and gamma-H2AX.

24. A stem cell comprising: an engineered polynucleotide encoding a BCL2 protein and an engineered polynucleotide encoding a protein selected from BOLL, HOXB5, and MEIOC.

25. The stem cell of claim 24, wherein the stem cell is a pluripotent stem cell (PSC).

26. The stem cell of claim 25, wherein the PSC is an induced PSC.

27. An induced pluripotent stem cell comprising: a BCL2 protein and a protein selected from BOLL, HOXB5, and MEIOC.

28. The stem cell of any one of claims 24-27, wherein the stem cell is a human stem cell.

29. The stem cell of any one of claims 24-28, wherein the stem cell expresses:(a) BCL2 and BOLL;(b) BCL2 and HOXB5;(c) BCL2 and MEIOC;(d) BCL2, BOLL, and HOXB5;(e) BCL2, BOLL, and MEIOC;(f) BCL2, HOXB5, and MEIOC; or(g) BCL2, BOLL, HOXB5, and MEIOC.

30. Meiotic cells produced by the method of any one of claims 1-23.

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