{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:17:59Z","timestamp":1785403079858,"version":"3.56.0"},"reference-count":52,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,4,14]],"date-time":"2026-04-14T00:00:00Z","timestamp":1776124800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Taubman Institute Innovation Projects"},{"name":"NIH","award":["R01AG079163"],"award-info":[{"award-number":["R01AG079163"]}]},{"name":"NIH","award":["R01DK133448"],"award-info":[{"award-number":["R01DK133448"]}]},{"name":"NIH","award":["U01HL137182"],"award-info":[{"award-number":["U01HL137182"]}]},{"name":"NIH","award":["UH3CA268091"],"award-info":[{"award-number":["UH3CA268091"]}]},{"name":"NIH","award":["P30AG024824"],"award-info":[{"award-number":["P30AG024824"]}]},{"name":"NIH","award":["P30AG013283"],"award-info":[{"award-number":["P30AG013283"]}]},{"name":"NIH","award":["P30DK034933"],"award-info":[{"award-number":["P30DK034933"]}]},{"name":"NIH","award":["P30DK089503"],"award-info":[{"award-number":["P30DK089503"]}]},{"name":"NIH","award":["P30CA046592"],"award-info":[{"award-number":["P30CA046592"]}]},{"name":"NIH","award":["P30AR069620"],"award-info":[{"award-number":["P30AR069620"]}]},{"name":"NIH","award":["U2CDK110768"],"award-info":[{"award-number":["U2CDK110768"]}]},{"name":"Taiwanese Government Fellowship"},{"DOI":"10.13039\/100014989","name":"Chan Zuckerberg Initiative","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100014989","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/02qenvm24","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Glenn Foundation Core grants"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Aging is a regulated process marked by the accumulation of senescent cells, which remain viable but no longer divide. Senescent cells contribute to age-associated phenotypes and diseases, including osteoarthritis, dementia, and cancer, but their scarcity and heterogeneity have limited study. Here, we developed a fluorescence-guided high-resolution spatial transcriptomic profiling approach to precisely locate and profile p21-reporter-positive cells in aged liver. This method enabled unbiased detection of a p21-associated, senescence-enriched cell population and revealed its diverse cellular identities, including hepatocytes, macrophages, neutrophils, and plasma cells. Our analysis further showed that activated macrophages and hepatic stellate cells were more likely to exhibit a p21 positive (p21+) state than their resting counterparts. Transcriptomic profiling of p21-expressing cells indicated heterogeneous senescence-associated secretory phenotype (SASP) programs, with distinct inflammatory and remodeling signatures across cell identities and their spatial positions. In parallel, we identified an aggregation of interferon-stimulated gene (ISG)-expressing cells with limited overlap with p21 positivity, suggesting a distinct aging-associated stress program. Taken together, our fluorescence-guided spatial transcriptomic framework enables high-resolution, single-cell mapping of senescence in situ, delineating both senescent cell type specificity and cell identity\u2013independent senescence programs, thereby advancing a more comprehensive understanding of regulatory mechanisms underlying aging.<\/jats:p>","DOI":"10.3390\/biom16040579","type":"journal-article","created":{"date-parts":[[2026,4,14]],"date-time":"2026-04-14T10:29:08Z","timestamp":1776162548000},"page":"579","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Fluorescence-Guided Spatial Mapping of p21-Expressing Senescent Cells in Aged Liver at Single-Cell Resolution"],"prefix":"10.3390","volume":"16","author":[{"given":"Jer-En","family":"Hsu","sequence":"first","affiliation":[{"name":"Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0339-7483","authenticated-orcid":false,"given":"Binsheng","family":"Wang","sequence":"additional","affiliation":[{"name":"Masonic Institute on the Biology of Aging and Metabolism, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongha","family":"Hwang","sequence":"additional","affiliation":[{"name":"Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA"},{"name":"Space Planning and Analysis, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weiqiu","family":"Cheng","sequence":"additional","affiliation":[{"name":"Department of Biostatistics, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-4549-3104","authenticated-orcid":false,"given":"Qingyang","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yichen","family":"Si","sequence":"additional","affiliation":[{"name":"Department of Biostatistics, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ming","family":"Xu","sequence":"additional","affiliation":[{"name":"Masonic Institute on the Biology of Aging and Metabolism, University of Minnesota, Minneapolis, MN 