Engraftment of human mesenchymal stem cells in a severely immunodeficient mouse

Engraftment of human mesenchymal stem cells in a severely immunodeficient mouse | 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 Research Article Engraftment of human mesenchymal stem cells in a severely immunodeficient mouse Yuko Kato, Ryoji Ito, Takeshi Taketani, Yumi Matsuzaki, Satoru Miyagi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4452493/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Sep, 2024 Read the published version in Inflammation and Regeneration → Version 1 posted 5 You are reading this latest preprint version Abstract The transplantation of human mesenchymal stromal/stem cells (hMSCs) has potential as a curative and permanent therapy for congenital skeletal diseases. However, the self-renewal and differentiation capacities of hMSCs markedly vary. Therefore, cell proliferation and trilineage differentiation capacities were tested in vitro to characterize hMSCs before their clinical use. However, it remains unclear whether the ability of hMSCs in vitro accurately predicts that in living animals. The xenograft model is an alternative method for validating clinical MSCs. Nevertheless, the protocol still needs improvement and it has yet to be established whether hMSCs, which are expanded in culture for clinical use, retain the ability to engraft and differentiate into adipogenic, osteogenic, and chondrogenic lineage cells in transplantation settings. In the present study, to establish a robust xenograft model of MSCs, we examined the delivery routes of hMSCs and the immunological state of recipients. The intra-arterial injection of hMSCs into X-ray-irradiated (IR) NOG, a severely immunodeficient mouse, achieved the highest engraftment, but failed to sustain long-term engraftment. We demonstrated that graft cells localized to a collagenase-released fraction (CR), in which endogenous colony-forming cells reside. We also showed that Pdgfrα+Sca1+ MSCs (PαS), which reside in the CR fraction, resisted IR. These results show that our protocol enables hMSCs to fulfill a high level of engraftment in mouse bone marrow in the short term. In contrast, long-term reconstitution was restricted, at least partially, because of IR-resistant endogenous MSCs. Mesenchymal stem cells Mesenchymal stromal cells Xenograft model NOG Transplantation Regenerative medicine Figures Figure 1 Figure 2 Figure 3 Full Text Cite Share Download PDF Status: Published Journal Publication published 26 Sep, 2024 Read the published version in Inflammation and Regeneration → Version 1 posted Editorial decision: Major revision 08 Jun, 2024 Reviewers agreed at journal 23 May, 2024 Reviewers invited by journal 23 May, 2024 Editor assigned by journal 21 May, 2024 First submitted to journal 20 May, 2024 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. 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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-4452493","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":305804814,"identity":"d794e166-be69-4d7d-8b58-6f5006e7325c","order_by":0,"name":"Yuko Kato","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yuko","middleName":"","lastName":"Kato","suffix":""},{"id":305804815,"identity":"eef03f0f-5f7f-4644-a6dd-34d0a060f1f3","order_by":1,"name":"Ryoji Ito","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ryoji","middleName":"","lastName":"Ito","suffix":""},{"id":305804816,"identity":"5710998f-13b4-4940-ba5b-77bc51d24655","order_by":2,"name":"Takeshi Taketani","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Taketani","suffix":""},{"id":305804817,"identity":"55fd6e81-1f23-4894-bc1a-01c1fb46cea9","order_by":3,"name":"Yumi Matsuzaki","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yumi","middleName":"","lastName":"Matsuzaki","suffix":""},{"id":305804818,"identity":"55f5ea80-1cc7-4fac-afcf-899296061cc5","order_by":4,"name":"Satoru Miyagi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIiWNgGAWjYDACZhBhYAMkEoBsAyQZxga8WtJI0QIBh6FaiAG67TyGH78UnI/mZ09g/lxQsE3enL2BTYKhxo6BeTZ2a8wO8xhLyxjczp3Z84BNeobBbcOdPQeAWo4lMzDOOYBLi4G0BFDLhhsJbMw8BrcZN9zI/ybBwHaAgXFGAk5bfksYnMvdfwPoMKAW+w33HwBt+YdXi5nkB4MDuRskEhikgVoSN9xgYJNgbMOnha3MmsEgOXfGmYdtIC3JG84kMFsk9iXz4PTL+cObb/74Y5fb3558+DPPn9u2G44fYLzx4ZudnCGOEGNg4DBg5gEzkKMO6CQewxk4dDCwP2D8gVVCXgKXllEwCkbBKBhhAADepF4oXRhERQAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Satoru","middleName":"","lastName":"Miyagi","suffix":""}],"badges":[],"createdAt":"2024-05-21 06:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4452493/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4452493/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s41232-024-00353-2","type":"published","date":"2024-09-26T15:58:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57943312,"identity":"841e05a2-1e3b-497d-a9bb-5d98688e14a6","added_by":"auto","created_at":"2024-06-07 19:05:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2114761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe CA injection improves MSC engraftment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Experimental design. (B) One day after GFP+ REC transplantation into C57BL6, CR (Left panel) or marrow (Right panel) fractions were prepared from a pair of tibia and femur and stained with the indicated antibodies for the FCM analysis. (C) Representative FCM profiles of the CR fraction from C57BL6 recipients transplanted with