Engineering synthetic signaling receptors to enable erythropoietin-free erythropoiesis

Engineering synthetic signaling receptors to enable erythropoietin-free erythropoiesis | 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 Engineering synthetic signaling receptors to enable erythropoietin-free erythropoiesis M. Cromer, Aadit Shah, Kiran Majeti, Freja Ekman, Sridhar Selvaraj, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4986623/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jan, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Blood transfusion plays a vital role in modern medicine. However, availability is contingent on donated blood, and frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor O-negative pluripotent stem cells emerges as a solution, yet the high cost of recombinant cytokines required for ex vivo erythroid differentiation remains a major barrier. Erythropoietin (EPO) signaling through the EPO receptor is indispensable to RBC development, and EPO is one of the most expensive components in erythroid-promoting media. Here, we used design-build-test cycles to develop highly optimized small molecule-inducible synthetic EPO receptors (synEPORs) which were integrated at a variety of genomic loci using homology-directed repair genome editing. We found that integration of synEPOR at the endogenous EPOR locus in an induced pluripotent stem cell producer line enabled culture with small molecule to yield equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production compared to cells cultured with EPO. Due to the dramatically lower cost of small molecules vs. recombinant cytokines, these efforts eliminate one of the most expensive elements of ex vivo culture media—EPO cytokine. Because dependence on cytokines is a common barrier to ex vivo cell production, these strategies could improve scalable manufacturing of a wide variety of clinically relevant cell types. More broadly, this work showcases how protein engineering and genome engineering may be combined to introduce precisely regulated and tunable behavior into cells, an advancement which will pave the way for increasingly sophisticated synthetic biology applications. Biological sciences/Developmental biology/Haematopoiesis/Erythropoiesis/Haematopoietic stem cells Biological sciences/Genetics/CRISPR-Cas systems/CRISPR-Cas9 genome editing Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells Biological sciences/Biotechnology/Molecular engineering/Synthetic biology Biological sciences/Developmental biology/Differentiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Additional Declarations Yes there is potential Competing Interest. M.H.P. is a member of the scientific advisory board of Allogene Therapeutics. M.H.P. has equity in CRISPR Tx and Kamau Tx. C.T.C., M.H.P., and M.K.C. have filed provisional patent no. PCT/US2023/076969. Supplementary Files ExtendedDatasynEPORDEGs.xls Dataset 1 ExtendedDatasynEPORTPMsv3.xlsx Dataset 2 ExtendedDataiEPORGOAnalysis.xlsx Dataset 3 ExtendedDatasynEPORSpearmanCorrelation.xls Dataset 4 NCBsynEPORSupplementalFiguresv2.pdf Cite Share Download PDF Status: Published Journal Publication published 29 Jan, 2025 Read the published version in Nature Communications → 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-4986623","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":367641319,"identity":"dd355987-8779-48bd-8321-32723d7d0a41","order_by":0,"name":"M. Cromer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBACCQbmBhib8QGQ4AdiAwJaGOFamEFKJRtI0cImQZQWyRmJjQ8Yau7J6bb3PqvmqamTYGBv3iaBT4u0RGKzAcOxYmOzM8fNbvMcOyzBwHOsDK8WOYnENgnGhoTEbTfS2G7zNhyoY5DIMSOkpf0HWMv9Z2zFvA1Ah8m/wa8F6LA2BogtbGzMvA3MEgwSPPi1SPY8bJZIOJYA9Esas+QcoF/YeNKKLfBpkTiefPDDh5oEObPjxxg/vAGGGD/74Y038GlhEEhgYEhAFmDDqxwE+A8QVDIKRsEoGAUjHQAA2q5ChhJIhtUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-8198-5010","institution":"University of California, San Francisco","correspondingAuthor":true,"prefix":"","firstName":"M.","middleName":"","lastName":"Cromer","suffix":""},{"id":367641320,"identity":"0f7cd2e7-6bc9-453a-b72b-edf6a206a1b3","order_by":1,"name":"Aadit