Compositions and methods for producing primordial germ cells

Abstract

The present disclosure relates to the generation of primordial germ cell-like cells from pluripotent stem cells. The disclosure also provides methods useful for producing such cells.

Description

VITRA.003WO PATENTCOMPOSITIONS AND METHODS FOR PRODUCING PRIMORDIAL GERM CELLSINCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 772,049, filed March 14, 2025, which is hereby expressly incorporated by reference in its entirety.BACKGROUND OF THE INVENTIONField of the Invention

[0002] The present disclosure relates to the generation of primordial germ celLlike cells. For example, the present disclosure relates to methods of producing primordial germ cells from pluripotent stem cells in vitro.Description of the Related Art

[0003] Primordial germ cells (PGCs) are the precursors of gametes and have utility in infertility and reproductive toxicity research and in developing clinical interventions, hr the last decade, several labs have published differentiation protocols to generate primordial germ cell-like cells (PGCLCs) from pluripotent stem cells (PSCs) in vitro, thereby recapitulating the first stage of oogenesis. However, these differentiation protocols are notoriously challenging and unreproducible, and often require pre-conditioning steps which change the ground state of the PSCs. Furthermore, most of these protocols require aggregation of PSCs into 3D aggregates with very high (200-500 ng / ml) levels of signaling proteins, driving up cost per differentiation, and making it difficult, non-automatable, and inefficient to derive PGCLCs in high cell numbers (> IM cells / batch of differentiation). The ability to easily, reproducibly, efficiently, and affordably generate many PGCLCs at once is necessary for uncovering the factors necessary to push these gamete precursors further down the oogenesis trajectory. Performing these differentiations using adherent cultures is preferable, because these types of cultures are amenable to automation by liquid handlers, increasing the reproducibility, efficiency, and cost per differentiation.

[0004] The disclosure provided herein are automatable, GMP-grade generation of primordial germ cells via non-transwell and non-matrigel overlay approaches.SUMMARY OF THE INVENTION

[0005] The present disclosure demonstrates generation of primordial germ cell-like cells from pluripotent stem cells in vitro.

[0006] Provided herein is a method of producing primordial germ cell-like cells (PGCLCs) from pluripotent stem cells (PSCs). The method includes (i) contacting a population of PSCs with a minimal media and (ii) contacting the apical membrane of the population of PSCs with a BMP and a ROCK inhibitor after the minimal media is removed.

[0007] In some embodiments, the population of PSCs is in a monolayer.

[0008] In some embodiments, the minimal media does not comprise calcium and magnesium.

[0009] In some embodiments, the contacting of step (i) is for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, or at least 11 minutes.

[0010] In some embodiments, the BMP is selected from the group consisting of BMP2, BMP4, BMP5, BMP6, BMP7, and combinations thereof. In some embodiments, the BMP is BMP4.

[0011] In some embodiments, the BMP is at a concentration of at least 0.1 ng / ml, or at least 1 ng / ml, or at about 2 ng / ml or at least 5 ng / ml, or at least 10 ng / ml or at least 20 ng / ml, or at least 25 ng / ml, or at least 30 ng / ml, or at least 35 ng / ml, or at least 40 ng / ml, or at least 50 ng / ml.

[0012] In some embodiments, step (ii) further comprises stem cell factor (SCP), epidermal growth factor (EGP), leukemia inhibitory factor (LIP), fibroblast growth factor (PGP), or combinations thereof.

[0013] In some embodiments, step (ii) comprises removing the minimal media from the PSCs.

[0014] In some embodiments, the method further includes (iii) isolating the PGCLCs. In some embodiments, the PGCLCs are isolated by fluorescence-activated cell sorting (PACS). In some embodiments, the PGCLCs are isolated based on positive surfaceexpression of OCT4+, ITGA6+PDPN+, CXCR4+PDPN+, and / or CD9+PDPN+ ITGA6+CXCR4+, ITGA6+CD9+, or CXCR4+CD9+.

[0015] In some embodiments, the pluripotent stem cells are human pluripotent stem cells.

[0016] In some embodiments, the pluripotent stem cells are autologous or allogenic to a selected subject.

[0017] In some embodiments, the pluripotent stem cells are induced pluripotent stem cells.

[0018] In some embodiments, the ROCK inhibitor is selected from the group consisting of Y-27632, CEPT, Chroman-1, Pasudil, Blebbistatin, SMIPH2, C3 transferase, H-1152, NSC23766, ML141, RKI-1447, GSK429286A, and BMS-5.

[0019] In some embodiments, at least 20% of the PGCLCs express OCT4, TPAP2C, SOX 17, or a combination thereof.

[0020] Also provided herein is an isolated population of PGCLCs expressing OCT4, TPAP2C, and SOX17.

[0021] Also provided herein is a composition comprising a substantially pure population of PGCLCs expressing OCT4, TFAP2C, and SOX17.

[0022] Also provided herein is a PGCLC produced by the method of the present disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0024] FIG. 1A shows immunofluorescence images showing DAPI, pSMADl, and TJP1 staining between O and 48 hours in 3 distinct conditions. iPSCs (-BMP4): standard culture (no CALO), no BMP4; iPSC (+BMP4): standard culture (no CALO), with 50 ng / ml BMP4; iPSC (CALO) (+BMP4): CALO pre-treatment, with 50 ng / ml BMP4.

[0025] FIG. IB shows quantification and comparison of % pSMAD! positive cells between 1 and 48 hours after iPSC (+BMP4) standard culture (no CALO), with 50 ng / ml BMP4; iPSC (CALO) (+BMP4): CALO pre-treatment, with 50 ng / ml. BMP4.

