{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T10:19:46Z","timestamp":1787566786356,"version":"build-2736575974"},"reference-count":23,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T00:00:00Z","timestamp":1787184000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012190","name":"Ministry of Science and Higher Education of the Russian Federation","doi-asserted-by":"crossref","award":["075-15-2025-464"],"award-info":[{"award-number":["075-15-2025-464"]}],"id":[{"id":"10.13039\/501100012190","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Because human infertility can arise from genetic, molecular, cellular, anatomical, and endocrine abnormalities, in vitro gametogenesis has become an important area of reproductive research. New developing technology involving the generation of human primordial germ cell-like cells (hPGCLCs) from induced pluripotent stem cells (hIPSCs) might assist with understanding early germ cell development (specification, migration, gametogenesis, and epigenetic reconstitution), as well as provide a solution for infertility and hereditary disorders. Given that human primordial germ cells (PGCs) are still not well characterized at a molecular level, we present a practical workflow to robustly and efficiently induce hPGCLCs from human pluripotent stem cells (hPSCs) XX and XY cell lines. This protocol describes the hPGCLC specification of hPSCs through sequential induction with Activin A for 2 days and BMP4 for 6 days in 2D and 3D culture systems. Induction of hPSCs into hPGCLCs demonstrated expression of early primordial germ cell markers, including PRDM1, NANOS3, DAZL, STELLA, SOX17, SSEA1, and cKIT, on the 8th day of hPGCLC generation. We described the protocol for generating early hPGCLCs, which provides an opportunity for further investigations into maturation into late germ cells and a chance to overcome a meiotic block to obtain haploid gametes in vitro.<\/jats:p>","DOI":"10.3390\/biology15161436","type":"journal-article","created":{"date-parts":[[2026,8,20]],"date-time":"2026-08-20T12:57:53Z","timestamp":1787230673000},"page":"1436","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Protocol of Human Primordial Germ Cell-like Cell Generation from Pluripotent Stem Cells in 2D and 3D Culture Systems"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5697-3774","authenticated-orcid":false,"given":"Vepa K.","family":"Abdyev","sequence":"first","affiliation":[{"name":"Koltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova, 26, 119334 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9026-6827","authenticated-orcid":false,"given":"Polina I.","family":"Sirotkina","sequence":"additional","affiliation":[{"name":"Department of Biology, Lomonosov Moscow State University, 1-12 Leninskie Gory, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Evgeniia D.","family":"Erofeeva","sequence":"additional","affiliation":[{"name":"Koltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova, 26, 119334 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-7613-3282","authenticated-orcid":false,"given":"Mariia A.","family":"Erokhina","sequence":"additional","affiliation":[{"name":"Koltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova, 26, 119334 Moscow, Russia"},{"name":"Department of Biology, Lomonosov Moscow State University, 1-12 Leninskie Gory, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-3522-7014","authenticated-orcid":false,"given":"Nikita Y.","family":"Grudinin","sequence":"additional","affiliation":[{"name":"Koltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova, 26, 119334 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5405-0212","authenticated-orcid":false,"given":"Ekaterina A.","family":"Vorotelyak","sequence":"additional","affiliation":[{"name":"Koltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova, 26, 119334 Moscow, Russia"},{"name":"Department of Biology, Lomonosov Moscow State University, 1-12 Leninskie Gory, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrey V.","family":"Vasiliev","sequence":"additional","affiliation":[{"name":"Koltzov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova, 26, 119334 Moscow, Russia"},{"name":"Department of Biology, Lomonosov Moscow State University, 1-12 Leninskie Gory, 119991 Moscow, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,20]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1016\/j.fertnstert.2025.04.040","article-title":"Human and Non-Human Primate Female in Vitro Gametogenesis toward Meiotic Entry: A Systematic Review","volume":"124","author":"Czukiewska","year":"2025","journal-title":"Fertil. Steril."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"hoaf056","DOI":"10.1093\/hropen\/hoaf056","article-title":"Generating Human Primordial Germ Cell-like Cells from Pluripotent Stem Cells: A Scoping Review of in Vitro Methods","volume":"2025","author":"Lakshmipathi","year":"2025","journal-title":"Hum. Reprod. Open"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"727","DOI":"10.1093\/hmg\/ddh088","article-title":"Spontaneous Differentiation of Germ Cells from Human Embryonic Stem Cells in Vitro","volume":"13","author":"Clark","year":"2004","journal-title":"Hum. Mol. 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J."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1883","DOI":"10.1089\/scd.2012.0412","article-title":"Passaging Techniques and ROCK Inhibitor Exert Reversible Effects on Morphology and Pluripotency Marker Gene Expression of Human Embryonic Stem Cell Lines","volume":"22","author":"Holm","year":"2013","journal-title":"Stem Cells Dev."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1016\/j.jbiosc.2014.10.024","article-title":"Maintenance of an Undifferentiated State of Human Induced Pluripotent Stem Cells through Migration-Dependent Regulation of the Balance between Cell-Cell and Cell-Substrate Interactions","volume":"119","author":"Kim","year":"2015","journal-title":"J. Biosci. Bioeng."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"5670","DOI":"10.1016\/j.biomaterials.2014.03.085","article-title":"Switching between Self-Renewal and Lineage Commitment of Human Induced Pluripotent Stem Cells via Cell-Substrate and Cell-Cell Interactions on a Dendrimer-Immobilized Surface","volume":"35","author":"Kim","year":"2014","journal-title":"Biomaterials"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"59","DOI":"10.4161\/cam.19583","article-title":"Role of E-Cadherin and Other Cell Adhesion Molecules in Survival and Differentiation of Human Pluripotent Stem Cells","volume":"6","author":"Li","year":"2012","journal-title":"Cell Adhes. Migr."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2442","DOI":"10.1016\/j.bpj.2010.02.029","article-title":"Individual Cell Movement, Asymmetric Colony Expansion, Rho-Associated Kinase, and E-Cadherin Impact the Clonogenicity of Human Embryonic Stem Cells","volume":"98","author":"Li","year":"2010","journal-title":"Biophys. J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1016\/j.stem.2020.11.005","article-title":"Capture of Mouse and Human Stem Cells with Features of Formative Pluripotency","volume":"28","author":"Kinoshita","year":"2021","journal-title":"Cell Stem Cell"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1038\/s41467-023-43871-2","article-title":"Derivation of Human Primordial Germ Cell-like Cells in an Embryonic-like Culture","volume":"15","author":"Esfahani","year":"2024","journal-title":"Nat. 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