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Jaensch"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Bottom-up proteomics"],"software":[""],"submitter_keywords":["Cell surface proteomics","Rna-seq","Physiologic media","Hipsc","Surfaceome","Drug screening","Cell manufacturing","Macrophages"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD070961"],"tissue":["Monocyte"],"sample_protocol":["1x106 iPSC-Mac were terminally differentiated in a 6-well tissue culture plate as previously described. After three days, the different cultivation conditions were washed several times with PBS and lysed in 2% SDS/PBS. Lysates were boiled for 5 – 10 min and stored at -80 °C until further use. Glycoprotein enrichment was performed as described previously[39]. Briefly, equal amounts of protein dissolved in 100 µL 2% SDS/PBS were centrifuged for 20 min at 20,000 xg. The supernatant was subjected to Pierce™ polyacrylamide spin-desalting columns (Thermo Scientific) equilibrated with 150 mM sodium acetate (pH 5.5) for buffer exchange. Carbohydrates were oxidized with 10 mM sodium periodate (Sigma-Aldrich) and incubated 30 min in the dark. Subsequently, another buffer exchange was performed with polyacrylamide spin-desalting columns equilibrated with PBS. The oxidized glycoproteins incubated overnight with Ultralink™ hydrazide resin beads (Thermo Scientific) in the presence of aniline as catalyst. The beads were washed five times with 0.5% SDS/PBS and one time with 8M Urea/100 mM Tris-HCl pH 7.8. For the reduction and alkylation step, proteins incubated one hour at 37 °C with 50 mM DTT and 30 min with 50 mM iodoacetamide in the dark. To remove non-glycoproteins from covalently linked glycoproteins, the beads were stringently washed: 3x 8M urea/100 mM Tris-HCl pH 7.8, 1x 100 mM Tris-HCl (pH 7.8), 3x 3 M NaCl in 100 mM Tris-HCl (pH 7.8), 3x 100 mM sodium acetate 150 mM/NaCl (pH 3.0), 2x H2O, 3x 30% acetonitrile, 2x 50 mM ammonium hydrogencarbonate (ABC). After the final wash, the beads were resuspended in 20 µL ABC and digested with 1 µg trypsin overnight at 37 °C. The peptide-containing eluate was collected. The beads were eluted again with 50 mM ABC and 30% acetonitrile. All eluate collections were combined and dried. The beads were washed again as described above and digested with 1U PNGase F (Promega) at 37 °C overnight. The eluate containing deglycosylated N-linked glycopeptides was collected and dried as a separate fraction."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["Samples were dissolved in 0.1% formic acid. 250 ng of the tryptic fraction and 15 ng of the PNGase Fraction were injected to a Dionex Ultimate 3000 HPLC system coupled to an Orbitrap Fusion™ mass spectrometer (Thermo Scientific). The general set-up was: spray voltage of 1.8 kV, heated capillary temperature of 300 °C, S-lens RF level of 60%, ion selection threshold of 50,000 counts for HCD. All peptides were loaded to a 200 cm C18 µPac™ analytical HPLC column (PharmaFluidics). Tryptic fractions were separated over a 160 min non-linear gradient (3%-90% acetonitrile) with a flow rate of 300 nL/min. Overview spectra were recorded with a cycle time of 1 s and a maximum injection time of 50 ms. The scan window was set to 400-1600 m/z with a resolution of 120,000. Selected precursors were fragmented with a higher energy collisional dissociation (HCD) of 30%. Ions were collected for MS2 spectra with a maximum injection time of 300 ms and AGC 2.5 × 104. The PNGase fraction was analyzed with the following changes: Peptides were eluted from the analytical columns in an 80 min non-linear gradient. Selected precursors for MS2 spectra were collected with a maximum injection time of 500 ms and an AGC of 1.2 × 104. Raw files were analyzed using the MaxQuant software (version 2.4.2.0) with default settings if not stated otherwise. For the protein identification and label-free quantification (LFQ), carbamidomethylation on cysteines was considered as fixed and methionine oxidation as variable modification. Additionally, asparagine deamidation was enabled for PNGase fractions. All peptides were used for protein quantification. Bioinformatic analysis was performed with Perseus software version 2.1.1.0. For this, identified and LFQ-quantified proteins were filtered to remove potential contaminants, reversed proteins and proteins only identified by site. Only proteins stably identified in both replicates of any condition were kept for further analysis. Missing values were imputed by normal distribution with a width of 0.3 and a down shift of 1.8. Gene ontology (GO) annotations for human proteins were added and pair-wise comparisons of the different culture media was performed. Proteins were considered as significantly altered if the p-value was below 0.05."],"omics_type":["Proteomics"],"labhead":["Lothar Jänsch"],"instrument_platform":[""],"labhead_affiliation":["Cellular Proteome Research, Helmholtz-Centre for Infection Research, Braunschweig, Germany"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"publication":["41964059 Schinke M, Gensch I, Rietschel T, Nguyen AHH, Slevogt H, Jänsch L, Lachmann N. Defined culture conditions improve functional properties of mature iPSC-derived macrophages for therapeutic screening. Stem Cell Res Ther. 2026 17(1):134 10.1186/s13287-026-05009-1"],"submitter_mail":["lothar.jaensch@helmholtz-hzi.de"],"submitter_affiliation":["HZI"],"submitter_country":["Germany"],"pubmed_abstract":["<h4>Background</h4>Generation and use of human macrophages in vitro are essential for a variety of (therapeutic) applications. Recently, there has been growing interest in macrophages derived from induced pluripotent stem cells (iPSC-Mac), which has led to the development of numerous differentiation protocols. These protocols typically involve a stepwise differentiation process using well-defined culture media, though the final differentiation stage often relies on undefined animal serum components, such as fetal bovine serum (FBS). This study aimed to elucidate protocol-dependent effects on iPSC-Mac usability.