[0026] FIG. 2A shows immunofluorescence images showing PGC markers OCT4, TFAP2C. SOX17, and DAPI staining at 96 hours in 3 distinct conditions. iPSCs (-BMP4): standard culture (no CALO), no BMP4; iPSC (+BMP4): standard culture (no CALO), with 10 ng / ml BMP4; iPSC (CALO) (+BMP4): CALO pre-treatment, with 10 ng / ml BMP4.

[0027] FIG. 2B shows quantification and of % OCT4 / SOX17 / TFAP2C positive cells at 96 hours after iPSCs (-BMP4): standard culture (no CALO), no BMP4; iPSC (+BMP4) standard culture (no CALO), with 10 ng / ml BMP4; iPSC (CALO) (+BMP4): CALO pretreatment, with 10 ng / ml BMP4.

[0028] FIG. 2C shows fluorescence activated cell sorting (FACS) analysis of OCT4 on iPSCs (-BMP4): standard culture (no CALO) (left) and iPSC (CALO) (+BMP4): CALO pre-treatment, with 10 ng / ml BMP4.

[0029] FIG. 2D shows fluorescence activated cell sorting (FACS) analysis of ITGA6 and PDPN, CXCR4 and PDPN, and CD9 and PDPN on PGCs derived with CALO pretreatment. FACS analysis of the same markers on iPSCs are shown in the lower right quadrants.DETAILED DESCRIPTION

[0030] The present disclosure provides, among others, a method of differentiating primordial germ cells in adherent culture without using traditional methods that include transwell membranes or matrigel overlay. These traditional methods, which have been previously described, function mechanistically by either circumventing the cell-cell contact barrier, or by inverting a cell’s polarity. In either case, these interventions render the cells more receptive to signaling proteins in the media. As described herein, the present method directly weakens the cell-cell contact banner by inhibiting tight junction structure via a brief exposure to a minimal media. Actin polymerization needed for tight junction re-assembly is the inhibited by adding a small molecule inhibitor (such as Y -27632) to the media in combination with BMP which induces PGCLC differentiation. After 4 days of contact with BMP and ROCKi, PGCLCs are derived at efficiencies of -20%.

[0031] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0032] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.Definitions

[0033] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B.”

[0034] It is understood that aspects and embodiments of the disclosure described herein include “comprising”, “consisting”, and “consisting essentially of’ aspects and embodiments. As used herein, “comprising” is synonymous with “including”, “containing”, or “characterized by”, and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0035] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where thestated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0036] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and so forth. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art. a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0037] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0038] Although features of the disclosures may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the disclosures may be described herein in the context of separate embodiments for clarity, the disclosures may also be implemented in a single embodiment. Any published patent applications and any other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference for any purpose. In the case of conflict, the present specification, includingdefinitions, will control. Tn addition, the materials, methods, and examples are illustrative only and not intended to be limiting.Methods Of The Disclosure

[0039] As described in greater detail below, one aspect of the present disclosure relates to a method of producing primordial germ cell-like cells (PGCLCs) from pluripotent stem cells (PSCs). The method involves contacting a population of PSCs with a minimal media and contacting the apical membrane of the population of PSCs with a BMP and a ROCK inhibitor after the minimal media is removed.Primordial Germ Cells

[0040] Primordial germ cells (PGCs) are the embryonic precursors of gametes (sperm and eggs), which generate a new organism that is capable of creating endless new generations through germ cells. PGCs represent the founder cells of the germline. PGCs are specified during early mammalian postimplantation development and are uniquely programmed for transmission of genetic and epigenetic information to subsequent generations. Primordial germ cells are single cells that under certain culture conditions can form colonies of cells which morphologically resemble undifferentiated embryonic stem cells.

[0041] As used herein, primordial germ cell-like cells (PGCLCs) are cells derived from pluripotent stem cells in vitro that transcriptionally show high resemblance to in vivo PGCs. Primordial germ cells and PGCLCs express a number of different biomarkers that can be used to distinguish PGCLCs from other cell types. For example, PGCLCs cells are typically positive for Nanas C2HC-Type Zinc Finger 3 (NAN0S3), SRY-Box Transcription Factor 17 (SOX17), Transcription Factor AP-2 Gamma (TFAP2C), PR / SET Domain 1 (PRDM1), Octamer Binding Transcription Factor 4 (OCT4), and / or negative for SRY-Box Transcription Factor 2 (SOX2).Pluripotent Stem Cells

[0042] The PGCLCs provided herein are differentiated from pluripotent stem cells (PSCs). Pluripotent stem cells are 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 the primordial germ cells, and therefore into all cells of the adult body, but not extra- embryonic tissues such as the placenta (Shi et al. 2017).

[0043] Non-limiting examples of pluripotent stem cells for use in the methods described herein include induced pluripotent cell (iPSCs), 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, the pluripotent cells are human pluripotent cells.

[0044] In some embodiments, the pluripotent stem cells are embryonic stem cells, such as a human embryonic stem cell. “Embryonic stem cell” is a general term for pluripotent stem cells 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.

[0045] In some embodiments, the pluripotent stem cells are induced pluripotent stem cells. Induced pluripotent stem cells are reprogrammed mature cells that have the capacity to differentiate into different mature cell types.