Shah","email":"","orcid":"https://orcid.org/0000-0001-8672-6643","institution":"Stanford 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Francisco","correspondingAuthor":false,"prefix":"","firstName":"Tammy","middleName":"","lastName":"Tran","suffix":""},{"id":367641333,"identity":"b3b888e4-4db2-46d8-a524-67c4e75a0df4","order_by":14,"name":"Simon Chu","email":"","orcid":"https://orcid.org/0000-0003-3863-1548","institution":"University of California, San Francisco","correspondingAuthor":false,"prefix":"","firstName":"Simon","middleName":"","lastName":"Chu","suffix":""},{"id":367641334,"identity":"98d6caaf-ccc8-469e-a49e-0137ede1a024","order_by":15,"name":"Matthew Porteus","email":"","orcid":"https://orcid.org/0000-0002-3850-4648","institution":"Stanford University","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Porteus","suffix":""}],"badges":[],"createdAt":"2024-08-27 19:50:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4986623/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4986623/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-56239-5","type":"published","date":"2025-01-29T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":67351257,"identity":"8f44585d-b3e2-4b24-8310-2698f6cc964d","added_by":"auto","created_at":"2024-10-24 04:01:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":210197,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of FKBP-EPOR chimeras to facilitate EPO-free erythroid differentiation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Schematic of chimeric FKBP-EPOR transgenes integrated at the \u003cem\u003eCCR5\u003c/em\u003elocus via CRISPR/AAV-mediated editing. Red boxes represent the location of FKBP within the EPOR.\u003c/p\u003e\n\u003cp\u003eB: Schematic of HSPC editing and subsequent erythroid differentiation.\u003c/p\u003e\n\u003cp\u003eC: Percentage of edited HSPCs that acquired erythroid markers (CD34-/CD45-/CD71+/GPA+) +/-BB normalized to unedited cells +EPO at d14 of differentiation. Bars represent median +/-SEM; * = p\u0026lt;0.05 by unpaired t-test.\u003c/p\u003e\n\u003cp\u003eD: Percent edited alleles over the course of differentiation +/-BB and +/-EPO. Bars represent median +/-SEM.\u003c/p\u003e\n\u003cp\u003eE: Representative flow cytometry staining and gating scheme for synEPOR 1.5-edited HSPCs at d14 of differentiation -EPO and +/-BB. Arrows indicate that only gated cells are displayed on the subsequent plot.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-4986623/v1/799c45b9d08a2b84922918f3.png"},{"id":67351258,"identity":"07b0bbf6-82c7-494a-8314-eb9963d83a77","added_by":"auto","created_at":"2024-10-24 04:01:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":232224,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulation of synEPOR effect by addition of signal peptide \u0026amp; EPOR truncation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Schematic of second-generation synEPORs integrated at \u003cem\u003eCCR5\u003c/em\u003elocus. Red boxes represent the FKBP domain; yellow and green triangles indicate EPOR and IL6 SPs, respectively; dashed line represents EPOR truncation.\u003c/p\u003e\n\u003cp\u003eB: Percentage of edited HSPCs that acquired erythroid markers (CD34-/CD45-/CD71+/GPA+) +/-BB normalized to unedited cells +EPO at d14 of differentiation. synEPOR 1.5 data from Fig. 1C shown for comparison. Bars represent median +/-SEM; * = p\u0026lt;0.05, ** = p\u0026lt;0.01, and **** = p\u0026lt;0.0001 by unpaired t-test.\u003c/p\u003e\n\u003cp\u003eC: Percent edited alleles over the course of differentiation +/-BB and +/-EPO. Bars represent median +/-SEM; * = p\u0026lt;0.05 and **** = p\u0026lt;0.0001 comparing d0 vs. d14 within treatment by unpaired t-test.\u003c/p\u003e\n\u003cp\u003eD: Representative flow cytometry staining and gating scheme for synEPOR-edited HSPCs at d14 of differentiation -EPO and +/-BB. Arrows indicate that only gated cells are displayed on the subsequent plot.