Heparan sulfate Sulfatases are essential for patterning of human stem cell-derived midbrain dopaminergic neurons

Heparan sulfate Sulfatases are essential for patterning of human stem cell-derived midbrain dopaminergic neurons | 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 Heparan sulfate Sulfatases are essential for patterning of human stem cell-derived midbrain dopaminergic neurons Chiara Tremolanti, Carmen Abaurre, Yu-Chun Chien, Carmen Salto, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7190215/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 Midbrain dopaminergic neurons (mDA) are selectively lost in Parkinson’s disease (PD), driving sustained efforts to generate bona fide mDA neurons from human-induced pluripotent stem cells (iPSCs) for replacement therapy. While morphogen gradients and transcription factors have been extensively studied, extracellular regulators remain largely overlooked. Here, we identify the heparan sulfate-modifying enzymes SULF1 and SULF2 as essential for establishing mDA neuron identity in vitro. Using CRISPR/Cas9-engineered iPSCs, we show that loss of SULF1/2 increases 6-O-sulfation of heparan sulfate chains and disrupts anterior-posterior and dorsoventral patterning in cells exposed to a midbrain differentiation protocol. Double-knockout cells fail to acquire midbrain fate and instead adopt caudal and neural crest-like identities, as revealed by single-nucleus RNA sequencing. Mechanistically, we find enhanced FGF signaling and demonstrate that FGF inhibition redirects cells toward midbrain progenitors, without fully restoring ventral identity. These findings establish a critical role for SULF1/2 in human mDA neuron development and uncover a previously unrecognized layer of extracellular control over neuronal patterning, opening for novel strategies to refine differentiation protocols for PD and beyond. Biological sciences/Developmental biology/Differentiation Biological sciences/Cell biology/Glycobiology Full Text Additional Declarations There is NO Competing Interest. Ernest Arenas was co-founder, held equity in, and was an advisor of Cholestenix. Carmen Salto holds equity in Cholestenix. The remaining authors declare no competing interests. Supplementary Files SupplementaryInformationTremolantietal.pdf Supplementary information 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. 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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-7190215","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":497792781,"identity":"e71364b7-9e60-4b80-9280-297948ed0461","order_by":0,"name":"Chiara Tremolanti","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/klEQVRIiWNgGAWjYDACCcYGBOcBELOxtzEcIEaLBJiTAGSw8RwjpAWZAmlhkEjD7y7+2c2NnytqGOrM27sTHyTU3Knjk3yWeIDhjw1uS+4cbJY8c4xBQubM2c0GCceeSbBJpx04wNiG2yoDicQGyQY2BgkJidxtEglsh4Fa0hsOMDYcxqel+WfDP6AW+bdALf+AWiSPNwAd9h+fljbJxjaQLbzbgGygFgm2AwcY2A7g9suNxDbLxj4JyRk8uZsNEvsOS7bxpCUcSGxLxqmFf0b645sN32z4JdjPbnzw4dthfvn2Y8YfPvyxw6kFZhkaP4GQhlEwCkbBKBgFeAEA1X5TN80mVNIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3113-7099","institution":"Karolinska Institutet","correspondingAuthor":true,"prefix":"","firstName":"Chiara","middleName":"","lastName":"Tremolanti","suffix":""},{"id":497792782,"identity":"8d9713b5-7d3f-4fdc-8f77-dfec9e455b12","order_by":1,"name":"Carmen Abaurre","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Abaurre","suffix":""},{"id":497792783,"identity":"ba2f9949-7df0-4da3-90c6-a8d6562033f8","order_by":2,"name":"Yu-Chun Chien","email":"","orcid":"https://orcid.org/0000-0002-2485-2932","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Yu-Chun","middleName":"","lastName":"Chien","suffix":""},{"id":497792784,"identity":"3cb0077d-2e9c-4624-ae74-b00d6b5465a3","order_by":3,"name":"Carmen Salto","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Salto","suffix":""},{"id":497792785,"identity":"b57bb452-85bd-4a52-9389-a338c9037aa2","order_by":4,"name":"Conor Mitchell","email":"","orcid":"","institution":"University of Copenhagen","correspondingAuthor":false,"prefix":"","firstName":"Conor","middleName":"","lastName":"Mitchell","suffix":""},{"id":497792786,"identity":"3fa93609-eb7b-47b1-8df9-a4789ab15e05","order_by":5,"name":"Emma Jurmand","email":"","orcid":"","institution":"Institut de Biologia Evolutiva (CSIC - 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Carmen Salto holds equity in Cholestenix. The remaining authors declare no competing interests.","formattedTitle":"Heparan sulfate Sulfatases are essential for patterning of human stem cell-derived midbrain dopaminergic neurons","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":"","lastPublishedDoi":"10.21203/rs.3.rs-7190215/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7190215/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Midbrain dopaminergic neurons (mDA) are selectively lost in Parkinson’s disease (PD), driving sustained efforts to generate bona fide mDA neurons from human-induced pluripotent stem cells (iPSCs) for replacement therapy. While morphogen gradients and transcription factors have been extensively studied, extracellular regulators remain largely overlooked. Here, we identify the heparan sulfate-modifying enzymes SULF1 and SULF2 as essential for establishing mDA neuron identity in vitro. Using CRISPR/Cas9-engineered iPSCs, we show that loss of SULF1/2 increases 6-O-sulfation of heparan sulfate chains and disrupts anterior-posterior and dorsoventral patterning in cells exposed to a midbrain differentiation protocol. Double-knockout cells fail to acquire midbrain fate and instead adopt caudal and neural crest-like identities, as revealed by single-nucleus RNA sequencing. Mechanistically, we find enhanced FGF signaling and demonstrate that FGF inhibition redirects cells toward midbrain progenitors, without fully restoring ventral identity. These findings establish a critical role for SULF1/2 in human mDA neuron development and uncover a previously unrecognized layer of extracellular control over neuronal patterning, opening for novel strategies to refine differentiation protocols for PD and beyond.","manuscriptTitle":"Heparan sulfate Sulfatases are essential for patterning of human stem cell-derived midbrain dopaminergic neurons","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 10:13:43","doi":"10.21203/rs.3.rs-7190215/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d41bba81-75a1-4fd5-9f25-046a305df1f7","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":52883092,"name":"Biological sciences/Developmental biology/Differentiation"},{"id":52883093,"name":"Biological sciences/Cell biology/Glycobiology"}],"tags":[],"updatedAt":"2026-05-11T16:00:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-11 10:13:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7190215","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7190215","identity":"rs-7190215","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}