Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and trophoblast stem cells

Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and trophoblast stem cells | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and trophoblast stem cells Mana Parast, Robert Morey, Francesca Soncin, Sampada Kallol, Nirvay Sah, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6797967/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Cytotrophoblast (CTB) of the early gestation human placenta are bipotent progenitor epithelial cells, which can differentiate into invasive extravillous trophoblast (EVT) and multinucleated syncytiotrophoblast (STB). Trophoblast stem cells (TSC), derived from early first trimester placentae, have also been shown to be bipotential; however, their cell-of-origin has not been identified. In this study, we set out to probe the transcriptional diversity of early and late first trimester villous CTB (vCTB) and compare these to TSC. To this end, we performed single-cell RNA sequencing (scRNA-seq) on placental villous tissue from early (6-8 weeks) and late (12-14 weeks) first trimester placentae; we also evaluated CTB within basal (maternal) and chorionic (fetal) regions of early first trimester placenta, both by scRNA-seq and GeoMx-based spatial transcriptomics. Finally, we performed scRNA-seq on three TSC lines, derived from 6-8 week gestation placentae, as well as on early first trimester CTB at several timepoints during TSC derivation. We found notable distinctions within CTB clusters based on gestational age, further influenced by location near the basal or chorionic plates. We identified trophoblast states representing “initial state” vCTB (in vivo CTB progenitors), as well as additional CTB subtypes, precursor STB, and precursor and mature EVT. CTB progenitors were enriched in early first trimester placentae at the basal plate; overall, basal plate CTB were biased toward EVT, and chorionic plate CTB toward STB, precursors. Clustering and trajectory inference analysis indicated that TSC were most like EVT precursor cells. In fact, vCTB lost their in vivo “initial state” markers, including PAGE4, as they transitioned to TSC during in vitro culture. This was confirmed by flow cytometric analysis of 6 different TSC lines, which showed uniform expression of proximal column markers ITGA2 and ITGA5. Additionally, we found that ITGA5+ CTB could be plated in 2D, forming only EVT upon spontaneous differentiation, but failed to form self-renewing organoids; conversely, ITGA5- CTB could not be plated in 2D, but readily formed organoids. Our findings suggest that distinct CTB states exist in different regions of the placenta as early as six weeks gestation and that current TSC lines most closely resemble ITGA5+ CTB, biased toward the EVT lineage. Biological sciences/Developmental biology/Embryogenesis/Cell lineage Biological sciences/Stem cells/Multipotent stem cells Placenta Trophoblast Cytotrophoblast Trophoblast stem cells Full Text Additional Declarations There is NO Competing Interest. Supplementary Files tablescombinedJune25.xlsx Supplementary Tables 1-13 submissionJune25supplementaryfigures.pdf Supplementary Figures Cite Share Download PDF Status: Under Review 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 Advisory Board 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-6797967","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":468912670,"identity":"8ec74533-fe2e-4821-b5f0-81f38dfa05e2","order_by":0,"name":"Mana Parast","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYBADOQMGBsYDjA1gDpDNRlCHgTFQGcOBg6RoSdxAtBbd2c3PPvz48yd9O3vzg8MfdzAk9vcf3sDwoewwTi1md44Zz+xtM8jd2XPM4MDBMwyJM26kFTDOOIdHy40EYwbeBoPcDTcSgFraGBI3SPAYMPO24dOS/pnxzx+DdIP7zz9AtPCfMWD+i1dLjjEzD5tBgsENHqgtDDkGzIz4tNw5U8ws22ZsuOFMTsGBs20SxiC/HOw5l45by+32zYxv/sjJGxw/vvFBZZuNLDDENj74UWaNUwuDBDbuAdzqMbWMglEwCkbBKMAEAAhEYnOPKb0kAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5963-2246","institution":"University of California - San Diego","correspondingAuthor":true,"prefix":"","firstName":"Mana","middleName":"","lastName":"Parast","suffix":""},{"id":468912671,"identity":"44c77e6d-2f5d-4fba-9bac-78890fc552d0","order_by":1,"name":"Robert Morey","email":"","orcid":"https://orcid.org/0000-0001-8480-4564","institution":"University of California - San