Human Germ Cell Specification via Combinatorial Expression of Prioritized Transcription Factors

Human Germ Cell Specification via Combinatorial Expression of Prioritized Transcription Factors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Article Human Germ Cell Specification via Combinatorial Expression of Prioritized Transcription Factors Christian Kramme, Merrick Smela, Bennett Wolf, Patrick Fortuna, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2652895/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The generation of germline cell types from human induced pluripotent stem cells (hiPSCs) represents a key milestone toward in vitro gametogenesis, which has the potential to transform reproductive medicine. Methods to recapitulate germline cell specification in vitro have relied on extensive, long term culture methods, the most notable of which is a four-month culture protocol employing xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. Recently, transcription factor (TF)-based methods have demonstrated the feasibility of exogenous factor expression to directly differentiate hiPSCs into cell types of interest, including various ovarian cell types. The protocols leveraged in these studies, however, utilize more local methods of factor selection, such as basic differential gene expression analysis, and lower-throughput screening strategies via iterative testing of a small set of TFs. In this work, we integrate our recently-described, more globally-representative graph theory and highly-parallelized screening protocols to globally identify and screen 46 oogenesis-regulating TFs for their role in human germline formation. We identify DLX5, HHEX , and FIGLA whose individual overexpression enhances hPGCLC formation from hiPSCs, as well as an additional set of three TFs, ZNF281 , LHX8 , and SOHLH1 , whose combinatorial overexpression drives DDX4+ germ cell formation from hiPSCs. In contrast to previous methods, our protocol employs a simple four-day, feeder-free monolayer culture condition. We characterize these TF-based germ cells via gene expression analyses, and demonstrate their broad similarity to in vivo and in vitro -derived germ cells. Together, these results identify new regulatory factors that enhance in vitro human germ cell specification and further establish unique computational and experimental tools for human in vitro oogenesis research. Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells Biological sciences/Developmental biology/Stem cells/Stem-cell differentiation Full Text Additional Declarations Yes there is potential Competing Interest. P.C., C.K., M.P.S., and G.C. are listed as inventors for U.S. Provisional Application No. 63/326,656, entitled: “Methods and Compositions for Producing Primordial Germ Cell-Like Cells,” and U.S. Provisional Application No. 63/326,607, entitled: “Methods and Compositions for Producing Oogonia-Like Cells.” P.C. is a co-founder, scientific advisor to Gameto, Inc. C.K. is currently the VP of Cell Engineering of Gameto, Inc. G.M.C. serves on the scientific advisory board of Gameto, Inc., Colossal Biosciences, and GCTx. Supplementary Files krammeetaleggsupplement.pdf SupplementaryTable1PageRankofallTFs.csv Supplementary Dataset 1 SupplementaryTable2TFsutilizedforscreening.xlsx Supplementary Dataset 2 SupplementaryTable3NormalizedCountMatrix.csv Supplementary Dataset 3 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2652895","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":181900770,"identity":"9080ff9c-f850-458a-a828-7ec0ccb6fd4e","order_by":0,"name":"Christian Kramme","email":"","orcid":"","institution":"Harvard Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Kramme","suffix":""},{"id":181900771,"identity":"aea40bb7-a6c6-4697-83b1-3390c37c4621","order_by":1,"name":"Merrick Smela","email":"","orcid":"","institution":"Wyss Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Merrick","middleName":"","lastName":"Smela","suffix":""},{"id":181900772,"identity":"494012e2-09f6-44c7-a5ad-b0aefa690bcc","order_by":2,"name":"Bennett Wolf","email":"","orcid":"","institution":"Harvard Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bennett","middleName":"","lastName":"Wolf","suffix":""},{"id":181900773,"identity":"f2dd6158-089d-4415-a9e5-9b54680c3b47","order_by":3,"name":"Patrick Fortuna","email":"","orcid":"","institution":"Wyss Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Fortuna","suffix":""},{"id":181900774,"identity":"77ba64ba-5f8b-4417-a106-781d9ea208f2","order_by":4,"name":"Garyk Brixi","email":"","orcid":"https://orcid.org/0000-0002-1253-0522","institution":"Duke University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Garyk","middleName":"","lastName":"Brixi","suffix":""},{"id":181900775,"identity":"ce5dedd1-31cd-46f2-91cb-b2ea6ee57c9b","order_by":5,"name":"Tian-Lai