Abstract Background The secretome of mesenchymal stromal cells isolated from the amniotic membrane (hAMSCs) has been extensively studied for its in vitro immunomodulatory activity as well as for the treatment of several preclinical models of immune-related disorders. The bioactive molecules within the hAMSCs secretome are capable of modulating the immune response and thus contribute to stimulating regenerative processes. At present, only a few studies have attempted to define the composition of the secretome, and several approaches, including multi-omics, are underway in an attempt to precisely define its composition and possibly identify key factors responsible for the therapeutic effect. Methods In this study, we characterized the protein composition of the hAMSCs secretome by a filter-aided sample preparation (FASP) digestion and liquid chromatography-high resolution mass spectrometry (LC–MS) approach. Data were processed for gene ontology classification and functional protein interaction analysis by bioinformatics tools. Results Proteomic analysis of the hAMSCs secretome resulted in the identification of 1521 total proteins, including 662 unique elements. A number of 157 elements, corresponding to 23.7%, were found as repeatedly characterizing the hAMSCs secretome, and those that resulted as significantly over-represented were involved in immunomodulation, hemostasis, development and remodeling of the extracellular matrix molecular pathways. Conclusions Overall, our characterization enriches the landscape of hAMSCs with new information that could enable a better understanding of the mechanisms of action underlying the therapeutic efficacy of the hAMSCs secretome while also providing a basis for its therapeutic translation. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-023-03557-4. Keywords: Proteomics; Secretome; Human amniotic mesenchymal stromal cells; Filter-aided sample preparation, Immunomodulation, Regenerative medicine Introduction Mesenchymal stromal cells isolated from the amniotic membrane (hAMSCs) of human term placenta possess potent immunomodulatory activity. Unlike other MSC, these cells do not require priming to exert their action on the cells of the immune system [[43]1]. Furthermore, hAMSCs are readily available from biological waste at the time of delivery without posing any risk to the donor, and a large number of cells can be obtained from human term placenta [[44]2]. It has been widely demonstrated that hAMSCs modulate the immune response mediated by both innate and adaptive immune cells. Indeed, hAMSCs inhibit the proliferation and differentiation of T lymphocytes toward inflammatory and cytotoxic subsets while promoting polarization toward regulatory T cells [[45]3–[46]5]. Furthermore, hAMSCs also modulate the polarization of monocytes to antigen presenting cells (mature dendritic cells and inflammatory macrophages) by inducing the acquisition of features that are typical of M2 immunoregulatory macrophages [[47]4–[48]6]. In addition, hAMSCs suppress the proliferation and differentiation of B lymphocytes to plasma cells [[49]7]. The in vitro immunomodulatory properties exhibited by hAMSCs are reflected in their therapeutic efficacy in various disease models characterized by impaired immune responses. In both in vitro and in vivo studies, the administration of conditioned medium collected from hAMSCs demonstrated comparable therapeutic efficacy to that of hAMSCs themselves [[50]1, [51]4, [52]8–[53]12]. These findings emphasize how the therapeutic actions of hAMSCs are primarily determined by their paracrine activity. The mechanisms through which hAMSCs regulate the immune response and enable other cells to facilitate tissue repair during pathological processes are only partially understood. The factors produced by hAMSCs that orchestrate their beneficial properties currently remain an intriguing subject of investigation. To date, the characterization of the hAMSCs secretome is limited to a few studies that employ various methodological approaches. For example, a study using Enzyme-Linked Immunosorbent Assays (ELISA) quantified 200 soluble cytokines, receptors, chemokines, growth factors, and inflammatory factors, and recently demonstrated that the hAMSCs secretome contains molecules associated with the remodeling and homeostasis of the extracellular matrix (ECM) as well as in immunomodulation [[54]13]. Another study, employing ELISA and quantitative PCR, demonstrated the presence of pro-angiogenic factors such as HGF, EGF, and bFGF in the hAMSCs secretome [[55]14]. In addition, a proteomic analysis using in-solution digestion label-free mass spectrometry revealed that the hAMSCs secretome contains proteins associated with wound healing [[56]15]. In particular, the hAMSCs secretome contained high levels of proteins related to angiogenesis, cellular differentiation, immune response, cell motility, and wound healing, such as collagen triple helix repeat-containing protein 1 (CTHRC1), lysyl oxidase homolog 2 (LOXL2), A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), galectin-1 (LGALS1), complement C3 (C3), and CCN family member 1 (CYR61). These proteins are known to promote keratinocyte migration and differentiation [[57]15]. However, a comprehensive characterization of the hAMSCs secretome, capable of providing insights into the vast array of molecules secreted by hAMSCs and potentially implicated in their therapeutic properties, is still lacking. Such characterization would advance our understanding of the molecular mechanisms and the factors contributing to or responsible for the immunomodulatory properties of the hAMSCs secretome. This knowledge could also contribute to the development of new, cell-free therapeutic strategies and the engineering of medical devices for MSC secretome-based treatments in regenerative medicine. In this study, we characterized the protein content of the hAMSCs secretome using a proteomic approach based on Filter-Aided Sample Preparation (FASP) digestion coupled with Liquid Chromatography-high resolution Mass Spectrometry (LC–MS) analysis. We identified, with high confidence, a protein pattern consisting of 157 elements that consistently characterized the hAMSCs secretome. These elements were further investigated for gene ontology analysis, pathway classification, and relative label-free quantification. Methods Isolation of mesenchymal stromal cells from human amniotic membrane (hAMSCs) Human term placentas (N = 19) were collected after obtaining written informed consent from mothers after vaginal delivery or cesarean section. The study was conducted in accordance with the Declaration of Helsinki, and informed consent was obtained following the guidelines defined by the Brescia Provincial Ethics Committee (number NP 2243, 19/01/2016). Amniotic membrane mesenchymal stromal cells (hAMSC) were isolated as previously described [[58]16]. Membrane fragments were digested at 37 °C in dispase (2.5 U/mL, VWR, Radnor, PA, USA) for 9 min and then transferred to RPMI 1640 complete medium containing 10% heat-inactivated fetal bovine serum (FBS), 1% P/S, and 1% L-glutamine (all from Sigma-Aldrich, St. Louis, MO, USA) to block digestion. The fragments were subsequently incubated in collagenase 0.94 mg/mL and DNase I (both from Roche, Basel, Switzerland) for 2.5–3 h at 37 °C. After centrifugation at low g, the resulting supernatant was filtered through a 100-μm cell strainer (BD Falcon, Bedford, MA, USA), and the cells were harvested by centrifugation. Freshly isolated cells were expanded to passage 1 (p1) by plating