Diego","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Morey","suffix":""},{"id":468912672,"identity":"f67e5689-1ecf-48d0-a983-877a5ee00945","order_by":2,"name":"Francesca Soncin","email":"","orcid":"","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"","lastName":"Soncin","suffix":""},{"id":468912673,"identity":"bf756071-8cff-48ec-b730-a0724f202adf","order_by":3,"name":"Sampada Kallol","email":"","orcid":"","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Sampada","middleName":"","lastName":"Kallol","suffix":""},{"id":468912674,"identity":"22742ba2-6bfd-421c-b947-22ddd199bb46","order_by":4,"name":"Nirvay Sah","email":"","orcid":"","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Nirvay","middleName":"","lastName":"Sah","suffix":""},{"id":468912675,"identity":"4231742b-5de5-49e5-b7d4-19a07a873f48","order_by":5,"name":"Zoe Manalo","email":"","orcid":"","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Zoe","middleName":"","lastName":"Manalo","suffix":""},{"id":468912676,"identity":"f2405d1a-5866-45ad-a1d3-fe813a4d47a5","order_by":6,"name":"Tony Bui","email":"","orcid":"","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Tony","middleName":"","lastName":"Bui","suffix":""},{"id":468912677,"identity":"fbaf4446-f7d3-47d6-a3a1-f51cdeb48641","order_by":7,"name":"Jaroslav Slamecka","email":"","orcid":"https://orcid.org/0000-0003-3610-3223","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Jaroslav","middleName":"","lastName":"Slamecka","suffix":""},{"id":468912678,"identity":"d7513e9e-6db4-49b8-aa56-2cbb51034eb7","order_by":8,"name":"Virginia Cheung","email":"","orcid":"","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Virginia","middleName":"","lastName":"Cheung","suffix":""},{"id":468912679,"identity":"d2213b11-68dc-4521-8480-5b16eca4786b","order_by":9,"name":"Donald Pizzo","email":"","orcid":"","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Donald","middleName":"","lastName":"Pizzo","suffix":""},{"id":468912680,"identity":"7ec26ff9-1b93-47a4-ac1b-8d00d84a57b2","order_by":10,"name":"Daniela Requena","email":"","orcid":"","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Requena","suffix":""},{"id":468912681,"identity":"10bd1a60-63b4-47b7-8345-1802f8c99c65","order_by":11,"name":"Ching-Wen Chang","email":"","orcid":"","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Ching-Wen","middleName":"","lastName":"Chang","suffix":""},{"id":468912682,"identity":"2348d413-9056-4bf4-aaea-9aefe47d6927","order_by":12,"name":"Omar Farah","email":"","orcid":"","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Omar","middleName":"","lastName":"Farah","suffix":""},{"id":468912683,"identity":"1ce73e71-8e64-4262-a2b5-52f433a6fc58","order_by":13,"name":"Ryan Kittle","email":"","orcid":"https://orcid.org/0009-0007-1147-5190","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Ryan","middleName":"","lastName":"Kittle","suffix":""},{"id":468912684,"identity":"97357319-fd3b-4cd4-8f6e-4c5748e34a2b","order_by":14,"name":"Kelly Lam","email":"","orcid":"","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Kelly","middleName":"","lastName":"Lam","suffix":""},{"id":468912685,"identity":"928780b5-70fd-4e2b-9879-335dff3d029e","order_by":15,"name":"Morgan Meads","email":"","orcid":"","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Morgan","middleName":"","lastName":"Meads","suffix":""},{"id":468912686,"identity":"24af70b2-7a5f-4248-9261-cddcfc54f601","order_by":16,"name":"Mariko Horii","email":"","orcid":"https://orcid.org/0000-0002-4996-6792","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Mariko","middleName":"","lastName":"Horii","suffix":""},{"id":468912687,"identity":"824f7cd7-9c55-43d9-8fef-bec4d0eb4785","order_by":17,"name":"Kathleen Fisch","email":"","orcid":"https://orcid.org/0000-0002-0117-7444","institution":"University of California San Diego","correspondingAuthor":false,"prefix":"","firstName":"Kathleen","middleName":"","lastName":"Fisch","suffix":""}],"badges":[],"createdAt":"2025-06-02 00:15:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6797967/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6797967/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87699118,"identity":"0ba3ae1a-f09c-424e-a70d-2859a6db2a02","added_by":"auto","created_at":"2025-07-28 07:03:24","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7322922,"visible":true,"origin":"","legend":"Article File","description":"","filename":"submissionJune25.