\u003c/p\u003e\n\u003cp\u003eE: Representative hemoglobin tetramer HPLC plots at d14 of erythroid differentiation. +BB and -BB/-EPO conditions were from cells edited with synEPOR; +EPO condition was from unedited cells. All plots normalized to 1e6 cells.\u003c/p\u003e\n\u003cp\u003eF: AlphaFold2-based structure prediction of truncated EPOR and synEPOR. SP was removed since this sequence will be cleaved following translocation to the membrane. TMD labeled with an arrow as a reference point.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-4986623/v1/a1b5cf6b37ce3a25921d6ef7.png"},{"id":67352356,"identity":"a2f675a1-2a6b-4f3b-8a07-c51471967f49","added_by":"auto","created_at":"2024-10-24 04:17:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":207643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulation of synEPOR effect by genome engineering.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Schematic of third-generation synEPORs that drive expression from: 1) PGK promoter from \u003cem\u003eCCR5\u003c/em\u003e safe harbor site; 2) erythroid-specific \u003cem\u003eHBA1\u003c/em\u003elocus; and 3) endogenous \u003cem\u003eEPOR\u003c/em\u003elocus.\u003c/p\u003e\n\u003cp\u003eB: Percentage of edited HSPCs that acquired erythroid markers (CD34-/CD45-/CD71+/GPA+) +/-BB normalized to unedited cells +EPO at d14 of differentiation. SFFV(synEPOR) data from Fig. 2B shown here for comparison. Bars represent median +/-SEM; * = p\u0026lt;0.05, and **** = p\u0026lt;0.0001 by unpaired t-test.\u003c/p\u003e\n\u003cp\u003eC: Representative flow cytometry staining and gating scheme for edited HSPCs at d14 of differentiation -EPO and +/-BB. Arrows indicate that only gated cells are displayed on the subsequent plot.\u003c/p\u003e\n\u003cp\u003eD: Representative hemoglobin tetramer HPLC plot of edited HSPCs at d14 of differentiation -EPO and +/-BB.\u003c/p\u003e\n\u003cp\u003eE: Cumulative cell count fold change of edited HSPCs over the course of differentiation.\u003c/p\u003e\n\u003cp\u003eF: Dose response of edited HSPCs cultured over a range of [BB] at d14 of differentiation normalized to unedited cells +EPO. Bars represent median +/-SEM; *** = p\u0026lt;0.001 and **** = p\u0026lt;0.0001 comparing +EPO/+BB to +BB conditions by unpaired t-test.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-4986623/v1/474b2f8acbb40e25da9132f8.png"},{"id":67351260,"identity":"2bac732d-a7b2-4d47-bd5f-53f0c8c674d5","added_by":"auto","created_at":"2024-10-24 04:01:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":272761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome-wide analysis of synEPOR-edited cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Schematic of well-characterized endogenous EPO+EPOR signaling effects vs. undefined BB+synEPORsignaling effects.\u003c/p\u003e\n\u003cp\u003eB: Transcripts per million (TPM) from RNA-Seq with annotation for globin, \u003cem\u003eEPOR\u003c/em\u003e, and \u003cem\u003esynEPOR\u003c/em\u003egenes.\u003c/p\u003e\n\u003cp\u003eC: Volcano plot comparing unedited and edited HSPCs at d14 of differentiation v. unedited HSPCs at d0. Dashed lines are drawn at +/-1 log2 fold change and adjusted P-value = 0.01. Total number of significantly down- and upregulated genes is shown in top left and top right of each plot, respectively.\u003c/p\u003e\n\u003cp\u003eD: Volcano plot comparing edited HSPCs at d14 +BB v. unedited HSPCs at d14 +EPO. Dashed lines are drawn at +/-1 log2 fold change and adjusted P-value = 0.01. Total number of significantly down- and upregulated genes is shown in top left and top right of each plot, respectively.\u003c/p\u003e\n\u003cp\u003eE: Principal component analysis of all conditions with covariance ellipses.\u003c/p\u003e\n\u003cp\u003eF: Summary of gene ontology (GO) analysis comparing all d14 conditions v. d0 control. Differentially expressed genes (P\u003cu\u003e\u0026lt;\u003c/u\u003e0.01 and log2 fold change \u003cu\u003e\u0026gt;\u003c/u\u003e2) were used as input. Plotted are significantly enriched GO pathways (adjusted P-value \u003cu\u003e\u0026lt;\u003c/u\u003e0.05) that were binned into broader categories with enrichment score derived by Enrichrsoftware. Count refers to the number of genes within each GO pathway that contributed to enrichment.