them at a density of 10^4 cells/cm^2 in Chang Medium C (Irvine Scientific, Santa Ana, CA, USA) supplemented with 2 mM L-glutamine at 37 °C in a 5% CO2 incubator. hAMSCs at p1 were phenotypically characterized as previously reported [[59]16]. The hAMSCs used in this study met the minimal criteria for consideration as MSCs, namely, the expression of MSC markers CD13 (97.7 ± 1.6%; mean ± SD), CD73 (88.3 ± 6.4%), and CD90 (94.8 ± 7.4%), and the lack of hematopoietic markers such as CD45 (1.8 ± 1.0%), CD66b (0%), and the epithelial marker CD324 (1.7 ± 1.0%) [[60]2, [61]17, [62]18]. Preparation of conditioned medium (CM), lyophilization and reconstitution hAMSC at p1 were cultured for 5 days in 24-well plates (Corning, NY, USA) at a density of 5 × 10^5 cells/well in 0.5 mL of DMEM-F12 medium (Sigma-Aldrich) without serum, supplemented with 2 mM L-glutamine (Sigma-Aldrich) and 1% P/S as previously described [[63]1]. After the incubation period, CM was collected, centrifuged at 300 × g, filtered through a 0.2-μm sterile filter (Sartorius Stedim, Florence, Italy) and stored at − 80 °C. The frozen CM was then lyophilized as previously described [[64]4] using a Lyophilizer Pilot MAX MX 8556 (Millrock Technology, USA), following the previously described procedure [[65]4]. This process involved freezing the samples at − 40 °C per 4 h and then at − 45 °C under vacuum. A first dry cycle, consisting of 7 steps at increasing temperatures was performed for 13 h under vacuum. Subsequently, a second dry cycle was performed under vacuum at 30 °C for one hour. Lyophilization was complete when the product reached 25 °C for at least 1 h. Prior to use, the lyophilized CM was reconstituted with 2.5 mL of sterile water and filtered through a 0.2-μm sterile filter (Sartorius Stedim, Florence, Italy). A total of 17 placentae were used to produce CM from hAMSC at p1. Each experiment was performed using a mix of CM derived from at least 3 different hAMSC donors, which were previously validated for their immunomodulatory activity, as reported in [[66]4]. Proteomic analysis Chemicals All organic solvents were of LC–MS grade. Iodoacetamide (IAA), D,L-dithiothreitol (DTT), ammonium bicarbonate (AMBIC), bovine serum albumin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water and formic acid (FA) were obtained from Merck (Darmstadt, Germany). Trypsin (Gold MS Grade) was supplied from Promega (Madison, WI, USA), and acetonitrile (ACN) was supplied from Merck (Darmstadt, Germany). Treatment of secretome samples and protein quantification Four different batches lyophilized hAMSCs secretome (Pools 1–4), with a total volume of 2500 μL each, and three DMEMF12 culture medium samples, used as reference controls (CTRL 1–3), were solubilized in 250 μL of LC–MS grade water to obtain a 10 × concentration. The samples were gently vortexed to facilitate resolubilization. Total protein content was measured using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA) with a UV–Vis spectrophotometer (8453 UV–Vis Supplies, Agilent Technologies, Waldbronn, Germany), using BSA as the protein of reference. FASP protein digestion protocol Filter-aided sample preparation (FASP) was employed using centrifugal Microcon filtration devices (Millipore) equipped with a 10 kDa molecular mass cut-off filter membrane for sample purification, concentration, and proteins digestion [[67]19, [68]20] for a MS-based proteomic analysis. The FASP method provides a higher number of identifications in comparison to in-solution digestion. This makes it a formidable choice for sample preparation in proteomic analysis, especially when dealing with secretomes characterized by a low or diluted protein content. FASP devices, equipped with a molecular cut-off membrane filter, allow efficient protein purification and enzymatic digestion through a series of steps involving buffer exchange, reagent addition, centrifugation, and ultimately protein concentration. This approach proves advantageous for the analysis of conditioned medium. A secretome volume corresponding to 50 µg of total protein content was mixed with 8 M urea in 0.1 M Tris/HCl buffer at pH 8.5 (Urea Buffer solution), transferred to the filter device, and centrifuged at 14,000 rpm for 15 min. The concentrated sample was diluted in the device with Urea Buffer solution and centrifuged once more. Subsequently, the supernatant was treated with 8 mM DTT in Urea Buffer solution (DTT solution) to reduce disulfide bridges. It was then incubated at 37 °C for 15 min and centrifuged again. Any excess DTT was eliminated through washings with Urea Buffer followed by centrifugations. The supernatant was then treated for thiols carboxamide methylation with 50 mM iodoacetamide (IAA) solution in Urea Buffer. The mixture was incubated in the dark at room temperature (RT) for 15 min, followed by centrifugation. Excess IAA was removed by incubating the sample with DTT solution at 37 °C for 15 min, followed by washes in Urea Buffer solution and then in ammonium bicarbonate for buffer exchange. Sample digestion was carried out overnight at 37 °C using trypsin 1 µg/µL in 1:100 (w/w) in ammonium bicarbonate buffer 50 mM. Enzymatic digestion was stopped by the addition of 1% FA (final concentration). The proteolytic peptides were collected by centrifugation, lyophilized, and then dissolved in 0.1% FA water solution (v/v) for LC–MS analysis. Ultra-high-performance liquid chromatography-nanoESI mass spectrometry analysis (UHPLC-ESI–MS/MS) UHPLC-ESI–MS/MS analyses were performed for each sample in triplicate on UltiMate 3000 RSLCnano System coupled to Orbitrap Elite MS detector with EASY-Spray nanoESI source (Thermo Fisher Scientific, Waltham, MA, USA). Instrumental operation and data acquisition were performed using Thermo Xcalibur 2.2 computer program (Thermo Fisher Scientific). Each sample was analyzed in replicate chromatographic runs (n = 3) to ensure data repeatability, robustness, and the proper operation of the instruments. The proteomic analysis of different batches of the hAMSCs secretome, each with replicate (n = 3) LC–MS analyses, along with the parameters applied to software data elaboration and filtering, ensured the repeatability and robustness of protein and peptide identification data. Chromatographic separations were performed on a PepMap C18 (2 µm particles, 100 Å pore size) EASY-Spray column with a length of 15 cm and an internal diameter (ID) of 50 µm (Thermo Fisher Scientific). This column was coupled to an Acclaim PepMap100 nano-trap cartridge (C18, 5 µm, 100 Å, 300 µm, ID × 5 mm) (Thermo Fisher Scientific). The separation was performed at 40 °C using gradient elution. Eluent A consisted of 0.1% FA, while eluent B was an ACN/FA solution (99.9:0.1, v/v). The gradient elution protocol was as follows: (i) 5% B for 7 min, (ii) 5% to 35% B for 113 min, (iii) 35% B to 99% for 2 min, (iv) 99% B for 3 min, (v) 99% to 1.6% B for 2 min, (vi) 1.6% B for 3 min, (vii) 1.6% to 78% B for 3 min, (viii) 78% B for 3 min, (ix) 78% to 1.6% B for 3 min, (x) 1.6% B for 3 min, (xi) 1.6% to 78% B for 3 min, (xii) 78% B for 3 min, (xiii) 78% B to 5% B for 2 min, and (xiv) 5% B for 20 min. The mobile phase flow rate was 0.3 µL/min. The injection volume was 5 µL. The Orbitrap Elite instrument operated in positive ionization mode with a 60,000 full scan resolution, in 350–2000 m/z acquisition range. MS/MS fragmentation was obtained using collision-induced dissociation (CID) with a normalized collision energy of 35%. The instrument used a Data-Dependent Scan (DDS) mode to perform MS/MS on the 20 most intense signals from