GFP+ RECs through the CA or IV injection. (D) The frequencies of GFP+ cells in the TN (Left panel) or stromal fraction (Right panel) were calculated from the FCM profile in (C). (E) Time course changes in GFP positivity in the TN or stromal fraction from C57BL6 transplanted with GFP-labeled REC (clones #3, 5, or 13) (E) or MSCs (F) are shown. Data are shown as the mean ± SEM. *P \u0026lt; .05; **P \u0026lt; .01; ***P \u0026lt; .005; ****P \u0026lt; .0005; *****P \u0026lt; .00005 by the Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4452493/v1/6f17987484768b2e46d37bff.jpg"},{"id":57943313,"identity":"fe069751-e1ad-498e-b1b1-eab96d7cf693","added_by":"auto","created_at":"2024-06-07 19:05:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1509751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunosuppression improves MSC engraftment.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Frequencies of GFP+ cells in the TN (Left panel) or stromal fraction (Right panel) from mice transplanted with GFP-labeled RECs in the presence or absence of TAC on Day 7. (B) Experimental design. (C) GFP-labeled RECs were transplanted into NOG mice with or without semi-lethal IR through the CA or IV injection. Recipients were analyzed on Days 7 and 28. (D) The frequencies of GFP+ cells in the TN (Left panel) or CD90+ stromal fraction (Right panel) were calculated from the FCM profile in (C). (E) Number of GFP+ in a pair of tibia and femur. Data are shown as the mean ± SEM. *P \u0026lt; .05; **P \u0026lt; .01; ***P \u0026lt; .005; ****P \u0026lt; .0005; *****P \u0026lt; .00005 by the Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4452493/v1/ad8ad2ab42de0ae6a36e0b52.jpg"},{"id":57943315,"identity":"6f335efb-1e1f-4bc6-ab95-b7fa108c9fba","added_by":"auto","created_at":"2024-06-07 19:05:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2510267,"visible":true,"origin":"","legend":"\u003cp\u003ePαS cells show IR resistance.\u003c/p\u003e\n\u003cp\u003eC57BL6 mice were X-ray irradiated and the frequencies of PαS and CAR/LepR+ MSCs were examined on Days 0, 3, 7, and 10. (A, B) Representative FCM profiles of CAR/LepR+ MSCs (A) and the cellularity of the marrow fraction, frequencies, and absolute number of CAR/LepR+ MSCs (B) are shown. (C, D) Representative FCM profiles of PαS cells (C) and the cellularity of the CR fraction, frequencies, and absolute number of PαS cells (B) are shown. Data are shown as the mean ± SEM. *P \u0026lt; .05; **P \u0026lt; .01; ***P \u0026lt; .005; ****P \u0026lt; .0005; *****P \u0026lt; .00005 by the Student’s t-test.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4452493/v1/4442cdfd0a26e9260e44c569.jpg"},{"id":65627361,"identity":"57c29b3c-506a-4f63-a915-db95bed6006c","added_by":"auto","created_at":"2024-09-30 16:15:26","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6473596,"visible":true,"origin":"","legend":"","description":"","filename":"Katoetal.final.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4452493/v1_covered_6e705d11-e25c-4526-85d2-00d344f2482c.pdf"}],"financialInterests":"","formattedTitle":"Engraftment of human mesenchymal stem cells in a severely immunodeficient mouse","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammation-and-regeneration","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ireg","sideBox":"Learn more about [Inflammation and Regeneration](http://inflammregen.biomedcentral.com/)","snPcode":"41232","submissionUrl":"https://www.editorialmanager.com/ireg/default2.aspx","title":"Inflammation and Regeneration","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Mesenchymal stem cells, Mesenchymal stromal cells, Xenograft model, NOG, Transplantation, Regenerative medicine","lastPublishedDoi":"10.21203/rs.3.rs-4452493/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4452493/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The transplantation of human mesenchymal stromal/stem cells (hMSCs) has potential as a curative and permanent therapy for congenital skeletal diseases. However, the self-renewal and differentiation capacities of hMSCs markedly vary. Therefore, cell proliferation and trilineage differentiation capacities were tested in vitro to characterize hMSCs before their clinical use. However, it remains unclear whether the ability of hMSCs in vitro accurately predicts that in living animals. The xenograft model is an alternative method for validating clinical MSCs. Nevertheless, the protocol still needs improvement and it has yet to be established whether hMSCs, which are expanded in culture for clinical use, retain the ability to engraft and differentiate into adipogenic, osteogenic, and chondrogenic lineage cells in transplantation settings. In the present study, to establish a robust xenograft model of MSCs, we examined the delivery routes of hMSCs and the immunological state of recipients. The intra-arterial injection of hMSCs into X-ray-irradiated (IR) NOG, a severely immunodeficient mouse, achieved the highest engraftment, but failed to sustain long-term engraftment. We demonstrated that graft cells localized to a collagenase-released fraction (CR), in which endogenous colony-forming cells reside. We also showed that Pdgfrα+Sca1+ MSCs (PαS), which reside in the CR fraction, resisted IR. These results show that our protocol enables hMSCs to fulfill a high level of engraftment in mouse bone marrow in the short term. 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