55455, USA"},{"name":"Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hyun-Min","family":"Kang","sequence":"additional","affiliation":[{"name":"Department of Biostatistics, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2200-6011","authenticated-orcid":false,"given":"Jun Hee","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Molecular & Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"813","DOI":"10.1016\/j.cell.2019.10.005","article-title":"Cellular Senescence: Defining a Path Forward","volume":"179","author":"Gorgoulis","year":"2019","journal-title":"Cell"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/s43587-021-00121-8","article-title":"Strategies for Targeting Senescent Cells in Human Disease","volume":"1","author":"Gasek","year":"2021","journal-title":"Nat. Aging"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"958","DOI":"10.1038\/s41580-024-00727-x","article-title":"The senescence-associated secretory phenotype and its physiological and pathological implications","volume":"25","author":"Wang","year":"2024","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_4","first-page":"2853","article-title":"Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor","volume":"6","author":"Patil","year":"2008","journal-title":"PLoS Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"4150","DOI":"10.1016\/j.cell.2024.05.059","article-title":"Guidelines for Minimal Information on Cellular Senescence Experimentation in vivo","volume":"187","author":"Ogrodnik","year":"2024","journal-title":"Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1006\/excr.1994.1063","article-title":"Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen","volume":"211","author":"Noda","year":"1994","journal-title":"Exp. Cell Res."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1795","DOI":"10.1016\/j.cmet.2024.07.006","article-title":"Intermittent clearance of p21-highly-expressing cells extends lifespan and confers sustained benefits to health and physical function","volume":"36","author":"Wang","year":"2024","journal-title":"Cell Metab."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"962","DOI":"10.1038\/s43587-021-00107-6","article-title":"An inducible p21-Cre mouse model to monitor and manipulate p21-highly-expressing senescent cells in vivo","volume":"1","author":"Wang","year":"2021","journal-title":"Nat. Aging"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1016\/j.cmet.2021.11.002","article-title":"Targeting p21Cip1 highly expressing cells in adipose tissue alleviates insulin resistance in obesity","volume":"34","author":"Wang","year":"2022","journal-title":"Cell Metab."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2381","DOI":"10.1038\/s41588-025-02314-y","article-title":"Advancing biological understanding of cellular senescence with computational multiomics","volume":"57","author":"Li","year":"2025","journal-title":"Nat. Genet."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"776","DOI":"10.1038\/s43587-023-00446-6","article-title":"Spatial mapping of cellular senescence: Emerging challenges and opportunities","volume":"3","author":"Gurkar","year":"2023","journal-title":"Nat. Aging"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2283","DOI":"10.1038\/s41467-025-57632-w","article-title":"Senescent-like microglia limit remyelination through the senescence associated secretory phenotype","volume":"16","author":"Gross","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1038\/s43587-024-00755-4","article-title":"Clearance of p21 highly expressing senescent cells accelerates cutaneous wound healing","volume":"5","author":"Gasek","year":"2025","journal-title":"Nat. Aging"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3559","DOI":"10.1016\/j.cell.2021.05.010","article-title":"Microscopic examination of spatial transcriptome using Seq-Scope","volume":"184","author":"Cho","year":"2021","journal-title":"Cell"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1038\/s41596-024-01065-0","article-title":"Seq-Scope: Repurposing Illumina sequencing flow cells for high-resolution spatial transcriptomics","volume":"20","author":"Kim","year":"2025","journal-title":"Nat. Protoc."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"eabo2787","DOI":"10.1126\/sciimmunol.abo2787","article-title":"TREM2 macrophages induced by human lipids drive inflammation in acne lesions","volume":"7","author":"Do","year":"2022","journal-title":"Sci. Immunol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1093\/bioinformatics\/bts635","article-title":"STAR: Ultrafast universal RNA-seq aligner","volume":"29","author":"Dobin","year":"2013","journal-title":"Bioinformatics"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"3573","DOI":"10.1016\/j.cell.2021.04.048","article-title":"Integrated analysis of multimodal single-cell data","volume":"184","author":"Hao","year":"2021","journal-title":"Cell"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1888","DOI":"10.1016\/j.cell.2019.05.031","article-title":"Comprehensive Integration of Single-Cell Data","volume":"177","author":"Stuart","year":"2019","journal-title":"Cell"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"e167835","DOI":"10.1172\/JCI167835","article-title":"Serum amyloid A expression in liver promotes synovial macrophage activation and chronic arthritis via NFAT5","volume":"134","author":"Li","year":"2024","journal-title":"J. Clin. Investig."