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ESC culture conditions were first tuned by incorporating 2i factors to obtain and validate pluripotent ESC clones. During induction, bFGF, activin A, and 1% KSR were used to generate epiblast-like cells (EpiLCs), confirmed by qRT-PCR expression patterns. Subsequent EpiLC-to-PGCLC transformation tested combinations of BMP4, BMP8b, EGF, LIF, and SCF, yielding differentiated PGCLCs with characteristic marker expression and migration to gonads after embryo injection.",{"@graph":69,"@context":122},[70,84,105],{"@type":71,"itemListElement":72},"BreadcrumbList",[73,77,79,82],{"item":74,"name":75,"@type":76,"position":8},"https://docshare.wps.com","Home","ListItem",{"item":78,"name":9,"@type":76,"position":14},"https://docshare.wps.com/document/",{"item":80,"name":40,"@type":76,"position":81},"https://docshare.wps.com/document/research-report/",3,{"item":83,"name":65,"@type":76,"position":19},"https://docshare.wps.com/document/establishment-and-optimization-of-the-two-step-induction-system-for-generating-primordial-germ-cell-like-cells-from-chicken-embryonic-stem-cells/439807/",{"url":83,"name":65,"@type":85,"image":86,"author":91,"headline":65,"publisher":94,"fileFormat":97,"inLanguage":63,"description":67,"dateModified":98,"datePublished":99,"encodingFormat":97,"isAccessibleForFree":100,"interactionStatistic":101},"DigitalDocument",{"url":87,"@type":88,"width":89,"height":90},"https://docshare.wps.com/thumbnails/establishment-and-optimization-of-the-two-step-induction-system-for-generating-primordial-germ-cell-like-cells-from-chicken-embryonic-stem-cells/439807.png","ImageObject",300,407,{"name":92,"@type":93},"Stanley","Person",{"url":74,"name":95,"@type":96},"DocShare","Organization","application/pdf","2026-10-02","2026-09-29",true,{"@type":102,"interactionType":103,"userInteractionCount":14},"InteractionCounter",{"@type":104},"ViewAction",{"@type":106,"mainEntity":107},"FAQPage",[108,114,118],{"name":109,"@type":110,"acceptedAnswer":111},"What is the two-step induction system designed to produce?","Question",{"text":112,"@type":113},"It is designed to generate primordial germ cell-like cells (PGCLCs) from chicken embryonic stem cells (ESCs).","Answer",{"name":115,"@type":110,"acceptedAnswer":116},"How were ESC culture conditions optimized in the first step?",{"text":117,"@type":113},"The ESC culture conditions were optimized by incorporating 2i factors, enabling yield and validation of pluripotent ESC clones.",{"name":119,"@type":110,"acceptedAnswer":120},"Which factors were evaluated to improve the EpiLC-to-PGCLC transformation efficiency?",{"text":121,"@type":113},"The study evaluated combinations of BMP4, BMP8b, EGF, LIF, and SCF and assessed induction efficiency using flow cytometry, qRT-PCR, and immunofluorescence.","https://schema.org",{"og:url":83,"og:type":124,"og:title":65,"og:site_name":95,"og:description":67},"article",{"robots":126,"canonical":83},"index,follow",{"doc_id":128,"site_id":62},439807,1790971564,{"code":4,"msg":5,"data":131},{"doc_id":128,"user_id":132,"nickname":92,"user_avatar":133,"doc_module":4,"category_id":39,"category_name":40,"doc_title":65,"doc_description":67,"doc_content":134,"file_id":135,"file_url":136,"file_type":137,"file_size":138,"view_count":14,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":139,"language":140,"language_code":63,"site_id":62,"html_lang":63,"table_of_contents":141,"faqs":142,"seo_title":143,"seo_description":67,"update_tm":144,"read_time":145},2336477405376,"https://ap-avatar.wpscdn.com/davatar_29158cc5080c5b710cf443261637dec0","RESEARCH ARTICLE  \nEstablishment and optimization of the two-step induction system for generating primordial germ cell-like cells from chicken embryonic stem cells  \nZeyu Li 1,2,3 , XianShuai Xu 1,2,3, GuangZheng Liu 1,2,3, XiaoQian Lv 1,2,3, JiuZhou Song4, HongYan Sun 1,2,3, YingJie Niu 1,2,3, QiSheng Zuo 1,2,3,5, Wei Han6, BiChun Li 1,2,3,5,7 and Kai Jin 1,2,3,5   \n1 Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, China  \n2 Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, China  \n3 Institutes of Agricultural Science and Technology Development, Yangzhou University, China  \n4 Department of Animal & Avian Sciences, University of Maryland, College Park, MD, USA  \n5 College of Bioscience and Biotechnology, Yangzhou University, China  \n6 Poultry Institute, Jiangsu Institute of Poultry Sciences/Chinese Academy of Agricultural Sciences, Yangzhou, China  \n7 College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China  \nKeywords  \nchicken; embryonic stem cells; optimization; primordial germ cell-like cells; two-step induction system  \nCorrespondence Bichun Li  \nE-mail: [yubcli@yzu.edu.cn](yubcli@yzu.edu.cn)  \n[Correction added on 29 September 2025 after first online publication: Corresponding author has been corrected to “Bichun Li” in this version.]  \n(Received 2 December 2024, revised 12 July 2025, accepted 25 August 2025)  \ndoi:10.1002/2211-5463.70116  \nEdited by Alberto Alape-Giron  \nPrimordial germ cells (PGCs) are the progenitor cells of sperm and eggs. Xenotransplantation of chicken PGCs can achieve germline transmission. However, there are still challenges in obtaining many PGCs from endangered birds in vitro. In this study, at first, by incorporating 2i factors, the embryonic stem cells (ESCs) culture conditions were optimized, successfully yielding and validating pluripotent ESCs clones. Then, during induction ESCs, bFGF, activin A, and 1% KSR were added to Epiblast-like cells (EpiLCs) . Quantitative real-time polymerase chain reaction (qRT-PCR) showed Pax6, Eomes, and Vimentin expression patterns similar to primary epiblast, indicating successful EpiLCs induction. During EpiLCs to Primordial germ cell-like cells (PGCLCs) transformation, we evaluated BMP4, BMP8b, EGF, LIF, and SCF combinations’ impact on induction efficiency. Flow cytometry, qRTPCR, and immunofluorescence showed high expression of Cvh, C-kit, Dazl, CVH, and DAZL in PGCLCs, suggesting successful EpiLCs differentiation. Induced PGCLCs injected into 2.5-day chick embryos migrated to gonads by day 7–7 . 5, demonstrating migration and colonization. This study optimized a two-step protocol for in vitro differentiation of chicken ESCs into PGCLCs. This research’s results not only provide a reference for obtaining many PGCLCs in vitro but also open up a new approach for the development and application of genetic resource preservation technology in domestic chickens.  \nAbbreviations  \nAKP, alkaline phosphatase; BMP4, bone morphogenetic protein 4; BMP8b, bone morphogenetic protein 8b; cESCs, chicken embryonic stem cells; CVH, chicken Vasa homolog; DAPI, 40 ,6-diamidino-2-phenylindole; DAZL, deleted in azoospermia like; EGF, epidermal growth factor; EpiLCs, epiblast-like cells; ERK, extracellular signal-regulated kinase; ESCs, embryonic stem cells; FBS, fetal bovine serum; FGFR, fibroblast growth factor receptor; KIT, cluster of differentiation 117; KSR, knockout serum replacement; LIF, leukemia inhibitory factor; MEK, mitogen-activated protein kinase kinase; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline with tween-20; PGCLCs, primordial germ cell-like cells; PGCs, primordial germ cells; qRT-PCR, quantitative real-time polymerase chain reaction; SCF, stem cell factor; SSEA-1, stage-specific embryonic antigen-1; TGFb, transforming growth facto","cbCaipDnuVPe7ATf","https://ap.wps.com/l/cbCaipDnuVPe7ATf","pdf",2650218,17,"English","# Establishment and optimization of the two-step induction system\n## Optimization of ESC culture and validation of pluripotency\n## Induction of EpiLCs and marker characterization\n## Transformation to PGCLCs and evaluation of growth factor combinations\n## In vivo migration and colonization after embryo injection\n## Background and rationale for two-step conversion","[{\"question\":\"What is the two-step induction system designed to produce?\",\"answer\":\"It is designed to generate primordial germ cell-like cells (PGCLCs) from chicken embryonic stem cells (ESCs).\"},{\"question\":\"How were ESC culture conditions optimized in the first step?\",\"answer\":\"The ESC culture conditions were optimized by incorporating 2i factors, enabling yield and validation of pluripotent ESC clones.\"},{\"question\":\"Which factors were evaluated to improve the EpiLC-to-PGCLC transformation efficiency?\",\"answer\":\"The study evaluated combinations of BMP4, BMP8b, EGF, LIF, and SCF and assessed induction efficiency using flow cytometry, qRT-PCR, and immunofluorescence.\"}]","Establishment and optimization of the two-step induction system for generating primordial germ cell-like cells from chicken embryonic stem cells | PDF",1790690281,43]