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-4986623/v1/073e2137783aed8a3479c825.png"},{"id":67352076,"identity":"54c50fe8-c039-43ba-a7ba-869878e71f28","added_by":"auto","created_at":"2024-10-24 04:09:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":200637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentiation of iPSCs into erythroid cells using synEPOR+BB.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Schematic of iPSC-to-erythroid cell differentiation strategy and subsequent analysis.\u003c/p\u003e\n\u003cp\u003eB: Percentage of cells that acquired erythroid markers (CD34-/CD45-/CD71+/GPA+) over the course of differentiation. Bars represent mean +/-SEM; ns = not statistically significant, * = p\u0026lt;0.05, ** = p\u0026lt;0.01, *** = p\u0026lt;0.001, and **** = p\u0026lt;0.0001 comparing +BB to +EPO conditions by unpaired t-test.\u003c/p\u003e\n\u003cp\u003eC: Percentage of total cell proliferation normalized to clones cultured +EPO over the course of differentiation. Bars represent mean +/SEM; ns = not statistically significant, * = p\u0026lt;0.05, ** = p\u0026lt;0.01, *** = p\u0026lt;0.001, and **** = p\u0026lt;0.0001 comparing +BB to +EPO conditions by unpaired t-test.\u003c/p\u003e\n\u003cp\u003eD: Representative hemoglobin tetramer HPLC plots of edited and unedited iPSC-derived erythroid cells at end of differentiation.\u003c/p\u003e\n\u003cp\u003eE: Ratio of HbF production in +BB v. +EPO conditions of synEPOR-edited iPSC-derived erythroid cells at end of differentiation.\u003c/p\u003e\n\u003cp\u003eF: Cost comparison of EPO and BB (lowest price per mg commercially available for purchase as of 2/9/24).\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-4986623/v1/97593f3e83f7bb558bb29e67.png"},{"id":75077004,"identity":"5e8e2e40-8fb3-43fc-afc4-e48ae3252dd6","added_by":"auto","created_at":"2025-01-30 08:08:10","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1000209,"visible":true,"origin":"","legend":"","description":"","filename":"NCBsynEPORManuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4986623/v1_covered_9e30170d-41c5-4d39-a91b-1f72cde218ba.pdf"},{"id":67351263,"identity":"d3f95382-62dc-4803-8ed2-f9d86d60e655","added_by":"auto","created_at":"2024-10-24 04:01:23","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16239616,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"ExtendedDatasynEPORDEGs.xls","url":"https://assets-eu.researchsquare.com/files/rs-4986623/v1/25042c5acb1d21878284f6f7.xls"},{"id":67351265,"identity":"8037c32e-b9e6-467c-b3b5-5b9b618c00a5","added_by":"auto","created_at":"2024-10-24 04:01:24","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3035447,"visible":true,"origin":"","legend":"\u003cp\u003eDataset 2\u003c/p\u003e","description":"","filename":"ExtendedDatasynEPORTPMsv3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4986623/v1/f755c4b341b314f0727a316a.xlsx"},{"id":67352074,"identity":"43aefcd2-7786-43d4-91c4-03ceaaeba307","added_by":"auto","created_at":"2024-10-24 04:09:23","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":168461,"visible":true,"origin":"","legend":"Dataset 3","description":"","filename":"ExtendedDataiEPORGOAnalysis.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4986623/v1/68b7f35c5578f13c950c4764.xlsx"},{"id":67352073,"identity":"4a09b4ce-36ee-4e26-aeb9-4921469eb23e","added_by":"auto","created_at":"2024-10-24 04:09:23","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":36352,"visible":true,"origin":"","legend":"Dataset 4","description":"","filename":"ExtendedDatasynEPORSpearmanCorrelation.xls","url":"https://assets-eu.researchsquare.com/files/rs-4986623/v1/9fee92f7e057b4e17c7f5bd5.xls"},{"id":67351266,"identity":"897fb5e1-91dc-4367-ad1f-c57724528c56","added_by":"auto","created_at":"2024-10-24 04:01:24","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":5072864,"visible":true,"origin":"","legend":"","description":"","filename":"NCBsynEPORSupplementalFiguresv2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4986623/v1/89d2dfe76ffadebbbf5f9ccf.