each MS spectrum. The minimum signal threshold was set to 500.0, and an isolation width of 2 m/z was applied, with a default charge state to + 2. MS/MS spectra acquisition was performed in the linear ion trap at a normal scan rate. Data analysis LC–MS and MS/MS raw data were processed using two software tools: the HPLC–MS apparatus management software (Xcalibur 2.0.7 SP1, Thermo Fisher Scientific), and the Proteome Discoverer 1.4 software (version 1.4.1.14, Thermo Fisher Scientific) that was used for protein identification based on the SEQUEST HT cluster as search engine against the Homo Sapiens (UniProtKB/Swiss-Prot protein knowledgebase released in 2021_4), and Bos taurus (UniProtKB/Swiss-Prot protein knowledgebase released in 2022_02). The signal to Noise (S/N) threshold was set to 1.5. Trypsin was used for cleavage with a maximum of 2 missed cleavage sites, and the minimum and maximum peptide length were set to 6 and 144 residues, respectively. Tolerance settings for the analysis included a mass tolerance 10 ppm, fragment mass tolerance of 0.5 Da and 0.02 Da; both “use average precursor mass” and “use average fragment mass” were set to “False”. Methionine oxidation (+ 15.99 Da) and N-Terminal acetylation (+ 42.011 Da) were set as dynamic modifications, while carbamidomethylation of cysteine (+ 57.02 Da) was set as a static modification. Validation of protein and peptide identifications was performed through a decoy database search and calculation of the False Discovery Rate (FDR) statistical value using the Percolator node in Proteome Discoverer workflow. We set strict and relaxed FDR target values at 0.01 and 0.05, respectively. The FDR measures the confidence in the identification by estimating the number of false positive identifications among all the identifications found by a peptide identification search. The protein identification results from each secretome pool (Pools 1–4) were analyzed as a multireport data file, combining data from three analytical replicates (runs 1–3). The data were filtered to ensure high confidence peptide identification, requiring a minimum of at least 2 peptides per protein, each with a minimum peptide length of 9 amino acids and peptide rank 1, according to the Human Proteome Project (HPP) Mass Spectrometry Data Interpretation Guidelines [[69]21]. Gene ontology (GO) analysis and classification were conducted using Reactome ([70]https://reactome.org) [[71]22] and Protein Analysis Through Evolutionary Relationships (PANTHER, [72]http://www.pantherdb.org) by applying the Fisher’s Exact test type with false discovery rate (FDR) correction for statistical test of over-representation [[73]23]. The protein expression data were sourced from The Human Protein Atlas ([74]https://www.proteinatlas.org) [[75]24, [76]25]. Functional protein interaction networks were analyzed using the STRING tool [[77]26] with the highest confidence level (0.900), and Cytoscape ([78]https://cytoscape.org/). Sample data grouping analysis was performed using the Venn diagram tool ([79]https://bioinfogp.cnb.csic.es/tools/venny). Results The proteomic profiles of four different hAMSCs-CM samples obtained from serum-free cell cultures were characterized using LC–MS analysis, after Filter-Aided Sample Preparation (FASP) digestion with trypsin. To identify common protein elements in comparison to a cell-free DMEMF12 culture medium used as a control, the data obtained for each pool were subjected to grouping analysis. The resulting list of proteins, which consistently characterized the hAMSCs secretome, and could thus be associated with its biological activities, was obtained by Proteome Discoverer data elaboration after validation by the Percolator node and FDR statistical value calculation and filtered for high confidence identification according to the Human Proteome Project Mass Spectrometry Data Interpretation Guidelines [[80]21]. Bioinformatics tools were applied to investigate the gene ontology classification, pathway over-representation, and protein functional interactions of the identified proteins. Proteomic characterization of the hAMSCs secretome The total protein content of each hAMSC-CM pool was determined using the Bradford assay. The mean value for the 4 pools was 0.49 ± 0.23 mg/ml. This measurement was obtained after re-dissolving the lyophilized pools to a 10 × concentration in water. For proteomic analysis, an aliquot of the each pool, equivalent to 50 μg of total protein, underwent FASP digestion and LC–MS analysis in triplicate runs for proteomic characterization. The Proteome Discoverer software processed the LC–MS data and identified a total of 1521 proteins in the hAMSCs secretome with a high level of confidence. These proteins were distributed differently among the analyzed pools, with 355, 436, 242, and 488 proteins identified in pools 1 through 4, respectively (protein identification data in Additional file [81]1: Table S1). Grouping analysis revealed a total of 662 unique elements with 157 proteins shared by all the hAMSCs-CM pools (Fig. [82]1 and Table [83]1). These 157 proteins, corresponding to 23.7%, of the total unique elements, characterize the hAMSCs secretome and thus may be associated to the biological activities exerted by hAMSCs-CM. Fig. 1. Fig. 1 [84]Open in a new tab Venn diagram resulting from grouping analysis of the proteins identified in the hAMSCs-CM pools 1–4 (list of identifications per pool in Additional file [85]1: Table S1) Table 1. List of the 157 proteins elements commonly identified in hAMSCs secretome pools 1–4 Uniprot accession Protein name Gene name MW [kDa] [86]P62328 Thymosin beta-4 TYB4 5,0 [87]P02795 Metallothionein-2 MT2 6,0 [88]P05387 60S acidic ribosomal protein P2 RLA2 11,7 [89]P99999 Cytochrome c CYC 11,7 [90]O75368 SH3 domain-binding glutamic acid-rich-like protein SH3L1 12,8 [91]P09382 Galectin-1 LEG1 14,7 [92]P0DP25 Calmodulin-3 CALM3 16,8 [93]P60660 Myosin light polypeptide 6 MYL6 16,9 [94]P15531 Nucleoside diphosphate kinase A NDKA 17,1 [95]P62937 Peptidyl-prolyl cis–trans isomerase A PPIA 18,0 [96]P19105 Myosin regulatory light chain 12A ML12A 19,8 [97]Q99497 Parkinson disease protein 7 PARK7 19,9 [98]P21291 Cysteine and glycine-rich protein 1 CSRP1 20,6 [99]P55145 Mesencephalic astrocyte-derived neurotrophic factor MANF 20,7 [100]Q06830 Peroxiredoxin-1 PRDX1 22,1 [101]P37802 Transgelin-2 TAGL2 22,4 [102]Q01995 Transgelin TAGL 22,6 [103]P80723 Brain acid soluble protein 1 BASP1 22,7 [104]P01033 Metalloproteinase inhibitor 1 TIMP1 23,2 [105]P09211 Glutathione S-transferase P GSTP1 23,3 [106]P23284 Peptidyl-prolyl cis–trans isomerase B PPIB 23,7 [107]P16035 Metalloproteinase inhibitor 2 TIMP2 24,4 [108]P09936 Ubiquitin carboxyl-terminal hydrolase isozyme L1 UCHL1 24,8 [109]P62906 60S ribosomal protein L10a RL10A 24,8 [110]P20618 Proteasome subunit beta type-1 PSB1 26,5 [111]P60174 Triosephosphate isomerase TPIS 26,7 [112]P48307 Tissue factor pathway inhibitor 2 TFPI2 26,9 [113]P78417 Glutathione S-transferase omega-1 GSTO1 27,5 [114]P52823 Stanniocalcin-1 STC1 27,6 [115]P63104 14–3-3 protein zeta/delta 1433Z 27,7 [116]P61981 14–3-3 protein gamma 1433G 28,3 [117]P25788 Proteasome subunit alpha type-3 PSA3 28,4 [118]P67936 Tropomyosin alpha-4 chain TPM4 28,5 [119]Q16270 Insulin-like growth factor-binding protein 7 IBP7 29,1 [120]P62258 14–3-3 protein epsilon 1433E 29,2 [121]P28070 Proteasome subunit beta type-4 PSB4 29,2 [122]O00584 Ribonuclease T2 RNT2 29,5 [123]P25786 Proteasome subunit alpha type-1 PSA1 29,5 [124]P47756 F-actin-capping protein subunit beta CAPZB 31,3 [125]P29966 Myristoylated