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2630","DOI":"10.1002\/eji.200323985","article-title":"Tissue-specific regulation of the human acute-phase serum amyloid A genes, SAA1 and SAA2, by glucocorticoids in hepatic and epithelial cells","volume":"33","author":"Thorn","year":"2003","journal-title":"Eur. J. Immunol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.bbrc.2022.11.079","article-title":"Neutralizing serum amyloid a protects against sinusoidal endothelial cell damage and platelet aggregation during acetaminophen-induced liver injury","volume":"639","author":"You","year":"2023","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1146\/annurev-immunol-032713-120231","article-title":"Interferon-stimulated genes: A complex web of host defenses","volume":"32","author":"Schneider","year":"2014","journal-title":"Annu. Rev. Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"785","DOI":"10.1016\/j.celrep.2015.03.069","article-title":"DNA damage-induced type I interferon promotes senescence and inhibits stem cell function","volume":"11","author":"Yu","year":"2015","journal-title":"Cell Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1117","DOI":"10.1038\/s43587-021-00151-2","article-title":"Senolytic vaccination improves normal and pathological age-related phenotypes and increases lifespan in progeroid mice","volume":"1","author":"Suda","year":"2021","journal-title":"Nat. Aging"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"ehae666.3654","DOI":"10.1093\/eurheartj\/ehae666.3654","article-title":"Vaccination targeting senescence-associated transmembrane protein alleviated cardiovascular pathology in the mice","volume":"45","author":"Hsiao","year":"2024","journal-title":"Eur. Heart J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"653","DOI":"10.1242\/jcs.188672","article-title":"The integrin\u2013collagen connection\u2014A glue for tissue repair?","volume":"129","author":"Zeltz","year":"2016","journal-title":"J. Cell Sci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1038\/cr.2009.139","article-title":"Complement and its role in innate and adaptive immune responses","volume":"20","author":"Dunkelberger","year":"2010","journal-title":"Cell Res."},{"key":"ref_29","first-page":"459","article-title":"The Lcn2-engineered HEK-293 cells show senescence under stressful condition","volume":"18","author":"Bahmani","year":"2015","journal-title":"Iran. J. Basic Med. Sci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3161","DOI":"10.1182\/blood-2006-09-003004","article-title":"The cytoprotective protein C pathway","volume":"109","author":"Mosnier","year":"2007","journal-title":"Blood"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2145","DOI":"10.1038\/s41598-024-52710-3","article-title":"Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) deletion in myeloid cells augments cholestatic liver injury","volume":"14","author":"Krishnan","year":"2024","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"648","DOI":"10.1002\/hep4.1069","article-title":"TRAIL deletion prevents liver inflammation but not adipose tissue inflammation during murine diet-induced obesity","volume":"1","author":"Hirsova","year":"2017","journal-title":"Hepatol. Commun."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1210","DOI":"10.1016\/j.celrep.2014.07.032","article-title":"Kcnn4 is a regulator of macrophage multinucleation in bone homeostasis and inflammatory disease","volume":"8","author":"Kang","year":"2014","journal-title":"Cell Rep."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Huang, C., Santofimia-Casta\u00f1o, P., and Iovanna, J. (2021). NUPR1: A Critical Regulator of the Antioxidant System. Cancers, 13.","DOI":"10.3390\/cancers13153670"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"14133","DOI":"10.1038\/ncomms14133","article-title":"SCIMP is a transmembrane non-TIR TLR adaptor that promotes proinflammatory cytokine production from macrophages","volume":"8","author":"Luo","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_36","first-page":"4080","article-title":"CRISPLD2 attenuates pro-inflammatory cytokines production in HMGB1-stimulated monocytes and septic mice","volume":"13","author":"Zhang","year":"2021","journal-title":"Am. J. Transl. Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1336","DOI":"10.1038\/s41418-025-01468-w","article-title":"The Trim32-DPEP2 axis is an inflammatory switch in macrophages during intestinal inflammation","volume":"32","author":"Zhan","year":"2025","journal-title":"Cell Death Differ."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"van Daalen, K.R., Reijneveld, J.F., and Bovenschen, N. (2020). Modulation of Inflammation by Extracellular Granzyme A. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.00931"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"6891","DOI":"10.1080\/21655979.2021.1973781","article-title":"Lumican silencing alleviates tumor necrosis factor-\u03b1-induced nucleus pulposus cell inflammation and senescence by inhibiting apoptosis signal regulating kinase 1\/p38 signaling pathway via inactivating Fas ligand expression","volume":"12","author":"Li","year":"2021","journal-title":"Bioengineered"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"e00586-18","DOI":"10.1128\/MCB.00586-18","article-title":"Uncoupling the Senescence-Associated Secretory Phenotype from Cell Cycle Exit via Interleukin-1 Inactivation Unveils Its Protumorigenic Role","volume":"39","author":"Lau","year":"2019","journal-title":"Mol. Cell. Biol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"12077","DOI":"10.1074\/jbc.M117.797126","article-title":"The intracellular chloride channel proteins CLIC1 and CLIC4 induce IL-1\u03b2 transcription and activate the NLRP3 inflammasome","volume":"292","author":"Coll","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"10112","DOI":"10.1038\/ncomms10112","article-title":"Leukotriene C4 is the major trigger of stress-induced oxidative DNA damage","volume":"6","author":"Dvash","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"e70000","DOI":"10.1096\/fj.202400808RR","article-title":"Research progress on interferon and cellular senescence","volume":"38","author":"Wang","year":"2024","journal-title":"FASEB J."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"596","DOI":"10.1038\/s41586-020-2499-y","article-title":"Ageing hallmarks exhibit organ-specific temporal signatures","volume":"583","author":"Schaum","year":"2020","journal-title":"Nature"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"101133","DOI":"10.1016\/j.xgen.2025.101133","article-title":"Cellular senescence in human liver under normal aging and cancer","volume":"6","author":"Karpova","year":"2026","journal-title":"Cell Genom."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Basisty, N., Kale, A., Jeon, O.H., Kuehnemann, C., Payne, T., Rao, C., Holtz, A., Shah, S., Sharma, V., and Ferrucci, L. (2020). A proteomic atlas of senescence-associated secretomes for aging biomarker development. PLoS Biol., 18.","DOI":"10.1371\/journal.pbio.3000599"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1038\/nrg3185","article-title":"Insights into the regulation of protein abundance from proteomic and transcriptomic analyses","volume":"13","author":"Vogel","year":"2012","journal-title":"Nat. Rev. Genet."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1720","DOI":"10.1128\/MCB.19.3.1720","article-title":"Correlation between protein and mRNA abundance in yeast","volume":"19","author":"Gygi","year":"1999","journal-title":"Mol. Cell. Biol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1097\/PSY.0000000000001320","article-title":"New Directions in Geroscience: Integrating Social and Behavioral Drivers of Biological Aging","volume":"86","author":"Nielsen","year":"2024","journal-title":"Psychosom. Med."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"e14034","DOI":"10.1111\/acel.14034","article-title":"Enabling translational geroscience by broadening the scope of geriatric care","volume":"23","author":"Ferrucci","year":"2024","journal-title":"Aging Cell"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1196\/annals.1354.010","article-title":"Aging in mouse and human systems: A comparative study","volume":"1067","author":"Demetrius","year":"2006","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.mad.2016.10.001","article-title":"Mouse models of ageing and their relevance to disease","volume":"160","author":"Dogan","year":"2016","journal-title":"Mech. Ageing Dev."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/4\/579\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,16]],"date-time":"2026-04-16T04:10:45Z","timestamp":1776312645000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/4\/579"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,14]]},"references-count":52,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2026,4]]}},"alternative-id":["biom16040579"],"URL":"https:\/\/doi.org\/10.3390\/biom16040579","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,14]]}}}