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nM.H.P. is a member of the scientific advisory board of Allogene Therapeutics. M.H.P. has equity in CRISPR Tx and Kamau Tx. C.T.C., M.H.P., and M.K.C. have filed provisional patent no. PCT/US2023/076969.","formattedTitle":"Engineering synthetic signaling receptors to enable erythropoietin-free erythropoiesis","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"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-4986623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4986623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Blood transfusion plays a vital role in modern medicine. However, availability is contingent on donated blood, and frequent shortages pose a significant healthcare challenge. Ex vivo manufacturing of red blood cells (RBCs) derived from universal donor O-negative pluripotent stem cells emerges as a solution, yet the high cost of recombinant cytokines required for ex vivo erythroid differentiation remains a major barrier. Erythropoietin (EPO) signaling through the EPO receptor is indispensable to RBC development, and EPO is one of the most expensive components in erythroid-promoting media. Here, we used design-build-test cycles to develop highly optimized small molecule-inducible synthetic EPO receptors (synEPORs) which were integrated at a variety of genomic loci using homology-directed repair genome editing. We found that integration of synEPOR at the endogenous EPOR locus in an induced pluripotent stem cell producer line enabled culture with small molecule to yield equivalent erythroid differentiation, transcriptomic changes, and hemoglobin production compared to cells cultured with EPO. Due to the dramatically lower cost of small molecules vs. recombinant cytokines, these efforts eliminate one of the most expensive elements of ex vivo culture media—EPO cytokine. Because dependence on cytokines is a common barrier to ex vivo cell production, these strategies could improve scalable manufacturing of a wide variety of clinically relevant cell types. More broadly, this work showcases how protein engineering and genome engineering may be combined to introduce precisely regulated and tunable behavior into cells, an advancement which will pave the way for increasingly sophisticated synthetic biology applications.","manuscriptTitle":"Engineering synthetic signaling receptors to enable erythropoietin-free erythropoiesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-24 04:01:18","doi":"10.21203/rs.3.rs-4986623/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":"535da294-1463-48ac-a0ee-501103bac030","owner":[],"postedDate":"October 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39121137,"name":"Biological sciences/Developmental biology/Haematopoiesis/Erythropoiesis/Haematopoietic stem cells"},{"id":39121138,"name":"Biological sciences/Genetics/CRISPR-Cas systems/CRISPR-Cas9 genome editing"},{"id":39121139,"name":"Biological sciences/Stem cells/Pluripotent stem cells/Induced pluripotent stem cells"},{"id":39121140,"name":"Biological sciences/Biotechnology/Molecular engineering/Synthetic biology"},{"id":39121141,"name":"Biological sciences/Developmental biology/Differentiation"}],"tags":[],"updatedAt":"2025-01-30T08:08:02+00:00","versionOfRecord":{"articleIdentity":"rs-4986623","link":"https://doi.org/10.1038/s41467-025-56239-5","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-01-29 05:00:00","publishedOnDateReadable":"January 29th, 2025"},"versionCreatedAt":"2024-10-24 04:01:18","video":"","vorDoi":"10.1038/s41467-025-56239-5","vorDoiUrl":"https://doi.org/10.1038/s41467-025-56239-5","workflowStages":[]},"version":"v1","identity":"rs-4986623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4986623","identity":"rs-4986623","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}