alanine-rich C-kinase substrate MARCS 31,5 [126]P17936 Insulin-like growth factor-binding protein 3 IBP3 31,7 [127]P00491 Purine nucleoside phosphorylase PNPH 32,1 [128]P09486 SPARC SPRC 34,6 [129]Q12841 Follistatin-related protein 1 FSTL1 35,0 [130]P40926 Malate dehydrogenase, mitochondrial MDHM 35,5 [131]P08758 Annexin A5 ANXA5 35,9 [132]P04406 Glyceraldehyde-3-phosphate dehydrogenase G3P 36,0 [133]P40925 Malate dehydrogenase, cytoplasmic MDHC 36,4 [134]P07195 L-lactate dehydrogenase B chain LDHB 36,6 [135]P00338 L-lactate dehydrogenase A chain LDHA 36,7 [136]O43852 Calumenin CALU 37,1 [137]P37837 Transaldolase TALDO 37,5 [138]P07858 Cathepsin B CATB 37,8 [139]Q9UBP4 Dickkopf-related protein 3 DKK3 38,4 [140]P07355 Annexin A2 ANXA2 38,6 [141]P04083 Annexin A1 ANXA1 38,7 [142]Q15293 Reticulocalbin-1 RCN1 38,9 [143]P04075 Fructose-bisphosphate aldolase A ALDOA 39,4 [144]P60709 Actin, cytoplasmic 1 ACTB 41,7 [145]P36222 Chitinase-3-like protein 1 CH3L1 42,6 [146]P07093 Glia-derived nexin GDN 44,0 [147]P08727 Keratin, type I cytoskeletal 19 K1C19 44,1 [148]P07339 Cathepsin D CATD 44,5 [149]P00558 Phosphoglycerate kinase 1 PGK1 44,6 [150]P05121 Plasminogen activator inhibitor 1 PAI1 45,0 [151]P50454 Serpin H1 SERPH 46,4 [152]P06733 Alpha-enolase ENOA 47,1 [153]Q8NBS9 Thioredoxin domain-containing protein 5 TXND5 47,6 [154]Q15113 Procollagen C-endopeptidase enhancer 1 PCOC1 47,9 [155]Q15084 Protein disulfide-isomerase A6 PDIA6 48,1 [156]P27797 Calreticulin CALR 48,1 [157]O95967 EGF-containing fibulin-like extracellular matrix protein 2 FBLN4 49,4 [158]P13489 Ribonuclease inhibitor RINI 49,9 [159]P26641 Elongation factor 1-gamma EF1G 50,1 [160]P68363 Tubulin alpha-1B chain TBA1B 50,1 [161]P31150 Rab GDP dissociation inhibitor alpha GDIA 50,6 [162]P50395 Rab GDP dissociation inhibitor beta GDIB 50,6 [163]Q01518 Adenylyl cyclase-associated protein 1 CAP1 51,9 [164]P05787 Keratin, type II cytoskeletal 8 K2C8 53,7 [165]P03956 Interstitial collagenase MMP1 54,0 [166]P09238 Stromelysin-2 MMP10 54,1 [167]P30101 Protein disulfide-isomerase A3 PDIA3 56,7 [168]Q16851 UTP–glucose-1-phosphate uridylyltransferase UGPA 56,9 [169]P07237 Protein disulfide-isomerase PDIA1 57,1 [170]P12081 Histidine–tRNA ligase, cytoplasmic HARS1 57,4 [171]P14618 Pyruvate kinase PKM KPYM 57,9 [172]P07602 Prosaposin SAP 58,1 [173]P13645 Keratin, type I cytoskeletal 10 K1C10 58,8 [174]P14314 Glucosidase 2 subunit beta GLU2B 59,4 [175]P35527 Keratin, type I cytoskeletal 9 K1C9 62,0 [176]P06744 Glucose-6-phosphate isomerase G6PI 63,1 [177]P14866 Heterogeneous nuclear ribonucleoprotein L HNRPL 64,1 [178]Q08380 Galectin-3-binding protein LG3BP 65,3 [179]P35908 Keratin, type II cytoskeletal 2 epidermal K22E 65,4 [180]P04264 Keratin, type II cytoskeletal 1 K2C1 66,0 [181]P29401 Transketolase TKT 67,8 [182]P02768 Albumin ALBU 69,3 [183]P0DMV8 Heat shock 70 kDa protein 1A HS71A 70,0 [184]P13797 Plastin-3 PLST 70,8 [185]P11142 Heat shock cognate 71 kDa protein HSP7C 70,9 [186]Q16881 Thioredoxin reductase 1, cytoplasmic TRXR1 70,9 [187]P11021 Endoplasmic reticulum chaperone BiP BIP 72,3 [188]P13667 Protein disulfide-isomerase A4 PDIA4 72,9 [189]P08253 72 kDa type IV collagenase MMP2 73,8 [190]P02545 Prelamin-A/C LMNA 74,1 [191]Q15582 Transforming growth factor-beta-induced protein ig-h3 BGH3 74,6 [192]P09871 Complement C1s subcomponent C1S 76,6 [193]P02787 Serotransferrin TRFE 77,0 [194]P00736 Complement C1r subcomponent C1R 80,1 [195]O00391 Sulfhydryl oxidase 1 QSOX1 82,5 [196]P08238 Heat shock protein HSP 90-beta HS90B 83,2 [197]O00469 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 PLOD2 84,6 [198]P06396 Gelsolin GELS 85,6 [199]Q9Y4K0 Lysyl oxidase homolog 2 LOXL2 86,7 [200]P05556 Integrin beta-1 ITB1 88,4 [201]P55072 Transitional endoplasmic reticulum ATPase TERA 89,3 [202]P14625 Endoplasmin ENPL 92,4 [203]Q15063 Periostin POSTN 93,3 [204]P13639 Elongation factor 2 EF2 95,3 [205]Q14764 Major vault protein MVP 99,3 [206]P12814 Alpha-actinin-1 ACTN1 103,0 [207]P55786 Puromycin-sensitive aminopeptidase PSA 103,2 [208]O43707 Alpha-actinin-4 ACTN4 104,8 [209]P10253 Lysosomal alpha-glucosidase LYAG 105,3 [210]Q9Y6C2 EMILIN-1 EMIL1 106,6 [211]P12109 Collagen alpha-1(VI) chain CO6A1 108,5 [212]P15144 Aminopeptidase N AMPN 109,5 [213]P27816 Microtubule-associated protein 4 MAP4 120,9 [214]P18206 Vinculin VINC 123,7 [215]P08123 Collagen alpha-2(I) chain CO1A2 129,2 [216]P07996 Thrombospondin-1 TSP1 129,3 [217]P35442 Thrombospondin-2 TSP2 129,9 [218]P02461 Collagen alpha-1(III) chain CO3A1 138,5 [219]P02452 Collagen alpha-1(I) chain CO1A1 138,9 [220]Q14112 Nidogen-2 NID2 151,2 [221]P08572 Collagen alpha-2(IV) chain CO4A2 167,4 [222]P11047 Laminin subunit gamma-1 LAMC1 177,5 [223]Q13219 Pappalysin-1 PAPP1 180,9 [224]P20908 Collagen alpha-1(V) chain CO5A1 183,4 [225]Q14766 Latent-transforming growth factor beta-binding protein 1 LTBP1 186,7 [226]P01024 Complement C3 CO3 187,0 [227]P46940 Ras GTPase-activating-like protein IQGAP1 IQGA1 189,1 [228]Q14767 Latent-transforming growth factor beta-binding protein 2 LTBP2 194,9 [229]P07942 Laminin subunit beta-1 LAMB1 197,9 [230]P35579 Myosin-9 MYH9 226,4 [231]Q9Y490 Talin-1 TLN1 269,6 [232]P02751 Fibronectin FINC 272,2 [233]O75369 Filamin-B FLNB 278,0 [234]P21333 Filamin-A FLNA 280,6 [235]Q13813 Spectrin alpha chain, non-erythrocytic 1 SPTN1 284,4 [236]Q14315 Filamin-C FLNC 290,8 [237]P35555 Fibrillin-1 FBN1 312,1 [238]Q99715 Collagen alpha-1(XII) chain COCA1 332,9 [239]P12111 Collagen alpha-3(VI) chain CO6A3 343,5 [240]P13611 Versican core protein CSPG2 372,6 [241]P98160 Basement membrane-specific heparan sulfate proteoglycan core protein PGBM 468,5 [242]Q07954 Prolow-density lipoprotein receptor-related protein 1 LRP1 504,3 [243]P62328 Thymosin beta-4 TYB4 5,0 [244]P02795 Metallothionein-2 MT2 6,0 [245]P05387 60S acidic ribosomal protein P2 RLA2 11,7 [246]P99999 Cytochrome c CYC 11,7 [247]O75368 SH3 domain-binding glutamic acid-rich-like protein SH3L1 12,8 [248]P09382 Galectin-1 LEG1 14,7 [249]P0DP25 Calmodulin-3 CALM3 16,8 [250]P60660 Myosin light polypeptide 6 MYL6 16,9 [251]P15531 Nucleoside diphosphate kinase A NDKA 17,1 [252]P62937 Peptidyl-prolyl cis–trans isomerase A PPIA 18,0 [253]P19105 Myosin regulatory light chain 12A ML12A 19,8 [254]Q99497 Parkinson disease protein 7 PARK7 19,9 [255]P21291 Cysteine and glycine-rich protein 1 CSRP1 20,6 [256]P55145 Mesencephalic astrocyte-derived neurotrophic factor MANF 20,7 [257]Q06830 Peroxiredoxin-1 PRDX1 22,1 [258]P37802 Transgelin-2 TAGL2 22,4 [259]Q01995 Transgelin TAGL 22,6 [260]P80723 Brain acid soluble protein 1 BASP1 22,7 [261]P01033 Metalloproteinase inhibitor 1 TIMP1 23,2 [262]P09211 Glutathione S-transferase P GSTP1 23,3 [263]P23284 Peptidyl-prolyl cis–trans isomerase B PPIB 23,7 [264]P16035 Metalloproteinase inhibitor 2 TIMP2 24,4 [265]P09936 Ubiquitin carboxyl-terminal hydrolase isozyme L1 UCHL1 24,8 [266]P62906 60S ribosomal protein L10a RL10A 24,8 [267]P20618 Proteasome subunit beta type-1 PSB1 26,5 [268]P60174 Triosephosphate isomerase TPIS 26,7 [269]P48307 Tissue factor pathway inhibitor 2 TFPI2 26,9 [270]P78417 Glutathione S-transferase omega-1 GSTO1 27,5 [271]P52823 Stanniocalcin-1 STC1 27,6 [272]P63104 14–3-3 protein zeta/delta 1433Z 27,7 [273]P61981 14–3-3 protein gamma 1433G 28,3 [274]P25788 Proteasome subunit alpha type-3 PSA3 28,4 [275]P67936 Tropomyosin alpha-4 chain TPM4 28,5 [276]Q16270 Insulin-like growth factor-binding protein 7 IBP7 29,1 [277]P62258 14–3-3 protein epsilon 1433E 29,2 [278]P28070 Proteasome subunit beta type-4 PSB4 29,2 [279]O00584 Ribonuclease T2 RNT2 29,5 [280]P25786 Proteasome subunit