[0046] In some embodiments, the induced pluripotent stem cells are human induced pluripotent stem cells. Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell artificially derived from a non-pluripotent cell. Examples include germ cells, keratinizing epithelial cells (e.g., keratinized epidermal cells), mucosal epithelial cells (e.g., epithelial cells of the superficial layer of tongue), exocrine gland epithelial cells (e.g., mammary gland cells), hormone- secreting cells (e.g., adrenomedullary cells), cells for metabolism or storage (e.g., liver cells), intimal epithelial cells constituting interfaces (e.g., type I alveolar cells), intimal epithelial cells of the obturator canal (e.g., vascular endothelial cells), cells having cilia with transporting capability (e.g., airway epithelial cells), cells for extracellular matrix secretion (e.g., fibroblasts), constrictive cells (e.g., smooth muscle cells), cells of the blood and the immune system (e.g., T lymphocytes), sense-related cells (e.g., bacillary cells), autonomic nervous system neurons (e.g., cholinergic neurons), sustentacular cells of sensory organs and peripheral neurons (e.g., satellite cells), nerve cells and glia cells of the central nervous system (e.g., astroglia cells), pigment cells (e.g., retinal pigment epithelial cells), progenitor cells thereof (tissue progenitor cells) and the like. There is no limitation on the degree of cell differentiation; even undifferentiated progenitor cells (including somatic stem cells) and finally differentiated mature cells can be used alike as sources ofsomatic cells in the present disclosure. Examples of undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as adipose-derived stromal (stem) cells, nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.

[0047] The starting cells can be induced to form pluripotent stem cells using either genetic or chemical induction methods. Examples of methods for generating human induced pluripotent stem cells include those described by US patent 8,058.065 (Yamanaka et al.), WO / 2019 / 165988 by Pei et al., and United States Patent Application No. 20190282624 by Deng et al. Induced PSC can also be generated through chemical reprogramming, via JNK pathway inhibition as illustrated by Guan et al. (Nature 605: 325-331 (2022)).

[0048] In some embodiments, the pluripotent stem cells can be obtained from a selected subject, for example a human subject desiring in vitro fertilization. Thus, in some embodiments, the pluripotent stem cells are autologous to a selected subject. In other embodiments, the pluripotent stem cells are allogeneic to a selected subject.Pluripotent Stem Cell Maintenance

[0049] In some embodiments, the method described herein includes culturing, in culture media, a population of pluripotent stem cells (PSCs) to produce an expanded population of PSCs prior to initiating the differentiation process into PGCLCs.

[0050] In the methods described herein, the starting population of PSCs can comprise various densities. In some embodiments, the starting population of PSCs comprises about 10,000 cells / mm2, about 11,000 cells / mm2, about 12,000 cells / mm2, about 13,000 cells / mm2, about 14,000 cells / mm2, or about 15,000 cells / mm2. In some embodiments, the starting population of PSCs comprises about 12,000 cells / mm2.

[0051] Pluripotent cells can be cultured and maintained in an undifferentiated state using a variety of methods. In some embodiments, the methods herein comprise culturing PSCs in a feeder-free, serum-free culture media. In some embodiments, pluripotent cells are cultured and maintained in an essentially undifferentiated state using defined, feeder-independent culture system, such as in a TeSR or ES medium {see, e.g., Chen et al. (2011). In some embodiments, culture media 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 p (rh TGFJ3) {e.g., 20 pM to25 pM). In some embodiments, culture media comprises mTeSR™ Plus medium (STEMCELL Technologies).

[0052] In some embodiments, the culture medium also comprises a ROCK inhibitor. The ROCK inhibitor can be Y-27632, which is a cell-permeable, highly potent and selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK). Y-27632 inhibits both ROCK1 (Ki=220nM)and ROCK2 (Ki=300nM). Use of Y-27632 can improve survival of stem cells when they are dissociated to single cells and after thawing the stem cells. Y-27632 can also reduce or block apoptosis of stem cells. Other exemplary ROCK inhibitors include, but are not limited to, thiazovivin, Y27632, Fasudil, AR122-86, Y27632 H-1152, Y-30141, Wf-536, HA-1077, hydroxyl-HA. -1077, GSK2699962A, SB-772077-B, N-(4-pyridyl)-N’-(2.4,6-trichlorophenyl) urea, 3-(4-pyridyl)-lH-indole, and (R) -(+)-trans-N-(4-pyridyl)-4-(l-aminoethyl)-cyclohexanecarboxamide, pyrintegrin, and blebbistatin.

[0053] The ROCK inhibitor can be used in the culture media in amounts of at least 0.5 pM, or at least 1.0 pM, or at least 2.0 pM, or at least 3.0 pM, or at least 4.0 pM, or at least 5.0 pM, or at least 6.0 pM, or at least 7.0 pM, or at least 8.0 pM, or at least 9.0 pM, or at about 10 pM. In general, the ROCK inhibitor is used in the culture media in amounts less than 30 pM, or less than 25 pM, or less than 20 pM, or less than 15 pM. In some embodiments, the ROCK inhibitor is used at 10 pM.

[0054] The ROCK inhibitor can be used in the culture media when the hiPSCs are initially seeded into the vessel (e.g., wells) where the primordial germ cell-like cells will be generated. In some embodiments, the ROCK inhibitor can be removed prior to the culture media being replaced with minimal media as described in more detail below.

[0055] When initially maintaining the PSC culture for use in the methods herein, a matrix component can be included in a defined media for culturing and maintaining pluripotent cells in a substantially or essentially undifferentiated state. Various matrix components may be used to culture and maintain pluripotent cells, such as hESC or iPSC. For example, Matrigel, Geltrex, collagen IV, fibronectin, laminin, and vitronectin in combination can be used to provide a solid support for embryonic cell culturing and maintenance.

[0056] It is appreciated that additional methods for the culture and maintenance of human embryonic stem cells, as would be known to one of skill in the art. may be used with the present embodiments.

[0057] 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.Differentiation

[0058] As described above, the method of the present disclosure involves contacting a population of PSCs with a minimal media and contacting the apical membrane of the population of PSCs with a BMP and a ROCK inhibitor after the minimal media is removed.