<h4>Methods</h4>We assessed the impact of a serum-supplemented medium (RPMI + FBS) compared to a fully defined medium (X-VIVO 15™) on the function of terminally differentiated human iPSC-Mac. Upon harvest, the cells were differentiated for three to four days in the respective media. Subsequently, phenotypic analysis was performed by microscopy and flow cytometry, as well as transcriptome and proteome analysis. Functional responsiveness to stimuli such as polarizing cytokines and lipopolysaccharide (LPS) was evaluated using flow cytometry and ELISA assay. The pharmaceutical screening potential was examined using secondary stimuli with dexamethasone.<h4>Results</h4>While both media compositions effectively produced fully functional human macrophages, we observed significant differences in their stage of activation and their functional responsiveness. Macrophages differentiated under the defined, serum-free conditions showed a more neutral activation state, an enhanced cholesterol metabolism, and were more sensitive to post pro- or anti-inflammatory stimulation compared to serum-grown cells. Most important, iPSC-Mac from these cultures also exhibit a more robust and reproducible response to co-stimulatory signals with IFNγ or LPS in combination with the immune-suppressive agent dexamethasone. In contrast, serum-grown iPSC-Mac demonstrated a more pre-activated state, with noteworthy background levels of interleukin 6 (IL-6). Upon stimulation, these cells showed reduced sensitivity and responsiveness towards secondary signals, underscoring potential challenges when using FBS-based media for drug screening or immunomodulatory assessments.<h4>Conclusions</h4>The enhanced responsiveness to co-stimulatory signals makes iPSC-Mac from defined cultures more suitable for testing immunomodulatory drugs, novel bio-assays and in vivo applications."],"pubmed_title":["Defined culture conditions improve functional properties of mature iPSC-derived macrophages for therapeutic screening."],"pubmed_authors":["Schinke Maximilian M, Gensch Ingrid I, Rietschel Tina T, Nguyen Ariane Hai Ha AHH, Slevogt Hortense H, Jänsch Lothar L, Lachmann Nico N"],"additional_accession":[]},"is_claimable":false,"name":"Generation of human iPSC-derived macrophages under animal-free conditions enables their application for safety and efficacy testing","description":"Generation and use of human macrophages in vitro are essential for a variety of (therapeutic) applications. Recently, there has been growing interest in macrophages derived from induced pluripotent stem cells (iPSC-Mac), which has led to the development of numerous differentiation protocols. These protocols typically involve a stepwise differentiation process using well-defined culture media, though the final differentiation stage often relies on undefined animal serum components, such as fetal bovine serum (FBS). In this study, we assessed the impact of a serum-containing medium in comparison to a fully defined medium (X-VIVO 15TM) on human iPSC-Mac function. While both media compositions effectively produced fully functional human macrophages, we observed significant differences in their stage of activation and their functional responsiveness. Macrophages differentiated under the defined, serum-free conditions showed a more neutral activation state, an enhanced cholesterol metabolism, and were more sensitive to post pro- or anti-inflammatory stimulation compared to serum-grown cells. Most important, iPSC-Mac from these cultures also exhibit a more robust and reproducible response to co-stimulatory signals with IFNγ or lipopolysaccharide (LPS) in combination with the immune-suppressive agent dexamethasone. In contrast, serum-grown iPSC-Mac demonstrated a more pre-activated state, with noteworthy background levels of interleukin 6 (IL-6). Upon stimulation, these cells showed reduced sensitivity and responsiveness towards secondary signals, underscoring potential challenges when using FBS-based media for drug screening or immunomodulatory assessments. Given the enhanced responsiveness to co-stimulatory signals makes iPSC-Mac from defined-cultures more suitable for testing immunomodulatory drugs, novel bio-assays and in vivo applications.","dates":{"publication":"2026-04-27","submission":"2025-11-19"},"accession":"PXD070961","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:2042546","NEWT:259447","NEWT:295546","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:1073882","NEWT:6819","NEWT:475174","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:135622","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:1584","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:7719","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:44685","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:2065263","NEWT:177437","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:485","NEWT:56636","NEWT:2499525","NEWT:2853422","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:1502","NEWT:128017","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1579","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:108458","NEWT:5936","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:5911","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:906914","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:548","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:2126348","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:2903","NEWT:185579","NEWT:13076","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:550","NEWT:33941","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:9995","NEWT:575412","NEWT:28112","NEWT:49012","NEWT:6493","NEWT:10832","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425","NEWT:104105","NEWT:216257","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:5147","NEWT:28108","NEWT:28104","NEWT:407821","NEWT:568703","NCBITaxon:2","NEWT:568708","NEWT:1613","NEWT:986","NEWT:52641","NEWT:28532","NEWT:353152","NEWT:298176","NEWT:353153","NEWT:227377","NEWT:1774284","NEWT:40674","NEWT:1194669","NEWT:74426","NEWT:443906","NEWT:13037","NEWT:519","NEWT:2510939","NEWT:7394","NEWT:6063","NEWT:1328388","NEWT:105487","NEWT:1548728","NEWT:667127","NEWT:9557","NEWT:377586","NEWT:300641","NEWT:39655","NEWT:9554"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