alpha type-1 PSA1 29,5 [281]P47756 F-actin-capping protein subunit beta CAPZB 31,3 [282]P29966 Myristoylated alanine-rich C-kinase substrate MARCS 31,5 [283]P17936 Insulin-like growth factor-binding protein 3 IBP3 31,7 [284]P00491 Purine nucleoside phosphorylase PNPH 32,1 [285]P09486 SPARC SPRC 34,6 [286]Q12841 Follistatin-related protein 1 FSTL1 35,0 [287]P40926 Malate dehydrogenase, mitochondrial MDHM 35,5 [288]P08758 Annexin A5 ANXA5 35,9 [289]P04406 Glyceraldehyde-3-phosphate dehydrogenase G3P 36,0 [290]P40925 Malate dehydrogenase, cytoplasmic MDHC 36,4 [291]P07195 L-lactate dehydrogenase B chain LDHB 36,6 [292]P00338 L-lactate dehydrogenase A chain LDHA 36,7 [293]O43852 Calumenin CALU 37,1 [294]P37837 Transaldolase TALDO 37,5 [295]P07858 Cathepsin B CATB 37,8 [296]Q9UBP4 Dickkopf-related protein 3 DKK3 38,4 [297]P07355 Annexin A2 ANXA2 38,6 [298]P04083 Annexin A1 ANXA1 38,7 [299]Q15293 Reticulocalbin-1 RCN1 38,9 [300]P04075 Fructose-bisphosphate aldolase A ALDOA 39,4 [301]P60709 Actin, cytoplasmic 1 ACTB 41,7 [302]P36222 Chitinase-3-like protein 1 CH3L1 42,6 [303]P07093 Glia-derived nexin GDN 44,0 [304]P08727 Keratin, type I cytoskeletal 19 K1C19 44,1 [305]P07339 Cathepsin D CATD 44,5 [306]P00558 Phosphoglycerate kinase 1 PGK1 44,6 [307]P05121 Plasminogen activator inhibitor 1 PAI1 45,0 [308]P50454 Serpin H1 SERPH 46,4 [309]P06733 Alpha-enolase ENOA 47,1 [310]Q8NBS9 Thioredoxin domain-containing protein 5 TXND5 47,6 [311]Q15113 Procollagen C-endopeptidase enhancer 1 PCOC1 47,9 [312]Q15084 Protein disulfide-isomerase A6 PDIA6 48,1 [313]P27797 Calreticulin CALR 48,1 [314]O95967 EGF-containing fibulin-like extracellular matrix protein 2 FBLN4 49,4 [315]P13489 Ribonuclease inhibitor RINI 49,9 [316]P26641 Elongation factor 1-gamma EF1G 50,1 [317]P68363 Tubulin alpha-1B chain TBA1B 50,1 [318]P31150 Rab GDP dissociation inhibitor alpha GDIA 50,6 [319]P50395 Rab GDP dissociation inhibitor beta GDIB 50,6 [320]Q01518 Adenylyl cyclase-associated protein 1 CAP1 51,9 [321]P05787 Keratin, type II cytoskeletal 8 K2C8 53,7 [322]P03956 Interstitial collagenase MMP1 54,0 [323]P09238 Stromelysin-2 MMP10 54,1 [324]P30101 Protein disulfide-isomerase A3 PDIA3 56,7 [325]Q16851 UTP–glucose-1-phosphate uridylyltransferase UGPA 56,9 [326]P07237 Protein disulfide-isomerase PDIA1 57,1 [327]P12081 Histidine–tRNA ligase, cytoplasmic HARS1 57,4 [328]P14618 Pyruvate kinase PKM KPYM 57,9 [329]P07602 Prosaposin SAP 58,1 [330]P13645 Keratin, type I cytoskeletal 10 K1C10 58,8 [331]P14314 Glucosidase 2 subunit beta GLU2B 59,4 [332]P35527 Keratin, type I cytoskeletal 9 K1C9 62,0 [333]P06744 Glucose-6-phosphate isomerase G6PI 63,1 [334]P14866 Heterogeneous nuclear ribonucleoprotein L HNRPL 64,1 [335]Q08380 Galectin-3-binding protein LG3BP 65,3 [336]P35908 Keratin, type II cytoskeletal 2 epidermal K22E 65,4 [337]P04264 Keratin, type II cytoskeletal 1 K2C1 66,0 [338]P29401 Transketolase TKT 67,8 [339]P02768 Albumin ALBU 69,3 [340]P0DMV8 Heat shock 70 kDa protein 1A HS71A 70,0 [341]P13797 Plastin-3 PLST 70,8 [342]P11142 Heat shock cognate 71 kDa protein HSP7C 70,9 [343]Q16881 Thioredoxin reductase 1, cytoplasmic TRXR1 70,9 [344]P11021 Endoplasmic reticulum chaperone BiP BIP 72,3 [345]P13667 Protein disulfide-isomerase A4 PDIA4 72,9 [346]P08253 72 kDa type IV collagenase MMP2 73,8 [347]P02545 Prelamin-A/C LMNA 74,1 [348]Q15582 Transforming growth factor-beta-induced protein ig-h3 BGH3 74,6 [349]P09871 Complement C1s subcomponent C1S 76,6 [350]P02787 Serotransferrin TRFE 77,0 [351]P00736 Complement C1r subcomponent C1R 80,1 [352]O00391 Sulfhydryl oxidase 1 QSOX1 82,5 [353]P08238 Heat shock protein HSP 90-beta HS90B 83,2 [354]O00469 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 PLOD2 84,6 [355]P06396 Gelsolin GELS 85,6 [356]Q9Y4K0 Lysyl oxidase homolog 2 LOXL2 86,7 [357]P05556 Integrin beta-1 ITB1 88,4 [358]P55072 Transitional endoplasmic reticulum ATPase TERA 89,3 [359]P14625 Endoplasmin ENPL 92,4 [360]Q15063 Periostin POSTN 93,3 [361]P13639 Elongation factor 2 EF2 95,3 [362]Q14764 Major vault protein MVP 99,3 [363]P12814 Alpha-actinin-1 ACTN1 103,0 [364]P55786 Puromycin-sensitive aminopeptidase PSA 103,2 [365]O43707 Alpha-actinin-4 ACTN4 104,8 [366]P10253 Lysosomal alpha-glucosidase LYAG 105,3 [367]Q9Y6C2 EMILIN-1 EMIL1 106,6 [368]P12109 Collagen alpha-1(VI) chain CO6A1 108,5 [369]P15144 Aminopeptidase N AMPN 109,5 [370]P27816 Microtubule-associated protein 4 MAP4 120,9 [371]P18206 Vinculin VINC 123,7 [372]P08123 Collagen alpha-2(I) chain CO1A2 129,2 [373]P07996 Thrombospondin-1 TSP1 129,3 [374]P35442 Thrombospondin-2 TSP2 129,9 [375]P02461 Collagen alpha-1(III) chain CO3A1 138,5 [376]P02452 Collagen alpha-1(I) chain CO1A1 138,9 [377]Q14112 Nidogen-2 NID2 151,2 [378]P08572 Collagen alpha-2(IV) chain CO4A2 167,4 [379]P11047 Laminin subunit gamma-1 LAMC1 177,5 [380]Q13219 Pappalysin-1 PAPP1 180,9 [381]P20908 Collagen alpha-1(V) chain CO5A1 183,4 [382]Q14766 Latent-transforming growth factor beta-binding protein 1 LTBP1 186,7 [383]P01024 Complement C3 CO3 187,0 [384]P46940 Ras GTPase-activating-like protein IQGAP1 IQGA1 189,1 [385]Q14767 Latent-transforming growth factor beta-binding protein 2 LTBP2 194,9 [386]P07942 Laminin subunit beta-1 LAMB1 197,9 [387]P35579 Myosin-9 MYH9 226,4 [388]Q9Y490 Talin-1 TLN1 269,6 [389]P02751 Fibronectin FINC 272,2 [390]O75369 Filamin-B FLNB 278,0 [391]P21333 Filamin-A FLNA 280,6 [392]Q13813 Spectrin alpha chain, non-erythrocytic 1 SPTN1 284,4 [393]Q14315 Filamin-C FLNC 290,8 [394]P35555 Fibrillin-1 FBN1 312,1 [395]Q99715 Collagen alpha-1(XII) chain COCA1 332,9 [396]P12111 Collagen alpha-3(VI) chain CO6A3 343,5 [397]P13611 Versican core protein CSPG2 372,6 [398]P98160 Basement membrane-specific heparan sulfate proteoglycan core protein PGBM 468,5 [399]Q07954 Prolow-density lipoprotein receptor-related protein 1 LRP1 504,3 [400]Open in a new tab As shown in Table [401]1, the 157 proteins shared by all hAMSCs-CM pools exhibited a wide range of molecular masses, spanning from 5 to 504 kDa. Interestingly, despite the cut-off of the FASP membrane filter, thymosin beta 4 and metallothionein 2, both with molecular masses < 10 kDa, were identified. One possible explanation could be the existence of protein complexes or dimeric peptides in hAMSCs-CM. Such complexes or dimers could account for their retention by the filter. Indeed, several reports have demonstrated the presence of actin-profilin complexes involving thymosin beta 4, the main G-actin sequestering agent, as well as the oligomerization of metallothioneins [[402]27, [403]28], which could support our hypothesis. The same proteomic analysis was applied to three different samples of cell free DMEMF12 culture medium, used as reference control samples (CTRL 1–3), in order to evaluate the influence of the medium’s composition on the proteome composition of hAMSCs-CM. Specifically for CTRL samples, protein identification was obtained by elaborating the LC–MS data against both the Homo sapiens and the Bos taurus protein databases (protein identification data of CTRL samples 1–3 are in Additional file [404]2: Table S2A-F). Additional file [405]2: Table S2G lists the proteins consistently identified in all DMEMF12 samples analyzed, therefore characterizing with repeatability the culture medium matrix, and representing the putative contaminants to be excluded from the analysis of proteins originating from the secretome. This list includes common contaminants such as keratins and albumin, which are frequently detected in proteomic analyses. Keratins are often associated with dust/contact-related contaminants, while albumin can