[0059] In some embodiments, prior to the population of PSCs being contacted in the methods described herein, the PSCs are passaged and are seeded at a density of about 10,000 cells / mm2, about 15,000 cells / mm2, about 20,000 cells / mm2, about 25,000 cells / mm2, about 30,000 cells / mm2, about 35,000 cells / mm2, about 40,000 cells / mm2, about 45,000 cells / mm2, about 50.000 cells / mm2, about 55,000 cells / mm2, about 60,000 cells / mm2, or about 65,000 cells / mm2.

[0060] The population of PSCs that is contacted in the methods described herein can be in a monolayer. The term “monolayer” as used herein can refer to cells that are attached to a solid support while growing under suitable culture conditions. Such cell populations can also be described as 2D as cells grow by attaching (adhering) to a substrate.

[0061] As described above, when the PSCs have been initially maintained in culture media containing a ROCK inhibitor, in some embodiments, the ROCK inhibitor is removed after seeding of the cells for the differentiation process. In some embodiments, the ROCK inhibitor is removed 24 hours after the PSCs are seeded for differentiation. This is Day 1 of the differentiation process (i.e., 24 hours after seeding of the cells).

[0062] The population of PSCs is next contacted with a minimal media. The term “minimal media” as used herein can refer to a cell culture media formulation that includes a small number of ingredients (e.g., 4, 3, 2, or 1 ingredients). A minimal media as used herein can include any media that contains neither signaling proteins nor calcium. In some embodiments, the minimal media does not contain calcium or magnesium. In some embodiments, the minimal media comprises DPBS. In some embodiments, the minimal media comprises DPBS without calcium and magnesium.

[0063] The minimal media as described herein also disrupts formation of tight junctions. In some embodiments, contacting the pluripotent stem cells as described herein results in the disruption of tight junction protein 1 (TJP1).

[0064] In some embodiments, the population of PSCs is contacted with the minimal media on Day 2 of differentiation (i.e., about 48 hours after the PSCs were seeded for the differentiation process).

[0065] In some embodiments, the population of PSCs can be contacted with the minimal media for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, or at least 11 minutes. In some embodiments, the population of PSCs is contacted with the minimal for 11 minutes. In some embodiments, the population of PSCs is contacted with DPBS without calcium and magnesium for 11 minutes.

[0066] Following exposure to the minimal media, the minimal media is removed from the population of PSCs and the apical membrane of the population of PSCs is contacted with a BMP and a ROCK inhibitor.

[0067] As used herein, the apical cell surface refers to the surface of a monolayer of cells that faces the culture medium. The apical surface does not include the cell surface that contacts the culture plate or the culture vessel or that contacts an aggregated cell mass.

[0068] In some embodiments, the BMP and a ROCK inhibitor are comprised within a cell culture medium. In some embodiments, the cell culture medium is a feeder-free, serum-free culture media. In some embodiments, the culture media comprises mTeSR™ Plus medium (STEMCELL Technologies).

[0069] ROCK inhibitors useful in the methods described herein are described above. In some embodiments, the ROCK inhibitor is Y-27632. The ROCK inhibitor can be used in the culture media in amounts of at least 0.5 pM, or at least 1.0 pM, or at least 2.0 pM, or at least 3.0 pM, or at least 4.0 pM, or at least 5.0 pM, or at least 6.0 pM, or at least 7.0 pM, or at least 8.0 pM, or at least 9.0 pM, or at about 10 pM. In general, the ROCK inhibitor is used in the culture media in amounts less than 30 pM, or less than 25 pM, or less than 20 pM, or less than 15 pM. In some embodiments, the ROCK inhibitor is used at 10 pM.

[0070] As used herein, “Bone Morphogenetic Protein (BMP)” refers to a polypeptide growth factor of the TGF-P superfamily. There are currently about 20 knownmembers in the BMP family. In some embodiments, the BMP is selected from BMP2, BMP4, BMP5, BMP6, BMP7, and combinations thereof. In some embodiments, the BMP is BMP4.

[0071] The BMP can be used in the culture media in various amounts. For example, BMP can be included in the culture media in amounts of at least 0.1 ng / ml, or at least 1 ng / ml, or at about 2 ng / ml or at least 5 ng / ml, or at least 10 ng / ml, or at least 20 ng / ml, or at least 25 ng / ml, or at least 30 ng / ml, or at least 35 ng / ml, or at least 40 ng / ml, or at least 50 ng / ml. In general, the BMP is used in the culture media in amounts less than 50 ng / ml, or less than 40 ng / ml, or less than 30 ng / ml, or less than 20 ng / ml, orless than 15 ng / ml. In some embodiments, the BMP is used at 10 ng / mL.

[0072] In addition to the ROCK inhibitor and the BMP, the population of PSCs can also be contacted with additional growth factors. According to some embodiments, the culture medium further includes at least one additional agent selected from the group consisting of stem cell factor (SCP), epidermal growth factor (EGP), leukemia inhibitory factor (LIP), fibroblast growth factor (PGP), or combinations thereof.

[0073] In some embodiments of the methods described herein, within 1 hour of exposure to BMP, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the pluripotent stem cells exhibit the activation of SMAD1, a protein that plays a crucial role in the differentiation of primordial germ cells (PGCs) by acting as key downstream signaling molecules in the Bone Morphogenetic Protein (BMP) pathway.

[0074] In some embodiments, the activation of SMAD1 in the pluripotent stem cells treated using the methods described herein is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold higher as compared to pluroptent stem cells that were not contacted with a minimal media as described herein.

[0075] The time for conversion of starting PSCs into primordial germ cells in the BMP-containing media can vary. In some embodiments, the population of PSCs are cultured in the presence of BMP and ROCK inhibitor for at least about 1 day, or for at least about 2 days, or for at least about 3 days, or for at least about 4 days, or for at least about 5 days, or forat least about 6 days. Tn some embodiments, the population of PSCs are cultured in the presence of BMP and ROCK inhibitor for at least about 6 days.