Zang","email":"","orcid":"","institution":"Duke University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tian-Lai","middleName":"","lastName":"Zang","suffix":""},{"id":181900776,"identity":"a6386cde-e693-412f-ac0d-18ec10f03f94","order_by":6,"name":"Sophia Vincoff","email":"","orcid":"","institution":"Duke University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sophia","middleName":"","lastName":"Vincoff","suffix":""},{"id":181900777,"identity":"881d8d84-5c6b-41ac-ba49-f76ad990bce4","order_by":7,"name":"Kalyan Palepu","email":"","orcid":"","institution":"Duke University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kalyan","middleName":"","lastName":"Palepu","suffix":""},{"id":181900778,"identity":"785b1dd6-a2df-401e-937a-bf5682846093","order_by":8,"name":"Sabrina Koseki","email":"","orcid":"https://orcid.org/0000-0001-7273-970X","institution":"MIT Media Lab","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sabrina","middleName":"","lastName":"Koseki","suffix":""},{"id":181900779,"identity":"1db5ac12-b66b-4ca0-b4f2-cbb7640c0f4d","order_by":9,"name":"Suhaas Bhat","email":"","orcid":"","institution":"Duke University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Suhaas","middleName":"","lastName":"Bhat","suffix":""},{"id":181900780,"identity":"9f59e0f9-19e3-436e-8cd5-d8efe5b1c857","order_by":10,"name":"Edward Dong","email":"","orcid":"","institution":"Wyss Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Edward","middleName":"","lastName":"Dong","suffix":""},{"id":181900781,"identity":"145bae17-8a20-4c82-baa4-12341d993dc5","order_by":11,"name":"Jessica Adams","email":"","orcid":"","institution":"Wyss Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"Adams","suffix":""},{"id":181900782,"identity":"0ba2a2ed-e0ad-44a3-a775-75ed86efbad2","order_by":12,"name":"Richie Kohman","email":"","orcid":"","institution":"Harvard Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Richie","middleName":"","lastName":"Kohman","suffix":""},{"id":181900783,"identity":"9ae1ae1b-f722-4bdd-9a23-85e5b3e49c96","order_by":13,"name":"Songlei Liu","email":"","orcid":"","institution":"Harvard Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Songlei","middleName":"","lastName":"Liu","suffix":""},{"id":181900784,"identity":"226a4e0e-adcd-4176-89bc-e6306a3aa5c3","order_by":14,"name":"Mutsumi Kobayashi","email":"","orcid":"","institution":"Massachusetts General Hospital Center for Cancer Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mutsumi","middleName":"","lastName":"Kobayashi","suffix":""},{"id":181900785,"identity":"8a27a75f-aa0f-4e1e-8414-47e273351612","order_by":15,"name":"Toshihiro Shioda","email":"","orcid":"","institution":"Massachusetts General Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Toshihiro","middleName":"","lastName":"Shioda","suffix":""},{"id":181900786,"identity":"6ec5329a-8dfa-4fc2-bdf1-e4f71707f9a0","order_by":16,"name":"George Church","email":"","orcid":"","institution":"Harvard Medical School","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"George","middleName":"","lastName":"Church","suffix":""},{"id":181900787,"identity":"be0a07da-9303-40a5-b091-524cc7a9db24","order_by":17,"name":"Pranam Chatterjee","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-3957-8478","institution":"Duke University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Pranam","middleName":"","lastName":"Chatterjee","suffix":""}],"badges":[],"createdAt":"2023-03-03 19:16:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2652895/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2652895/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":42007467,"identity":"f8261351-16f0-4ffc-9689-3d92645f698b","added_by":"auto","created_at":"2023-08-23 13:49:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1381803,"visible":true,"origin":"","legend":"","description":"","filename":"krammeetaleggmanuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2652895/v1_covered_47577a85-fd4b-418b-8067-ff1e0fb0baa6.pdf"},{"id":33995356,"identity":"90bbc7cf-6c3e-490b-bf33-2aed330a0162","added_by":"auto","created_at":"2023-03-09 05:40:30","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1271578,"visible":true,"origin":"","legend":"","description":"","filename":"krammeetaleggsupplement.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2652895/v1/d2a06545536c0556aeec9468.pdf"},{"id":33995358,"identity":"f3e892ab-ac41-45e3-9897-7a40b9e2aef4","added_by":"auto","created_at":"2023-03-09 05:40:30","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":37843,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Dataset 1\u003c/p\u003e","description":"","filename":"SupplementaryTable1PageRankofallTFs.csv","url":"https://assets-eu.researchsquare.com/files/rs-2652895/v1/ca24508c6080be21e4732eb6.csv"},{"id":33996142,"identity":"942038f4-e015-4c28-8d17-6bc94eaf7502","added_by":"auto","created_at":"2023-03-09 05:56:30","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15719,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Dataset 2\u003c/p\u003e","description":"","filename":"SupplementaryTable2TFsutilizedforscreening.