{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T10:08:37Z","timestamp":1779098917066,"version":"3.51.4"},"posted":{"date-parts":[[2026,5,13]]},"group-title":"Cell Biology","reference-count":24,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,13]],"date-time":"2026-05-13T00:00:00Z","timestamp":1778630400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"funder":[{"DOI":"10.13039\/100000072","name":"National Institute of Dental and Craniofacial Research","doi-asserted-by":"crossref","award":["R01DE032364"],"award-info":[{"award-number":["R01DE032364"]}],"id":[{"id":"10.13039\/100000072","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>This work aimed to establish a translationally viable, xeno-free, serum-free platform and protocol for the isolation and expansion of human salivary stem\/progenitor cells (hS\/PCs) suitable for regulatory qualification and future FDA-approved first-in-human autologous regenerative therapy trials for the treatment of hyposalivation disorders. Parotid gland specimens from non-cancerous regions\/tissues were collected from consented surgical patients. Primary hS\/PCs were isolated from tissue specimens, cultured in animal-component-free conditions, expanded to produce millions of cells, then enriched for CD44+ stem\/progenitor cells by magnetic cell sorting. Normal epithelial purity was assessed using cytokeratins 5\/14. Anti-CD133\/PROM1 (cancer marker) and anti- fibroblast (clone TE-7) antibodies were used to demonstrate a lack of contaminating cells. Phenotype validation was performed by flow cytometry and immunocytochemistry on both CD44+ sorted and unsorted populations. Senescence-associated beta-galactosidase (SA-\u03b2-gal) assays were performed across serial passages (P1\u2013P6). Pluripotency was demonstrated by culture under conditions supporting lineage-specific differentiation. Primary hS\/PCs demonstrated consistent expansion and epithelial morphology under serum-free conditions. CD44 expression remained high (&gt;95%) throughout expansion, with negligible detection of CD133 or fibroblast markers, confirming epithelial purity and absence of tumorigenic or stromal contamination. Immunocytochemistry corroborated these expression profiles. SA-\u03b2-gal staining revealed only a minor, passage-dependent increase (5\u201316%) in senescent cells from multiple donors, indicating retention of proliferative potential. Our defined, animal-free culture system supports stable expansion of pure low passage hS\/PCs under conditions compatible with good manufacturing practice (GMP).<\/jats:p>","DOI":"10.64898\/2026.05.12.724408","type":"posted-content","created":{"date-parts":[[2026,5,14]],"date-time":"2026-05-14T07:45:17Z","timestamp":1778744717000},"source":"Crossref","is-referenced-by-count":0,"title":["Optimizing Primary Human Salivary Stem\/Progenitor Cells for Tissue Engineering Applications"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0155-9781","authenticated-orcid":false,"given":"Thaise. C.","family":"Geremias","sequence":"first","affiliation":[{"name":"Diagnostic and Biomedical Sciences, School of Dentistry, The University of Texas Health Science Center at Houston, Houston, Texas"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2089-4051","authenticated-orcid":false,"given":"Fabio H. B.","family":"da Costa","sequence":"additional","affiliation":[{"name":"Diagnostic and Biomedical Sciences, School of Dentistry, The University of Texas Health Science Center at Houston, Houston, Texas"},{"name":"Department of Biosciences, Rice University, Houston, Texas"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nadia G.","family":"Mohyuddin","sequence":"additional","affiliation":[{"name":"Academic Institute Department of Otolaryngology Houston Methodist Hospital, Houston Methodist, Weill Cornell Medical College"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7547-6375","authenticated-orcid":false,"given":"Isabelle M.","family":"Lombaert","sequence":"additional","affiliation":[{"name":"Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Harbor"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4526-3088","authenticated-orcid":false,"given":"Mary C.","family":"Farach-Carson","sequence":"additional","affiliation":[{"name":"Diagnostic and Biomedical Sciences, School of Dentistry, The University of Texas Health Science Center at Houston, Houston, Texas"},{"name":"Department of Biosciences, Rice University, Houston, Texas"},{"name":"Bioengineering, Rice University, Texas"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3386-8239","authenticated-orcid":false,"given":"Danielle","family":"Wu","sequence":"additional","affiliation":[{"name":"Diagnostic and Biomedical Sciences, School of Dentistry, The University of Texas Health Science Center at Houston, Houston, Texas"},{"name":"Bioengineering, Rice University, Texas"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"issue":"3","key":"2026051802150643000_2026.05.12.724408v1.1","doi-asserted-by":"crossref","first-page":"e368","DOI":"10.1002\/wdev.368","article-title":"A critical look: Challenges in differentiating human pluripotent stem cells into desired cell types and organoids","volume":"9","year":"2020","journal-title":"Wiley Interdiscip Rev Dev Biol"},{"key":"2026051802150643000_2026.05.12.724408v1.2","doi-asserted-by":"crossref","unstructured":"Lombello, C.B. , et al., Principles of Tissue Engineering and Regenerative Medicine, in Current Trends in Biomedical Engineering, C.B. Lombello and P.A. da Ana , Editors. 