[0076] In some embodiments, the BMP and a ROCK inhibitor are removed and replaced with fresh BMP and a ROCK inhibitor (e.g., in a culture medium) between Days 3 to 5 of the differentiation process. In some embodiments, the BMP and a ROCK inhibitor are removed and replaced with fresh BMP and a ROCK inhibitor {e.g., in a culture medium) at Day 3. In some embodiments, the BMP and a ROCK inhibitor are removed and replaced with fresh BMP and a ROCK inhibitor (e.g., in a culture medium) at Day 4. In some embodiments, the BMP and a ROCK inhibitor are removed and replaced with fresh BMP and a ROCK inhibitor (e.g., in a culture medium) at Day 5.

[0077] In some embodiments, the population of PSCs differentiates into PGCLCs at an efficiency of about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29% after 4 days of BMP stimulation.

[0078] In some embodiments, the PSCs differentiate into PGCLCs which are OCT4+, SOX17+, and TFAP2C+. In some embodiments, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23% of the PGCLCs are OCT4+, SOX17+, and TFAP2C+.PGCLC Isolation

[0079] Following differentiation of the population of PSCs into PGCLCs, the PGCLCs can be isolated.

[0080] In some embodiments, the isolation of the PGCLCs is performed by sorting using a fluorescence activated cell sorter (FACS). Fluorescence activated cell sorting (FACS) is a well-known method for separating particles, including cells, based on the fluorescent properties of the particles (Kamarch, 1987, Methods Enzymol. 151:150-165). Laser excitation of fluorescent moieties in the individual particles results in a small electrical charge allowing electromagnetic separation of positive and negative particles from a mixture. In one embodiment, cell surface marker-specific antibodies or ligands are labeled with distinct fluorescent labels. Cells are processed through the cell sorter, allowing separation of cells based on their ability to bind to the antibodies used. FACS sorted particles may be directly deposited into individual wells of cell culture plates, such as 96-well or 384-well plates.

[0081] In some embodiments, the PGCLCs are sorted on the basis of expression of the markers podoplanin (PDPN), integrin alpha 6 (ITGA6), C-X-C chemokine receptor 4 (CXCR4), CD9, OCT4, and combinations thereof. In some embodiments, the cells are sorted based on the positive expression of both PDPN and ITGA6. In some embodiments, the cells are sorted based on the positive expression of both PDPN and CXCR4. In some embodiments, the cells are sorted based on the positive expression of both PDPN and CD9. In some embodiments, the cells are sorted based on the positive expression of both ITGA6 and CXCR4. In some embodiments, the cells are sorted based on the positive expression of both ITGA6 and CD9. In some embodiments, the cells are sorted based on the positive expression of both CXCR4 and CD9. In some embodiments, the cells are sorted based on the positive expression of OCT4.

[0082] With respect to antibody-mediated detection and sorting of PGCLCs, any antibody, specific for a particular marker, can be used, in combination with any fluorophore or other label suitable for the detection and sorting of cells (e.g., fluorescence-activated cell sorting).

[0083] PGCLCs can be labeled with an antibody to a single marker and detected and / sorted. PGCLCs can also be simultaneously labeled with multiple antibodies to different markers.

[0084] The sorted cells can be cultured and maintained using a variety of methods. In some embodiments, the methods herein comprise culturing PSCs with a matrix component in a defined media for culturing and maintaining the cells. Various matrix components may be used to culture and maintain PGCLCs, such as, for example, Matrigel, Geltrex, collagen IV, fibronectin, laminin, vitronectin, and combinations thereof.

[0085] The PGCLCs are cultured in a feeder-free, serum-free culture media. In some embodiments, PGCLCs are cultured and maintained in a TeSR or GK15 media. In some embodiments, culture media further comprises one or more growth factor, 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 0 (rh TGF0) (e.g., 20 pM to 25 pM). In some embodiments, culture media comprises mTeSR™ Plus medium (STEMCELL Technologies).

[0086] In some embodiments, the culture medium also comprises a BMP. Various BMPs useful in the methods herein are described in detail above. In some embodiments, the BMP is BMP4.

[0087] The BMP can be used in the culture media in various amounts. For example, BMP can be included in the culture media in amounts of at least 0.1 ng / ml, or at least 1 ng / ml, or at about 2 ng / ml or at least 5 ng / ml, or at least 10 ng / ml, or at least 20 ng / ml, or at least 25 ng / ml, or at least 30 ng / ml, or at least 35 ng / ml, or at least 40 ng / ml, or at least 50 ng / ml. In general, the BMP is used in the culture media in amounts less than 50 ng / ml, or less than 40 ng / ml, or less than 30 ng / ml, or less than 20 ng / ml, or less than 15 ng / ml. In some embodiments, the BMP is used at 10 ng / mL.

[0088] In some embodiments, the culture medium also comprises a ROCK inhibitor. ROCK inhibitors useful in the methods herein are described in detail above. In some embodiments, the ROCK inhibitor is Y-27632.

[0089] The ROCK inhibitor can be used in the culture media in amounts of at least 0.5 pM, or at least 1.0 pM, or at least 2.0 pM, or at least 3.0 pM, or at least 4.0 pM, or at least 5.0 pM, or at least 6.0 pM, or at least 7.0 pM, or at least 8.0 pM, or at least 9.0 pM, or at about 10 pM. In general, the ROCK inhibitor is used in the culture media in amounts less than 30 pM, or less than 25 pM, or less than 20 pM, or less than 15 pM. In some embodiments, the ROCK inhibitor is used at 10 pM.

[0090] In some embodiments, the PGCLCs are cultured in complete mTeSR-i-supplemented with 10 ng / mL BMP4 and ROCK inhibitor.

