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CysewskiMass SpectrometryBottom-up proteomicsAdipose-derived mesenchymal stem/stromal cellsCell therapyRegenerative medicineProteomicsImmuno-modulationOsteogenic differentiationhttps://www.ebi.ac.uk/pride/archive/projects/PXD073297Cell CultureApproximately 1 µg of peptides was separated on a Waters C18 column (0.3 mm × 150 mm) using a Micro M5 Sciex LC system at 5 µL/min. MS analysis was performed on a ZenoTOF 7600 equipped with an OptiFlow Turbo V ion source (spray voltage 5 kV, 200°C, curtain gas 35 psi, gas1 20 psi, gas2 60 psi). Data-dependent acquisition (top 20–100 pre-cursors) with dynamic background subtraction was employed. MS1 accumulation time was 100 ms, and MS/MS scans were acquired at 30 V CE with 5 ms accumulationPrideNot availableRaw MS data were processed in MaxQuant v2.6.5.0 against the UniProt human pro-teome (downloaded 01 June 2024; 83,413 entries) [108]. Protein identifications and LFQ values were obtained using standard parameters. Contaminants, reverse hits, proteins identified only by site or by a single peptide, and inconsistently quantified proteins were removed. LFQ intensities were log₂-transformed, and missing values imputed using the MNAR method (Gaussian distribution, shift 1.8 SD, width 0.3). Differential expression was as-sessed with limma (R Bioconductor), applying a Benjamini–Hochberg FDR ≤0.05 and |log₂FC| ≥1. MaxQuant analysis quantified 2.743 proteins, of which 324 were significant-ly regulated (FDR ≤0.05, |log₂FC| ≥1). For broader biological interpretation, a more per-missive cutoff (p <0.05, no FC threshold) was applied, yielding 854 proteins. Downstream functional analyses included Reactome pathway analysis and STRINGdb network analy-sisProteomicsDominik CysewskiPARTIALMedical University of BialystokHomo Sapiens (human)41972704 Szabłowska-Gadomska I, Rudziński S, Mroczko A, Mrozikiewicz-Rakowska B, Cysewski D, Gasperowicz P, Bocian K. Alterations in Immunomodulatory Potential of ADSCs Undergoing Osteogenic Differentiation in the Context of Future Therapeutic Applications. Cells. 2026 15(7):614 10.3390/cells15070614dominikcysewski@gmail.comMedical University of BialystokPoland<h4>Background</h4>Adipose-derived mesenchymal stem/stromal cells (ADSCs) are gaining recognition in regenerative medicine for their potential for adipogenic, osteogenic, and chondrogenic differentiation, as well as their immunomodulatory properties. However, ADSC-based therapies focus either on differentiation for tissue replacement or on counteracting unrestrained inflammation to prevent tissue destruction and initiate regeneration. Here, we aim to examine the immunomodulatory potential of osteogenically differentiated ADSCs by analyzing their proteomic profile.<h4>Methods</h4>Using LC-MS/MS, we generated the proteomic profiles of differentiated and undifferentiated ADSCs and compared them with the Reactome database. Transcriptomic analysis was also performed and compared with the proteomic profile.<h4>Results</h4>Comparison of the proteomic (499 up-regulated; 355 down-regulated) and transcriptomic (212 up-regulated; 232 down-regulated) profiles showed 60.1% concordance-both proteins and transcripts showed the same trend. Significantly upregulated proteins in differentiating ADSCs (-log<sub>10</sub><i>p</i> > 5 and >10) were grouped into four categories: propensity for osteogenic differentiation; immunomodulation/immune/inflammatory response; cell senescence; and cell cycle regulation. Among those proteins, thirteen were reported to play roles in processes such as immunomodulation, inflammatory signaling, or transplant rejection.<h4>Conclusions</h4>We observed that differentiating ADSCs might still exert immunomodulatory effects, which could be used in the treatment of, e.g., bone defects.Alterations in Immunomodulatory Potential of ADSCs Undergoing Osteogenic Differentiation in the Context of Future Therapeutic Applications.Szabłowska-Gadomska Ilona I, Rudziński Stefan S, Mroczko Agnieszka A, Mrozikiewicz-Rakowska Beata B, Cysewski Dominik D, Gasperowicz Piotr P, Bocian Katarzyna KfalseAlterations in immunomodulatory potential of ADSCs under-going osteogenic differentiation in the context of future thera-peutic applicationsFor proteomic analysis, ADSCs from four independent donors were analyzed (n = 4 biological replicates per condition, OM vs. SM). Cell pellets were lysed in 5% sodium de-oxycholate buffer containing TCEP and CAA, supplemented with protease and phospha-tase inhibitors, followed by sonication and incubation at 60 °C. Protein concentration was determined by BCA assay, and 25 µg of protein was processed with a modified FASP pro-tocol employing 30 kDa filters. After trypsin/LysC digestion, peptides were purified on Waters HLB plates, eluted with 70% acetonitrile/0.1% TFA, dried, and stored at −80 °C. Background: Adipose-derived mesenchymal stem/stromal cells (ADSCs) are gaining recognition in regenerative medicine thanks to their potential for adipogenic, osteogenic, and chondrogenic differentiation, as well as their immunomodulatory properties. How-ever, ADSC-based therapies focus either on differentiation for tissue replacements or on counteracting the unrestrained inflammation to prevent tissue destruction and initiate re-generation. Here, we aim to examine the immunomodulatory potential of osteogenically differentiated ADSCs by analyzing their proteomic profile. Methods: using the LC-MS/MS