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6797967/v1_covered_ea7e72a7-11fa-402f-9562-07696dc17509.pdf"},{"id":84356537,"identity":"c5c35cb8-4de4-4506-b58a-e859e281b6c4","added_by":"auto","created_at":"2025-06-11 03:07:23","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":147185,"visible":true,"origin":"","legend":"Supplementary Tables 1-13","description":"","filename":"tablescombinedJune25.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6797967/v1/e9d9c070d33b05ecaaa405ca.xlsx"},{"id":84356447,"identity":"c87a1012-fe93-43be-b2f4-5a421d255fa4","added_by":"auto","created_at":"2025-06-11 02:59:24","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10294376,"visible":true,"origin":"","legend":"Supplementary Figures","description":"","filename":"submissionJune25supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6797967/v1/a0d8440a19ce563afce09515.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and trophoblast stem cells","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Placenta, Trophoblast, Cytotrophoblast, Trophoblast stem cells","lastPublishedDoi":"10.21203/rs.3.rs-6797967/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6797967/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Cytotrophoblast (CTB) of the early gestation human placenta are bipotent progenitor epithelial cells, which can differentiate into invasive extravillous trophoblast (EVT) and multinucleated syncytiotrophoblast (STB). Trophoblast stem cells (TSC), derived from early first trimester placentae, have also been shown to be bipotential; however, their cell-of-origin has not been identified. In this study, we set out to probe the transcriptional diversity of early and late first trimester villous CTB (vCTB) and compare these to TSC. To this end, we performed single-cell RNA sequencing (scRNA-seq) on placental villous tissue from early (6-8 weeks) and late (12-14 weeks) first trimester placentae; we also evaluated CTB within basal (maternal) and chorionic (fetal) regions of early first trimester placenta, both by scRNA-seq and GeoMx-based spatial transcriptomics. Finally, we performed scRNA-seq on three TSC lines, derived from 6-8 week gestation placentae, as well as on early first trimester CTB at several timepoints during TSC derivation. We found notable distinctions within CTB clusters based on gestational age, further influenced by location near the basal or chorionic plates. We identified trophoblast states representing “initial state” vCTB (in vivo CTB progenitors), as well as additional CTB subtypes, precursor STB, and precursor and mature EVT. CTB progenitors were enriched in early first trimester placentae at the basal plate; overall, basal plate CTB were biased toward EVT, and chorionic plate CTB toward STB, precursors. Clustering and trajectory inference analysis indicated that TSC were most like EVT precursor cells. In fact, vCTB lost their in vivo “initial state” markers, including PAGE4, as they transitioned to TSC during in vitro culture. This was confirmed by flow cytometric analysis of 6 different TSC lines, which showed uniform expression of proximal column markers ITGA2 and ITGA5. Additionally, we found that ITGA5+ CTB could be plated in 2D, forming only EVT upon spontaneous differentiation, but failed to form self-renewing organoids; conversely, ITGA5- CTB could not be plated in 2D, but readily formed organoids. Our findings suggest that distinct CTB states exist in different regions of the placenta as early as six weeks gestation and that current TSC lines most closely resemble ITGA5+ CTB, biased toward the EVT lineage.","manuscriptTitle":"Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and trophoblast stem cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 02:59:19","doi":"10.21203/rs.3.rs-6797967/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c2b8e316-73cc-4619-884b-08cdb7a270aa","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":49783194,"name":"Biological sciences/Developmental biology/Embryogenesis/Cell lineage"},{"id":49783195,"name":"Biological sciences/Stem cells/Multipotent stem cells"}],"tags":[],"updatedAt":"2026-03-17T17:25:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-11 02:59:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6797967","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6797967","identity":"rs-6797967","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}