[0091] In some embodiments, the PGCLCs are cultured in GK15 medium OMEM supplemented with 15% KSR, 1 mM sodium pyruvate, 1 x NEAA, 2 mM L-glutamine, 100 uM beta- mercaptoethanol) with ROCK inhibitor.

[0092] The sorted PGCLCs can be cultured at various cell densities. In some embodiments, the sorted PGCLCs are cultured at a density of about 30,000 cells / cm2, about 32,000 cells / cm2, about 34,000 cells / cm2, about 36,000 cells / cm2, about 38,000 cells / cm2, about 40,000 cells / cm2, about 42,000 cells / cm2, about 44,000 cells / cm2, about 46,000 cells / cm2, about 48,000 cells / cm2, about 50,000 cells / cm2, about 52,000 cells / cm2, about 54,000 cells / cm2, about 58,000 cells / cm2, about 60,000 cells / cm2, about 62,000 cells / cm2, about 64,000 cells / cm2, about 66,000 cells / cm2, about 68,000 cells / cm2, about 70,000cells / cm2, about 72,000 cells / cm2, about 74,000 cells / cm2, about 76,000 cells / cm2, about 78,000 cells / cm2, about 80,000 cells / cm2, about 82,000 cells / cm2, about 84,000 cells / cm2, about 86,000 cells / cm2, about 88,000 cells / cm2, about 90,000 cells / cm2.

[0093] In some embodiments, culture media can be removed from the sorted PGCLCs within about 2 hours after plating. In some embodiments, the culture medium is removed and replaced with complete mTeSR-i- supplemented with 10 ng / mL BMP4 and ROCK inhibitor. In some embodiments, culture medium is removed and replaced with GK15 medium (G-MEM supplemented with 15% KSR, 1 mM sodium pyruvate, 1 x NEAA, 2 mM L-glutamine, 100 uM beta- mercaptoethanol) with ROCK inhibitor.

[0094] In some embodiments, the culture medium further comprises retinoic acid.Compositions Of The Disclosure

[0095] The present disclosure also provides an isolated population of PGCLCs expressing OCT4, TFAP2C, and SOX 17.

[0096] Also provided herein is a composition comprising a substantially pure population of PGCLCs expressing OCT4, TFAP2C, and SOX17.

[0097] The term “substantially pure”, with respect to a particular cell population, refers to a population of cells that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% pure, with respect to the cells making up a total cell population. With regard to a population of differentiated PGCLCs, the term “substantially pure” refers to a population of cells that contain fewer than about 30% of pluripotent stem cells. In some embodiments, the population of cells can contain fewer than about 25%, or 20%, or 15%, or 10%, or 8%, or 7%, or 5%, or 1% of pluripotent stem cells. Stated another way, the term “substantially pure” refers to a population of cells that contain at least about 70%. at least about 80%. at least about 90%. at least about 95%. at least about 97%, at least about 99%, or up to 100% of PGCLCs.

[0098] In some embodiments, OCT4, TFAP2C, and SOX17 are expressed in at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100% of the population of PGCLCs.

[0099] In some embodiments, the population of PGCLCs comprises at least about 2 x 106, 2.1 x 106, 2.2 x 106, 2.3 x 106, 2.4 x 106, 2.5 x 106, 2.6 x 106, 2.7 x 106, 2.8 x 106, 2.9 x 106, 3 x 106, 3.1 x 106, 3.2 x 106, 3.3 x 106, 3.4 x 106, 3.5 x 106, 3.6 x 106, 3.7 x 106, 3.8 x106, 3.9 x 106, 4 x IO6, 4.1 x 106, 4.2 x IO6, 4.3 x IO6, 4.4 x 106, 4.5 x IO6, 4.6 x 106, 4.7 x 1O6, 4.8 x 106, 4.9 x 106, 5 x 106cells.[O1OO] Also provided herein is a PGCLC or a population of PGCLCs produced by the methods described herein.NON-LIMITING EMBODIMENTS1. A method of producing primordial germ cell-like cells (PGCLCs) from pluripotent stem cells (PSCs), the method comprising:(i) contacting a population of PSCs with a minimal media and (ii) contacting the apical membrane of the population of PSCs with a BMP and a ROCK inhibitor after the minimal media is removed.2. The method of embodiment 1, wherein the population of PSCs is in a monolayer.3. The method of embodiment 1 or 2, wherein the minimal media does not comprise calcium and magnesium.4. The method of any one of embodiment 1 to 3, wherein the contacting of step (i) is for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, or at least 11 minutes.5. The method of any one of the preceding embodiments, wherein the BMP is selected from the group consisting of BMP2, BMP4, BMP5, BMP6, BMP7, and combinations thereof.6. The method of embodiment 5, wherein the BMP is BMP4.7. The method of any one of the preceding embodiments, wherein the BMP is at a concentration of at least 0.1 ng / ml, or at least 1 ng / ml, or at about 2 ng / ml or at least 5 ng / ml, or at least 10 ng / ml or at least 20 ng / ml, or at least 25 ng / ml, or at least 30 ng / ml, or at least 35 ng / ml, or at least 40 ng / ml, or at least 50 ng / ml.8. The method of any one of the preceding embodiments, wherein step (ii) further comprises stem cell factor (SCF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), fibroblast growth factor (FGF), or combinations thereof.9. The method of any one of the preceding embodiments, wherein step (ii) comprises removing the minimal media from the PSCs.10. The method of any one of the preceding embodiments, further comprising:(iii) isolating the PGCLCs.11. The method of embodiment 10, wherein the PGCLCs are isolated by fluorescence-activated cell sorting (FACS).12. The method of any one of embodiments 10-11. wherein the PGCLCs are isolated based on positive surface expression of OCT4+, ITGA6+PDPN+, CXCR4+PDPN+, and / or CD9+PDPN+ ITGA6+CXCR4+, ITGA6+CD9+, or CXCR4+CD9+.13. The method of any one of the preceding embodiments, wherein the pluripotent stem cells are human pluripotent stem cells.14. The method of embodiment 13, wherein the pluripotent stem cells are autologous or allogenic to a selected subject.15. The method of any one of the preceding embodiments, wherein the pluripotent stem cells are induced pluripotent stem cells.16. The method of any one of the preceding embodiments wherein the ROCK inhibitor is selected from the group consisting of Y-27632, CEPT, Chroman-I, Fasudil, Blebbistatin, SMIFH2, C3 transferase, H-1152, NSC23766, ML141, RKI-1447, GSK429286A. and BMS-5.17. The method of any one of preceding embodiments, wherein at least 20% of the PGCLCs express OCT4, TFAP2C. SOX 17, or a combination thereof.18. An isolated population of PGCLCs expressing OCT4, TFAP2C, and SOX17.19. A composition comprising a substantially pure population of PGCLCs expressing OCT4, TFAP2C, and SOX17.20. A PGCLC produced by the method of any one of embodiments 1 to 17.EXAMPLES