2023, Springer International Publishing: Cham. p. 127\u2013148.","DOI":"10.1007\/978-3-031-38743-2_8"},{"key":"2026051802150643000_2026.05.12.724408v1.3","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-3609-1_3"},{"key":"2026051802150643000_2026.05.12.724408v1.4","doi-asserted-by":"publisher","DOI":"10.5966\/sctm.2016-0083"},{"key":"2026051802150643000_2026.05.12.724408v1.5","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1007\/978-1-4939-8600-2_3","article-title":"Reassembly of Functional Human Stem\/Progenitor Cells in 3D Culture","volume":"1817","year":"2018","journal-title":"Methods Mol Biol"},{"key":"2026051802150643000_2026.05.12.724408v1.6","doi-asserted-by":"publisher","DOI":"10.1002\/lary.24297"},{"key":"2026051802150643000_2026.05.12.724408v1.7","doi-asserted-by":"publisher","DOI":"10.3389\/fmolb.2021.711602"},{"key":"2026051802150643000_2026.05.12.724408v1.8","doi-asserted-by":"publisher","DOI":"10.1177\/00220345221076122"},{"key":"2026051802150643000_2026.05.12.724408v1.9","doi-asserted-by":"publisher","DOI":"10.1177\/1535370213480714"},{"key":"2026051802150643000_2026.05.12.724408v1.10","doi-asserted-by":"publisher","DOI":"10.1038\/nature04372"},{"key":"2026051802150643000_2026.05.12.724408v1.11","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0050075"},{"key":"2026051802150643000_2026.05.12.724408v1.12","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-11-0179"},{"issue":"2","key":"2026051802150643000_2026.05.12.724408v1.13","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/j.fdj.2018.08.004","article-title":"Immunohistochemical evaluation of CD44 expression in mucoepidermoid carcinoma of human salivary glands","volume":"4","year":"2018","journal-title":"Future Dental Journal"},{"key":"2026051802150643000_2026.05.12.724408v1.14","doi-asserted-by":"publisher","DOI":"10.1111\/j.1601-0825.2011.01858.x"},{"issue":"6","key":"2026051802150643000_2026.05.12.724408v1.15","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1007\/s004180000220","article-title":"Expression of CD44 isoforms in normal salivary gland tissue: an immunohistochemical and ultrastructural study","volume":"114","year":"2000","journal-title":"Histochem Cell Biol"},{"issue":"17","key":"2026051802150643000_2026.05.12.724408v1.16","doi-asserted-by":"crossref","first-page":"7599","DOI":"10.7150\/thno.39893","article-title":"Microenvironmentally-driven Plasticity of CD44 isoform expression determines Engraftment and Stem-like Phenotype in CRC cell lines","volume":"10","year":"2020","journal-title":"Theranostics"},{"key":"2026051802150643000_2026.05.12.724408v1.17","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M112.339622"},{"key":"2026051802150643000_2026.05.12.724408v1.18","doi-asserted-by":"crossref","unstructured":"Pospieszna, J. , et al., Unmasking the Deceptive Nature of Cancer Stem Cells: The Role of CD133 in Revealing Their Secrets. Int J Mol Sci, 2023. 24(13).","DOI":"10.3390\/ijms241310910"},{"key":"2026051802150643000_2026.05.12.724408v1.19","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.canlet.2020.12.022","article-title":"An aptamer-based drug delivery agent (CD133-apt-Dox) selectively and effectively kills liver cancer stem-like cells","volume":"501","year":"2021","journal-title":"Cancer Lett"},{"issue":"3","key":"2026051802150643000_2026.05.12.724408v1.20","first-page":"299","article-title":"Clinical significance of stem cell marker CD133 expression in colorectal cancer","volume":"31","year":"2016","journal-title":"Histol Histopathol"},{"key":"2026051802150643000_2026.05.12.724408v1.21","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2009.191"},{"key":"2026051802150643000_2026.05.12.724408v1.22","doi-asserted-by":"publisher","DOI":"10.1006\/excr.2000.4875"},{"key":"2026051802150643000_2026.05.12.724408v1.23","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.92.20.9363"},{"issue":"1","key":"2026051802150643000_2026.05.12.724408v1.24","doi-asserted-by":"crossref","first-page":"36738","DOI":"10.1038\/s41598-025-20652-z","article-title":"Diversity of oxidative stress and senescence phenotypes induced by chemotherapeutic agents in HUVECs","volume":"15","year":"2025","journal-title":"Sci Rep"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.05.12.724408","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,18]],"date-time":"2026-05-18T09:15:47Z","timestamp":1779095747000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.05.12.724408"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,13]]},"references-count":24,"URL":"https:\/\/doi.org\/10.64898\/2026.05.12.724408","relation":{},"subject":[],"published":{"date-parts":[[2026,5,13]]},"subtype":"preprint"}}