derive from reagents and materials during the sample preparation step [[406]29–[407]31]. Since albumin was part of the 157 common protein elements of the hAMSCs secretome, we compared the pattern of the relative tryptic peptides identified either in the hAMSCs secretome or in DMEMF12, with both the Homo sapiens and Bos taurus databases (Additional file [408]2: Table S2H). The absence of peptides exclusively belonging to human albumin in the hAMSCs secretome suggests that the protein or its fragment peptides are probable contaminants rather than components of the secretome. Given the serum-free nature of the medium, it can be concluded that the contribution of the DMEMF12 to the proteome of the hAMSCs secretome is negligible. Gene ontology analysis of the 157 proteins characterizing the hAMSCs secretome Gene ontology classification and over-representation analysis of the molecular function, cellular component, and protein class of the 157 proteins which repeatedly characterize the hAMSCs secretome were performed using PANTHER ([409]www.pantherdb.org, accessed on August 23, 2022). Among the 157 proteins, different molecular functions were found to be significantly over- or under-represented (p value < 0.05, Fisher’s Exact statistical test type with FDR correction) when compared to the reference classification Homo sapiens (Table [410]2). The fold enrichment values revealed that the over-represented molecular functions with the highest values were disulfide isomerase activity, collagen and extracellular matrix binding, and disulfide oxidoreductase activity. Table 2. List of the molecular functions with statistically significant over-/under-representation in the group of the 157 common proteins of hAMSCs secretome pools 1–4 Molecular function Homo sapiens REFLIST (20,589) Client text box input (157) Client text box input (expected) Client text box input (over/ under) Client text box input (fold Enrichment) Client text box input (raw P-value) Client text box input (FDR) Protein disulfide isomerase activity (GO:0003756) 10 5 0.08  +  65.57 6.58E-08 3.59E-05 Collagen binding (GO:0005518) 8 2 0.06  +  32.79 2.46E-03 2.44E-02 Extracellular matrix binding (GO:0050840) 13 3 0.1  +  30.26 2.21E-04 4.03E-03 Disulfide oxidoreductase activity (GO:0015036) 18 4 0.14  +  29.14 2.08E-05 7.56E-04 Protein-disulfide reductase activity (GO:0015035) 15 3 0.11  +  26.23 3.19E-04 4.84E-03 Misfolded protein binding (GO:0051787) 16 3 0.12  +  24.59 3.77E-04 5.42E-03 Aminopeptidase activity (GO:0004177) 11 2 0.08  +  23.84 4.20E-03 3.76E-02 Oxidoreductase activity, acting on a sulfur group of donors (GO:0016667) 26 4 0.2  +  20.18 7.42E-05 1.84E-03 ATP binding (GO:0005524) 41 5 0.31  +  15.99 2.48E-05 7.98E-04 Isomerase activity (GO:0016853) 76 8 0.58  +  13.8 2.41E-07 6.58E-05 Serine-type endopeptidase inhibitor activity (GO:0004867) 40 4 0.31  +  13.11 3.39E-04 5.00E-03 Endopeptidase inhibitor activity (GO:0004866) 66 6 0.5  +  11.92 1.77E-05 7.42E-04 Heat shock protein binding (GO:0031072) 33 3 0.25  +  11.92 2.53E-03 2.46E-02 Peptidase inhibitor activity (GO:0030414) 66 6 0.5  +  11.92 1.77E-05 6.89E-04 Endopeptidase regulator activity (GO:0061135) 70 6 0.53  +  11.24 2.41E-05 8.23E-04 Peptidase regulator activity (GO:0061134) 72 6 0.55  +  10.93 2.80E-05 8.48E-04 Protease binding (GO:0002020) 78 6 0.59  +  10.09 4.26E-05 1.11E-03 Unfolded protein binding (GO:0051082) 69 5 0.53  +  9.5 2.46E-04 4.34E-03 Actin filament binding (GO:0051015) 135 9 1.03  +  8.74 1.54E-06 1.40E-04 Enzyme inhibitor activity (GO:0004857) 115 7 0.88  +  7.98 4.02E-05 1.16E-03 Metallopeptidase activity (GO:0008237) 116 7 0.88  +  7.91 4.24E-05 1.16E-03 Metalloendopeptidase activity (GO:0004222) 84 5 0.64  +  7.81 5.80E-04 7.19E-03 Serine-type endopeptidase activity (GO:0004252) 103 6 0.79  +  7.64 1.82E-04 3.68E-03 Serine hydrolase activity (GO:0017171) 107 6 0.82  +  7.35 2.21E-04 4.32E-03 Serine-type peptidase activity (GO:0008236) 107 6 0.82  +  7.35 2.21E-04 4.17E-03 Actin binding (GO:0003779) 199 11 1.52  +  7.25 6.06E-07 6.61E-05 Calcium ion binding (GO:0005509) 168 9 1.28  +  7.03 8.38E-06 5.08E-04 Cell adhesion molecule binding (GO:0050839) 141 6 1.08  +  5.58 8.98E-04 1.07E-02 Metal ion binding (GO:0046872) 268 11 2.04  +  5.38 9.44E-06 5.15E-04 Protein-containing complex binding (GO:0044877) 394 15 3  +  4.99 5.34E-07 7.28E-05 Endopeptidase activity (GO:0004175) 324 12 2.47  +  4.86 9.99E-06 4.96E-04 Cation binding (GO:0043169) 325 12 2.48  +  4.84 1.03E-05 4.68E-04 Peptidase activity (GO:0008233) 433 15 3.3  +  4.54 1.66E-06 1.30E-04 Cytoskeletal protein binding (GO:0008092) 411 14 3.13  +  4.47 4.52E-06 3.09E-04 Purine ribonucleotide binding (GO:0032555) 271 8 2.07  +  3.87 1.32E-03 1.47E-02 Carbohydrate derivative binding (GO:0097367) 345 10 2.63  +  3.8 3.84E-04 5.38E-03 Ribonucleotide binding (GO:0032553) 278 8 2.12  +  3.77 1.54E-03 1.68E-02 Ion binding (GO:0043167) 741 21 5.65  +  3.72 3.17E-07 5.77E-05 Purine nucleotide binding (GO:0017076) 288 8 2.2  +  3.64 1.91E-03 2.04E-02 Nucleoside phosphate binding (GO:1,901,265) 330 8 2.52  +  3.18 4.29E-03 3.78E-02 Nucleotide binding (GO:0000166) 330 8 2.52  +  3.18 4.29E-03 3.72E-02 Oxidoreductase activity (GO:0016491) 431 10 3.29  +  3.04 1.99E-03 2.09E-02 Anion binding (GO:0043168) 437 10 3.33  +  3 2.19E-03 2.26E-02 Small molecule binding (GO:0036094) 440 10 3.36  +  2.98 2.30E-03 2.33E-02 Enzyme regulator activity (GO:0030234) 472 10 3.6  +  2.78 3.75E-03 3.42E-02 Catalytic activity, acting on a protein (GO:0140096) 1473 25 11.23  +  2.23 2.50E-04 4.27E-03 Hydrolase activity (GO:0016787) 1739 26 13.26  +  1.96 1.29E-03 1.46E-02 Protein binding (GO:0005515) 2812 40 21.44  +  1.87 9.45E-05 2.15E-03 Catalytic activity (GO:0003824) 3916 50 29.86  +  1.67 1.38E-04 3.02E-03 Nucleic acid binding (GO:0003676) 1974 3 15.05 − 0.2 2.95E-04 4.60E-03 Molecular transducer activity (GO:0060089) 1085 1 8.27 − 0.12 3.51E-03 3.31E-02 Signaling receptor activity (GO:0038023) 1085 1 8.27 − 0.12 3.51E-03 3.25E-02 DNA binding (GO:0003677) 1361 1 10.38 − 0.1 5.15E-04 6.53E-03 Transporter activity (GO:0005215) 770 0 5.87 −  < 0.01 4.76E-03 4.06E-02 Transcription regulator activity (GO:0140110) 1265 0 9.65 −  < 0.01 7.79E-05 1.85E-03 cis-regulatory region sequence-specific DNA binding (GO:0000987) 810 0 6.18 −  < 0.01 3.15E-03 3.02E-02 DNA-binding transcription factor activity, RNA polymerase II-specific (GO:0000981) 984 0 7.5 −  < 0.01 9.51E-04 1.10E-02 RNA polymerase II cis-regulatory region sequence-specific DNA binding (GO:0000978) 798 0 6.09 −  < 0.01 5.09E-03 4.27E-02 RNA polymerase II transcription regulatory region sequence-specific DNA binding (GO:0000977) 1059 0 8.08 −  < 0.01 4.09E-04 5.59E-03 Transcription cis-regulatory region binding (GO:0000976) 1099 0 8.38 −  < 0.01 4.33E-04 5.77E-03 Double-stranded DNA binding (GO:0003690) 1164 0 8.88 −  < 0.01 1.79E-04 3.76E-03 Sequence-specific DNA binding (GO:0043565) 1148 0 8.75 −  < 0.01 2.85E-04 4.57E-03 DNA-binding transcription factor activity (GO:0003700) 1054 0 8.04 −  < 0.01 6.67E-04 8.09E-03 Sequence-specific double-stranded DNA binding (GO:1,990,837) 1117 0 8.52 −  < 0.01 2.71E-04 4.48E-03 Transcription regulatory region nucleic acid binding (GO:0001067) 1099 0 8.38 −  < 0.01 4.33E-04 5.63E-03 [411]Open in a new tab Figure [412]2 shows the results of the cellular components overrepresentation analysis (p value < 0.05, Fisher’s Exact test type with FDR correction), conducted using the 157 proteins characterizing the hAMSCs secretome (blue histograms), in comparison with the Homo sapiens list of genes used as a