[0101] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.EXAMPLE 1: METHODS

[0102] iPSC Maintenance. iPSCs were obtained from Applied StemCell. For some experiments, OCT4-GFP iPSCs were generated using the Invitrogen™ TrueDesign™ Genome Editor tool. Briefly, a P2A-GFP construct was knocked in at Exon 5 of the enogenous POU5F1 (OCT4 gene) locus, right before the stop codon on the C-terminus. P2A is a self cleaving peptide, so when the OCT4 gene is expressed, a GPF fluorescent protein is also co-expressed. iPSCs were cultured in complete mTeSR-i- medium (mTESR-i- supplemented with mTESR-i-supplement cocktail) and passaged every 3 - 4 days. To passage, iPSCs were briefly washed with DPBS (without calcium and magnesium) and detached and dissociated by Accutase treatment for 5 minutes at 37 °C. Next, cells were transferred to a 15-ml falcon tube, complete mTESR+ was added and cells were spun down at 700-900 x g for 3 to 5 minutes. Finally, medium was removed, and cells were resuspended in a fresh complete mTESR+ supplemented with 10 pM Rock inhibitor (Y-27632) and counted using either a hematocytometer, microscope or flow cytometry.

[0103] Next, iPSCs were seeded at a low density (12,000 live cells / cm2) into Geltrex®-coated (1:100 dilution) (or Matrigel®-coated at 1:60 dilution, or laminin / fibronectin-coated at 10 pg to 10 mg / ml) plates and overlaid with complete mTESR-i-medium containing 10 pM Rock inhibitor (Y-27632). Rock inhibitor was removed after 24-48 hours by changing the medium to complete mTeSR+.

[0104] Fluorescence-activated Cell Sorting (FACS). At Day 6 of differentiation, cells were briefly washed with DPBS and thereafter dissociated with Accutase for 15 - 20minutes at 37 °C. Cells were collected in a 15 ml tube and pelleted at 700 - 900 x g for 3 - 5 minutes. The supernatant was removed, and the pellet was resuspended in either PBS with 0.5% BSA or in complete mTeSR-i- medium.

[0105] Next, cells were stained for 30 minutes on ice with fluorophore-conjugated antibodies recognizing human Podoplanin (PDPN), Integrin alpha 6, CXC motif chemokine receptor type 4 (CXCR4), and CD9 (also known as Tetraspanin-29), at dilutions of 1:50 -1:200.

[0106] Thereafter cells were twice washed with either PBS with 0.5% BSA or complete mTeSR-i- and pelleted by centrifugation at 700 - 900 x g for 3 - 5 minutes in-between washes.

[0107] After the last wash, the cells were spun down as described above, and pellet was resuspended in complete mTeSR-i- medium with Rock inhibitor (Y-27632). Cells were thereafter stained through 35 pm mesh into a round-bottom polystyrene tube and DAPI was added at a concentration of 1:10,000.

[0108] Next, antibody- stained cells were loaded onto a BD FACS Ariail, and live PGCLCs (negative for DAPI) were identified by gating for single cells (gated using forward and side scatters) positive for OCT4 or double-positive for either PDPN and ITGA6, or PDPN and CXCR4, or PDPN and CD9, or ITGA6 and CXCR4, or ITGA6 and CD9, or CXCR4 and CD9.

[0109] The PGCLC population was thereafter sorted into plates coated with either Geltrex® or Matrigel® or human recombinant fibronectin or human recombinant laminin-521, and containing either complete mTeSR-i- supplemented with 10 ng / ml human recombinant BMP4 or GK15 medium (G-MEM supplemented with 15% KSR, 1 mM sodium pyruvate, 1 x NEAA, 2 mM L-glutamine, 100 uM beta-mercaptoethanol) with Rock inhibitor (Y-27632) at a density of 36,000 - 89,000 cells / cm2.

[0110] Two hours after plating the PGCLCs, the medium was removed and replaced with either fresh complete mTeSR-i- supplemented with 10 ng / ml human recombinant BMP4 and Rock inhibitor or GK.15 medium with Rock inhibitor and incubated overnight in 37 °Cat 5% CO2. Alternatively, retinoic acid was supplemented as well.EXAMPLE 2: GENERATION OF PRIMORDIAL GERM CELL-LIKE CELLS

[0111] This example demonstrates the generation of primordial germ cell-like cells from induced pluripotent stem cells.

[0112] iPSCs were passaged as described in Example 1. Passaged cells were seeded similarly as described, but at a density of 35,000-60,000 live cells / cm2.