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2652895/v1/a5672eeb2cac7b4df6c9a112.xlsx"},{"id":33995360,"identity":"8aa5fb9b-5adc-419a-8846-cf5c34c17dc1","added_by":"auto","created_at":"2023-03-09 05:40:30","extension":"csv","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10240875,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Dataset 3\u003c/p\u003e","description":"","filename":"SupplementaryTable3NormalizedCountMatrix.csv","url":"https://assets-eu.researchsquare.com/files/rs-2652895/v1/d393466d98686a006e4fc794.csv"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nP.C., C.K., M.P.S., and G.C. are listed as inventors for U.S. Provisional Application No. 63/326,656, entitled: “Methods and Compositions for Producing Primordial Germ Cell-Like Cells,” and U.S. Provisional Application No. 63/326,607, entitled: “Methods and Compositions for Producing Oogonia-Like Cells.” P.C. is a co-founder, scientific advisor to Gameto, Inc. C.K. is currently the VP of Cell Engineering of Gameto, Inc. G.M.C. serves on the scientific advisory board of Gameto, Inc., Colossal Biosciences, and GCTx.","formattedTitle":"Human Germ Cell Specification via Combinatorial Expression of Prioritized Transcription Factors","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2652895/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2652895/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe generation of germline cell types from human induced pluripotent stem cells (hiPSCs) represents a key milestone toward \u003cem\u003ein vitro\u003c/em\u003e gametogenesis, which has the potential to transform reproductive medicine. Methods to recapitulate germline cell specification \u003cem\u003ein vitro\u003c/em\u003e have relied on extensive, long term culture methods, the most notable of which is a four-month culture protocol employing xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. Recently, transcription factor (TF)-based methods have demonstrated the feasibility of exogenous factor expression to directly differentiate hiPSCs into cell types of interest, including various ovarian cell types. The protocols leveraged in these studies, however, utilize more local methods of factor selection, such as basic differential gene expression analysis, and lower-throughput screening strategies via iterative testing of a small set of TFs. In this work, we integrate our recently-described, more globally-representative graph theory and highly-parallelized screening protocols to globally identify and screen 46 oogenesis-regulating TFs for their role in human germline formation. We identify \u003cem\u003eDLX5, HHEX\u003c/em\u003e, and \u003cem\u003eFIGLA \u003c/em\u003ewhose individual overexpression enhances hPGCLC formation from hiPSCs, as well as an additional set of three TFs, \u003cem\u003eZNF281\u003c/em\u003e, \u003cem\u003eLHX8\u003c/em\u003e, and \u003cem\u003eSOHLH1\u003c/em\u003e, whose combinatorial overexpression drives DDX4+ germ cell formation from hiPSCs. In contrast to previous methods, our protocol employs a simple four-day, feeder-free monolayer culture condition. We characterize these TF-based germ cells via gene expression analyses, and demonstrate their broad similarity to \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e-derived germ cells. Together, these results identify new regulatory factors that enhance \u003cem\u003ein vitro\u003c/em\u003e human germ cell specification and further establish unique computational and experimental tools for human \u003cem\u003ein vitro\u003c/em\u003e oogenesis research.\u003c/p\u003e","manuscriptTitle":"Human Germ Cell Specification via Combinatorial Expression of Prioritized Transcription Factors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-09 05:40:25","doi":"10.21203/rs.3.rs-2652895/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e31280d3-60a8-43e2-ae45-2cb23405dec5","owner":[],"postedDate":"March 9th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":19767528,"name":"Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells"},{"id":19767529,"name":"Biological sciences/Developmental biology/Stem cells/Stem-cell differentiation"}],"tags":[],"updatedAt":"2023-08-23T13:40:44+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-09 05:40:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2652895","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2652895","identity":"rs-2652895","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}