technique, we created the proteomic profile of differentiated and undifferentiated ADSCs, and compared them with the Reactome database. The transcriptomic analysis was also performed and compared to the proteomic profile. Results: the comparison of proteomic (499 up-regulated; 355 down-regulated) and transcriptomic (212 up-regulated; 232 down-regulated) profiles showed 60,1% concordance – both proteins and transcripts showed the same trend. Significantly upregulated proteins in differentiating ADSCs (−log₁₀ p >5 and >10) were grouped into four categories: propensity for osteogenic differen-tiation; immunomodulation/immune/inflammatory response; cell senescence; cell cycle regulation. Among those proteins, thirteen were reported to play some role in processes such as immunomodulation, inflammatory signaling, transplant rejection, or graft-versus-host disease. Conclusions: we observed that differentiating ADSCs might still exert immunomodulatory effects, which could be used in the treatment of e.g., GvHD.2026-04-272026-01-20PXD073297NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:71647NEWT:2NEWT:157546NEWT:474186NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:1496NEWT:544496NEWT:2102NEWT:2042546NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:4513NEWT:180454NEWT:5722NEWT:1247NEWT:6938NEWT:1129NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:281395NEWT:10360NEWT:1211601NEWT:876138NEWT:47664NEWT:317548NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:272563NEWT:1147161NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:82688NEWT:95648NEWT:3885NEWT:3888NEWT:5821NEWT:1589NEWT:44139NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:913645NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4442NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NEWT:2164133NCBITaxon:183674NEWT:1316931NEWT:224308NEWT:167387NEWT:84645NEWT:44586NEWT:626528NEWT:3347NEWT:511145NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1282NEWT:1283NEWT:431241NEWT:931281NEWT:4550NEWT:1000561NEWT:2133NEWT:294381NEWT:197NEWT:1390363NEWT:77133NEWT:288705NEWT:29176NEWT:145481NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:556182NEWT:1270NEWT:374990NEWT:1392NEWT:498217NEWT:156471NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:156476NEWT:286NEWT:391619NEWT:360104NEWT:246196NEWT:287NEWT:246197NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:161934NEWT:1148NEWT:580240NEWT:5508NEWT:294128NEWT:5507NEWT:11676NEWT:55571NEWT:35500NEWT:1140NEWT:100226NEWT:4530NEWT:1143NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:11557NEWT:1094343NEWT:1336795NEWT:644042NEWT:296543NEWT:316435NEWT:42157NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:82380NEWT:1034304NEWT:105023NEWT:866628NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:9378NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NEWT:8175NCBITaxon:1313NEWT:43330NEWT:1603293NEWT:408169NEWT:44544NEWT:47946NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:1077286NEWT:943129NEWT:243277NEWT:990119NEWT:7067NEWT:2850NEWT:1111708NEWT:408172NEWT:227321NEWT:408170NEWT:493760NEWT:106590NEWT:260710NEWT:257313NEWT:3827NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:930945NEWT:106592NEWT:536231NEWT:46704NEWT:1436733NEWT:460519NEWT:1187947NEWT:572307NEWT:1432138NEWT:269796NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:1194599NEWT:272844NEWT:54571NEWT:9598NEWT:8030NEWT:1513458NCBITaxon:40559NEWT:1639NEWT:84588NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:759272NEWT:432359NEWT:46835NEWT:1182263NEWT:109757NEWT:943146NEWT:2711NEWT:300852NEWT:537457NEWT:1502NEWT:376686NEWT:95486NEWT:9103NEWT:508771NEWT:1883446NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:29158NEWT:644223NEWT:3635NEWT:5811NEWT:235443NEWT:180923NEWT:108458NEWT:272623NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:527796NEWT:225117NEWT:746128NEWT:499175NEWT:109779NEWT:43665NEWT:1266464NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:367830NEWT:1255228NEWT:178616NEWT:649908NEWT:410289NEWT:373153NEWT:375451NEWT:352472NEWT:357NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:411469NEWT:84023NEWT:1679NEWT:559292NEWT:39491NCBITaxon:5811NEWT:29491NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:1328426NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NCBITaxon:138NEWT:40483NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:38293NEWT:1428NEWT:327159NEWT:178876NEWT:1660NEWT:327160NEWT:573NEWT:9031NEWT:1872122NEWT:7091NEWT:108931NEWT:241368NEWT:1055524NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:7237NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:187878NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1821314NEWT:1249668NEWT:7108NEWT:40479NEWT:317NEWT:5286NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:273068NEWT:313625NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT:451516NEWT:63459NEWT:36185NEWT:588858NEWT:1225786NEWT:9639NCBITaxon:11071NEWT:242231NEWT:7574NEWT:1715256NEWT:197221NEWT:7215NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:2172103NEWT:6494NEWT:7460NEWT:6491NEWT:507601NEWT:434271NEWT:747679NEWT:643680NCBITaxon:6157NEWT:42752NEWT:746360NEWT:6239NEWT:89764NEWT:470150NEWT:162425NEWT:216257NEWT:102169NEWT:543734NEWT:9986NEWT:4054NEWT:73239NEWT:38018NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:118503NEWT:2059687NEWT:115357NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:12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