[0113] After 24 hours (at Day 1) Rock inhibitor was removed by changing the medium to complete mTeSR+. At Day 2, iPSCs were briefly washed with dPBS as a minimal media and treated for 11 minutes with dPBS without calcium and magnesium at 37 °C.

[0114] Next, dPBS without calcium and magnesium was removed, and iPSCs were overlaid with 0.6 ml / cm2of fresh complete mTESR-i- medium containing 10 ng / ml BMP4 and 10 pM Rock inhibitor (Y-27632).

[0115] At Days 3 to 5, medium was removed and replaced by 0.6 ml / cm2of fresh complete mTESR+ medium containing 10 ng / ml BMP4 and 10 pM Rock inhibitor (Y-27632).

[0116] At Day 6, cells were harvested and PGCLCs were collected by FACS for ITGA6+PDPN+, CXCR4+PDPN+, CD9+PDPN+ or OCT4+ as described in Example 1. FIG.2C shows that 28.6% of the differentiated iPSCs expressed OCT4 using an iPSC OCT4-GFP line generated as described above. FIG. 2D shows that the differentiated iPSCs contained 7.10% ITGA6+PDPN+ cells, 7.07% CXCR4+PDPN+ cells, and 6.28% CD9+PDPN+ cells.

[0117] Cells were then also analyzed activation of pSMADl as well as for expression of PGCLC markers at various time points.

[0118] As described above, the activation of pSMADl and presence of tight junction protein 1 (TJP1) was analyzed at various time points after additions of BMP4. As shown in FIG. 1A, iPSCs treated with BMP4 after CALO pre-treatment maintained a uniform activation of pSMADl over 48 hours along with a reduction in tight junction formation, demonstrating stability in signaling pathway activation.

[0119] As shown in FIG. IB, the percentage of pSMADl positive cells was significantly increased in cells briefly washed with dPBS as a minimal media and then treated with BMP4 for 1 - 48 hours. This level of activation persisted through at least 48 hours. This quantification further corroborates that CALO pre-treatment can induce stability in signaling pathway activation.

[0120] Next, the expression levels of the PGC markers OCT4, TFAP2C, and SOX17 were analyzed by fluorescence microscopy. As shown in FIGs. 2A and 2B, within 48 hours of exposure to BMP4, cells that were briefly washed with dPBS as a minimal media showed an increased amount of OCT4, TFAP2C, and SOX17 positive cells (FIG. 2A), and particularly an increase in OCT4 / TFAP2C / SOX17 triple positive cells (FIG. 2B) as compared to cells that were not. Triple positive expression of OCT4, TFAP2C, and SOX17 validates the identity of these cells, and is another method for quantification of the differentiation efficacy (% PGCLCs) derived by the CALO method.

[0121] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0122] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0123] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure and are to be included within the spirit and purview of this application.

[0124] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purpose.

Claims

WHAT IS CLAIMED IS:

1. A method of producing primordial germ cell-like cells (PGCLCs) from pluripotent stem cells (PSCs), the method comprising:(i) contacting a population of PSCs with a minimal media and (ii) contacting the apical membrane of the population of PSCs with a BMP and a ROCK inhibitor after the minimal media is removed.

2. The method of claim 1, wherein the population of PSCs is in a monolayer.

3. The method of claim 1, wherein the minimal media does not comprise calcium and magnesium.

4. The method of any claim 1, wherein the contacting of step (i) is for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, or at least 11 minutes.

5. The method of claim 1, wherein the BMP is selected from the group consisting of BMP2, BMP4, BMP5, BMP6, BMP7, and combinations thereof.

6. The method of claim 5, wherein the BMP is BMP4.

7. The method of claim 1, wherein the BMP is at a concentration of at least 0.1 ng / ml, or at least 1 ng / ml, or at about 2 ng / ml or at least 5 ng / ml, or at least 10 ng / ml or at least 20 ng / ml, or at least 25 ng / ml, or at least 30 ng / ml, or at least 35 ng / ml, or at least 40 ng / ml, or at least 50 ng / ml.

8. The method of claim 1, wherein step (ii) further comprises stem cell factor (SCF), epidermal growth factor (EGF), leukemia inhibitory factor (LIF), fibroblast growth factor (FGF), or combinations thereof.

9. The method of claim 1, wherein step (ii) comprises removing the minimal media from the PSCs.

10. The method of claim 1, further comprising:(iii) isolating the PGCLCs.

11. The method of claim 10, wherein the PGCLCs are isolated by fluorescence-activated cell sorting (FACS).

12. The method of any one of claims 10-11, wherein the PGCLCs are isolated based on positive surface expression of OCT4+, ITGA6+PDPN+. CXCR4+PDPN+, and / or CD9+PDPN+ ITGA6+CXCR4+, ITGA6+CD9+, or CXCR4+CD9+.

13. The method of claim 1, wherein the pluripotent stem cells are human pluripotent stem cells.

14. The method of claim 13. wherein the pluripotent stem cells are autologous or allogenic to a selected subject.

15. The method of claim 1, wherein the pluripotent stem cells are induced pluripotent stem cells.

16. The method of claim 1, wherein the ROCK inhibitor is selected from the group consisting of Y-27632, CEPT, Chroman-I, Fasudil, Blebbistatin, SMIFH2, C3 transferase, H-1152. NSC23766, ME141, RKI-1447, GSK429286A, and BMS-5.

17. The method of any one of preceding claims, wherein at least 20% of the PGCECs express OCT4, TFAP2C, SOX 17, or a combination thereof.

18. An isolated population of PGCECs expressing OCT4, TFAP2C, and SOX17.

19. A composition comprising a substantially pure population of PGCECs expressing OCT4, TFAP2C, and SOX17.

20. A PGCLC produced by the method of claim 1.

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