reference (orange histograms). While statistically significant, the fold enrichment values for cellular components were not as high as those observed for molecular functions, actin cytoskeleton, extracellular matrix, external encapsulating structure, endoplasmic reticulum, extracellular space and extracellular region cellular components, which showed the highest values. Fig. 2. [413]Fig. 2 [414]Open in a new tab Protein cellular component over-representation analysis of the157 proteins commonly identified in hAMSC-CM using Homo sapiens database as reference (Over-representation Test PANTHER GO-Slim Cellular Component, FISHER’s Exact Test Type with FDR correction, P < 0.05) Relative to the identified proteins in hAMSCs secretome pools, the highest % of genes belonged to extracellular matrix and structural proteins, chaperons, proteases, cytoskeletal proteins, and metalloproteases. This aligns with the biological activity of the hAMSCs secretome exerted during the proliferative and the remodeling phases of the healing process [[415]32]. The aldolase protein class, followed by the Hsp90 family and Hsp70 family chaperones, as well as extracellular matrix proteins, showed the highest fold enrichment values. Conversely, the protein classes of gene-specific transcriptional regulator, transmembrane signal receptor, and DNA-binding transcription factor resulted instead under-represented (Fig. [416]3). Fig. 3. [417]Fig. 3 [418]Open in a new tab Protein class over-representation analysis of the157 proteins commonly identified in hAMSC-CM using Homo sapiens database as reference (Over-representation Test PANTHER GO-Slim, FISHER’s Exact Test Type with FDR correction, P < 0.05) We finally investigated the predicted network of functional interactions between the 157 proteins common to all hAMSCs secretome pools using the STRING tool ([419]https://string-db.org, accessed on august 23, 2022) (Fig. [420]4). The network highlights functional and physical associations between the majority of the proteins and revealed 41 clusters significantly enriched, with the top ten listed in Table [421]3. It is noteworthy that some of these clusters are related to molecular processes involved in collagen formation and synthesis. Additionally, disease-gene associations inside the network revealed 21 significantly enriched diseases, that notably include different bone and cartilage related disorders (Table [422]4). Fig. 4. [423]Fig. 4 [424]Open in a new tab Protein–protein functional interaction network of the 157 proteins commonly identified in hAMSCs secretome (STRING tool analysis, highest confidence) Table 3. Top ten clusters significantly enriched in the 157 proteins that were repeatedly identified in hAMSC-CM (Fig. [425]4) #term Term description Observed gene count Background gene count Strength FDR CL:16,514 Collagen type I trimer, and lateral cystocele 5 5 2.1 1.28e-06 CL:17,150 EF-hand, Ca insensitive, and Vinculin 4 5 2.0 5.96e-05 CL:3175 Sequestering of calcium ion, and disulfide isomerase 4 5 2.0 5.96e-05 CL:16,512 Fibrillar collagen, C-terminal, and Lateral cystocele 6 12 1.79 7.21e-07 CL:16,612 Mixed, incl. g2 nidogen domain and fibulin, and collagen type xii trimer 3 6 1.79 0.0041 CL:17,130 Mixed, incl. profilin binding, and ef-hand, ca insensitive 7 17 1.71 1.31e-07 CL:11,557 Fructose 1,6-bisphosphate metabolic process, and xylulose biosynthetic process 4 10 1.7 0.00041 CL:11,589 Enolase, conserved site, and Phosphoglycerate mutase 1 3 8 1.67 0.0075 CL:16,595 Dissolution of Fibrin Clot, and positive regulation of sterol import 3 8 1.67 0.0075 CL:16,571 Mixed, incl. dissolution of fibrin clot, and negative regulation of metallopeptidase activity 7 20 1.64 2.98e-07 [426]Open in a new tab Table 4. Disease-gene associations significantly enriched in the proteins functional network (Fig. [427]4) of the 157 hAMSC secretome pools 1–4 common proteins #term ID Term description Observed gene count Background gene count Strength FDR Matching proteins in the network (gene name) DOID:7 Disease of anatomical entity 72 4452 0.3 8.22E-07 VCL,LGALS1,MYH9,MMP2,LAMB1,SERPINE1,COL1A1,GAPDH,TPI1,SPARC,CTSD,C3,KRT 9,KRT1,ACTN4,CHI3L1,LAMC1,LTBP2,YWHAE,VCAN,KRT10,PLOD2,UCHL1,CALM3,COL6 A3,ALB,COL1A2,PPIB,COL3A1,YWHAG,EEF2,EFEMP2,KRT2,CALR,COL12A1,FBN1,MDH2 ,FLNC,P4HB,PAPPA,TPM4,CTSB,FN1,ACTB,VCP,COL4A2,PNP,KRT19,COL6A1,THBS2,T AGLN2,LMNA,FLNA,COL5A1,SPTAN1,PGK1,HSPG2,HSPA1B,PSAP,ALDOA,YWHAZ,C1S,GP I,TGFBI,PARK7,FLNB,HARS,SERPINH1,MDH1,C1R,KRT8,PRKCSH DOID:13,359 Ehlers-Danlos syndrome 7 23 1.58 6.75E-06 COL1A1,COL1A2,COL3A1,COL12A1,COL5A1,C1S,C1R DOID:65 Connective tissue disease 24 715 0.62 6.78E-06 MMP2,COL1A1,SPARC,PLOD2,COL6A3,COL1A2,PPIB,COL3A1,EFEMP2,COL12A1,FBN1,P 4HB,VCP,COL6A1,THBS2,LMNA,FLNA,COL5A1,HSPG2,HSPA1B,C1S,FLNB,SERPINH1,C1 R DOID:12,347 Osteogenesis imperfecta 7 30 1.46 1.30E-05 COL1A1,SPARC,PLOD2,COL1A2,PPIB,P4HB,SERPINH1 DOID:4 Disease 81 5921 0.23 1.69E-05 VCL,LGALS1,MYH9,PSMA3,MMP2,LAMB1,SERPINE1,COL1A1,GAPDH,TPI1,SPARC,CTSD, C3,KRT9,KRT1,ACTN4,CHI3L1,LAMC1,LTBP2,YWHAE,VCAN,KRT10,PLOD2,UCHL1,CALM 3,COL6A3,ALB,COL1A2,PPIB,COL3A1,GAA,YWHAG,EEF2,EFEMP2,KRT2,CALR,COL12A1 ,FBN1,MDH2,FLNC,P4HB,PAPPA,UGP2,TPM4,CTSB,FN1,ACTB,VCP,COL4A2,PNP,KRT19 ,COL6A1,THBS2,TAGLN2,LMNA,FLNA,COL5A1,SPTAN1,PGK1,GSN,HSPG2,HSPA1B,IGFB P3,PSAP,ALDOA,YWHAZ,C1S,TKT,GDI1,GPI,TGFBI,PARK7,FLNB,HARS,SERPINH1,MDH 1,CALU,C1R,LDHA,KRT8,PRKCSH DOID:2256 Osteochondrodysplasia 10 108 1.06 2.23E-05 COL1A1,SPARC,PLOD2,COL1A2,PPIB,FBN1,P4HB,HSPG2,FLNB,SERPINH1 DOID:17 Musculoskeletal system disease 27 1074 0.5 8.88E-05 MMP2,COL1A1,SPARC,PLOD2,COL6A3,COL1A2,PPIB,COL3A1,EFEMP2,COL12A1,FBN1,F LNC,P4HB,ACTB,VCP,COL6A1,THBS2,LMNA,FLNA,COL5A1,HSPG2,HSPA1B,C1S,FLNB,H ARS,SERPINH1,C1R DOID:9120 Amyloidosis 8 70 1.15 9.57E-05 C3,KRT1,ALB,FN1,ACTB,GSN,HSPG2,TGFBI DOID:4603 Epidermolytic hyperkeratosis 4 4 2.1 0.00012 KRT9,KRT1,KRT10,KRT2 DOID:0050736 Autosomal dominant disease 27 1163 0.46 0.0003 MYH9,COL1A1,C3,KRT1,ACTN4,YWHAE,KRT10,CALM3,COL6A3,COL1A2,COL3A1,EEF2,K RT2,CALR,FBN1,FLNC,UGP2,VCP,COL4A2,COL6A1,LMNA,FLNA,GSN,HSPG2,TGFBI,FLN B,PRKCSH DOID:0080006 Bone development disease 11 220 0.79 0.00091 COL1A1,SPARC,PLOD2,COL1A2,PPIB,FBN1,P4HB,FLNA,HSPG2,FLNB,SERPINH1 DOID:0080001 Bone disease 16 523 0.58 0.0022 MMP2,COL1A1,SPARC,PLOD2,COL6A3,COL1A2,PPIB,FBN1,P4HB,VCP,COL6A1,THBS2,F LNA,HSPG2,FLNB,SERPINH1 DOID:0060877 Bullous congenital ichthyosiform erythroderma 3 3 2.1 0.003 KRT1,KRT10,KRT2 DOID:0060158 Acquired metabolic disease 12 320 0.67 0.0042 TPI1,C3,KRT1,ALB,FN1,ACTB,COL6A1,PGK1,GSN,HSPG2,TKT,TGFBI DOID:14,330 Parkinsons disease 5 51 1.09 0.0212 YWHAE,UCHL1,YWHAG,YWHAZ,PARK7 DOID:174 Acanthoma 3 8 1.67 0.0212 KRT1,KRT10,KRT2 DOID:863 Nervous system disease 34 2132 0.3 0.0212 MYH9,LAMB1,COL1A1,GAPDH,CTSD,C3,LAMC1,LTBP2,YWHAE,VCAN,UCHL1,COL6A3,ALB ,YWHAG,EEF2,FBN1,MDH2,CTSB,FN1,ACTB,VCP,COL4A2,LMNA,FLNA,COL5A1,SPTAN1, HSPG2,PSAP,YWHAZ,TGFBI,PARK7,HARS,MDH1,KRT8 DOID:0050739 Autosomal genetic disease 35 2323 0.27 0.0378 MYH9,PSMA3,COL1A1,C3,KRT1,ACTN4,YWHAE,KRT10,CALM3,COL6A3,COL1A2,PPIB,CO L3A1,EEF2,EFEMP2,KRT2,CALR,FBN1,FLNC,UGP2,VCP,COL4A2,PNP,COL6A1,LMNA,FL NA,GSN,HSPG2,IGFBP3,PSAP,TGFBI,PARK7,FLNB,HARS,PRKCSH DOID:0050557 Congenital muscular dystrophy 4 34 1.17 0.0459 COL6A3,COL12A1,COL6A1,LMNA DOID:630 Genetic disease 41 2962 0.24 0.0488 MYH9,PSMA3,COL1A1,GAPDH,CTSD,C3,KRT1,ACTN4,LTBP2,YWHAE,VCAN,KRT10,CALM3 ,COL6A3,COL1A2,PPIB,COL3A1,EEF2,EFEMP2,KRT2,CALR,FBN1,FLNC,PAPPA,UGP2,V CP,COL4A2,PNP,COL6A1,LMNA,FLNA,PGK1,GSN,HSPG2,IGFBP3,PSAP,TGFBI,PARK7,F LNB,HARS,PRKCSH DOID:90 Degenerative disc disease 3 13 1.46 0.0488 MMP2, COL1A1,THBS2 [428]Open in a new tab Next, we investigated the classification/prediction of secreted proteins among the 157 commonly-identified proteins, using the Human Protein Atlas (HPA) Subcellular Section—Secreted proteins (2793 genes) as a reference ([429]http://www.proteinatlas.org, accessed on October 13, 2022). Seventy-nine proteins (50.3%) were classified/predicted as secreted (Additional file [430]3: Table S3A). Among these, 27 were subclassified as “proteins secreted to extracellular matrix”, 27 as “proteins secreted to blood”, 20 as “intracellular and membrane proteins”, 3 as “proteins secreted in other tissues”, and 2 as “proteins secreted in female reproductive system” (data in Additional file [431]3: Table S3B). The remaining 78 proteins, which were not classified/predicted as secreted proteins, were further investigated for their potential functional relationships and for enriched clusters of interactions using STRING ([432]https://string-db.org, accessed on August 23, 2022). Notably, clusters related to carbon metabolism were among the top ten most significantly enriched (Fig. [433]5, Table [434]5). Fig. 5. [435]Fig. 5 [436]Open in a new tab Protein–protein functional interaction network of the 78 out of the 157 commonly identified proteins in hAMSCs secretome which were not classified as secreted proteins (STRING tool analysis, highest confidence) Table 5. List of the top ten clusters significantly enriched among the 78 proteins (Fig. [437]5) not classified as secreted proteins #term ID Term description Observed gene count Background gene count Strength FDR Matching proteins in your network (gene name) CL:11,548 Carbon metabolism, and Starch and sucrose metabolism 13 124 1.42 3.52E-11 TPI1,ENO1,GAA,PKM,TALDO1,MDH2,UGP2,PGK1,ALDOA,LDHB,TKT,MDH1,LDHA CL:17,048 RHO GTPases Activate WASPs and WAVEs, and actin filament organization 12 105 1.46 7.94E-11 VCL,ACTN4,IQGAP1,ANXA5,TLN1,PLS3,ACTB,FLNA,CAP1,TMSB4X, ACTN1,FLNB CL:11,549 Carbon metabolism, and Pyruvate metabolism 11 96 1.46 3.22E-10 TPI1,ENO1,PKM,TALDO1,MDH2,PGK1,ALDOA,LDHB,TKT,MDH1,LDHA CL:11,551 Pentose phosphate pathway, and Glycolysis 9 55 1.61 2.29E-09 TPI1,ENO1,PKM,TALDO1,PGK1,ALDOA,LDHB,TKT,LDHA CL:17,127 Mixed, incl. profilin binding, and profilin 7 33 1.73 9.01E-08 VCL,ACTN4,IQGAP1,TLN1,ACTB,TMSB4X, ACTN1 CL:17,130 Mixed, incl. profilin binding, and ef-hand, ca insensitive 6 17 1.95 1.27E-07 VCL,ACTN4,TLN1,ACTB,TMSB4X,ACTN1 CL:17,049 RHO GTPases Activate WASPs and WAVEs, and actin filament organization 9 96 1.37 1.28E-07 VCL,ACTN4,IQGAP1,TLN1,PLS3,ACTB,CAP1,TMSB4X,ACTN1 CL:11,554 Glycolysis, and Fructose-1,6-bisphosphatase 6 31 1.69 2.20E-06 TPI1,ENO1,TALDO1,PGK1,ALDOA,TKT CL:17,150 EF-hand, Ca insensitive, and Vinculin 4 5 2.3 8.11E-06 VCL,ACTN4,TLN1, ACTN1 CL:17,373 Muscle protein, and sarcomere organization 6 68 1.35 0.00012 MYH9,MYL12A,FLNC,TPM4,TAGLN,MYL6 [438]Open in a new tab Transcriptomics data revealed that 65% (n = 13,060) of all human proteins (n = 20,090) are expressed in placenta [[439]33], and 286 of these have an elevated expression when compared to other tissues. It was therefore interesting to determine how many of the 157 proteins that characterized the hAMSCs secretome were classified as placental proteins. To do this, we referred to the HPA placenta proteome database ([440]https://www.proteinatlas.org/humanproteome/tissue/placenta, accessed on October 13, 2022). The donut chart in Fig. [441]6 illustrates the distribution of the 157 proteins (outer blue ring) in relation to their classification as “elevated expression in placenta”, or “elevated in other tissues but expressed in placenta”, or “low tissue specificity but expressed in placenta”. The results demonstrate that 13 elements (8%) were classified to have an elevated expression in placenta, 57 (36%) with elevated expression in other tissues but expressed in placenta, and 84 elements (54%) expressed in the placenta but with low tissue specificity (data analysis in Additional file [442]4: Table S4). Only 3 out of the 157 proteins, namely keratins type I cytoskeletal 9, keratin type II cytoskeletal 2 epidermal, and complement C1r subcomponent, were not classified as genes expressed in placenta. Complement C1r subcomponent is involved in the assembling of complement C1, the first component of the classical pathway of the complement system of the innate immune system. On the other hand, both keratins were identified in the control medium (DMEMF12) and therefore they can be excluded from the components of the hAMSCs secretome. Fig. 6. [443]Fig. 6 [444]Open in a new tab Distribution of the 157 commonly identified proteins in all hAMSCs-CM based on placenta gene classification as i) elevated expression in placenta; ii) elevated expression in other tissues but expressed in placenta; iii) low tissue specificity but expression in placenta and iv) proteins identified in hAMSCs not classified as placenta proteins.The number of proteins and the relative percent value (versus total 157) are shown It is noteworthy that the 13 genes with elevated expression in placenta were all classified as secretome proteins in the HPA database (Venn diagram grouping analysis in Additional file [445]4: Table S4). These genes include tissue factor pathway inhibitor 2, pappalysin-1, glia-derived nexin, collagen alpha -1(III) chain, collagen alpha-2(IV) chain, fibrillin-1, fibronectin, insulin-like growth factor-binding protein 3, laminin subunit gamma-1, nidogen-2, plasminogen activator inhibitor 1, SPARC, and transforming growth factor-beta-induced protein ig-h3. SPARC is a 32 kDa calcium-binding matricellular multifunctional glycoprotein [[446]34], whose expression is closely associated with that of fibrillar collagens, such as type I collagen. This protein acts more as a regulator of cellular behavior rather than as a structural component of the ECM, and is involved in tissue remodeling, repair, development, and cellular turnover [[447]35]. Pathway enrichment analysis in the secretome of hAMSCs cells The molecular pathways over-representation analysis of the 157 commonly-identified proteins in the hAMSCs secretome was performed using Reactome ([448]https://reactome.org, accessed on September 21, 2022). Figure [449]7 shows the Voronoi diagram representation of the results obtained. The diagram highlights the over-represented hierarchical pathways, including the Immune system, Signal transduction, Gene expression (Transcription), Hemostasis, Developmental biology, DNA repair, Disease, Extracellular matrix organization, Cellular responses to stimuli, and Nephrin family interactions. Some of these pathways are extremely important in regenerative processes, thus we further investigated their protein elements. Fig. 7. [450]Fig. 7 [451]Open in a new tab Voronoi diagram representation of the hierarchical pathways overrepresentations analysis by Reactome of the 157 commonly-identified protein elements of hAMSC-CM s The Immune system pathway plays a critical role in directing tissue repair and regeneration outcomes [[452]36]. Sixty-three out of the 157 commonly-identified proteins were involved in this pathway (list in Table [453]6). The disease gene annotation performed using the STRING tool showed an enrichment of genes associated with various conditions, including Ehlers-Danlos syndrome, Amyloidosis, Connective tissue disease, Disease of anatomical entity, Autosomal dominant disease, Osteogenesis imperfecta type 1, Osteogenesis imperfecta type 4, and Otopalatodigital syndrome type. Table 6. List of the 63 proteins involved in the Immune system pathway Protein ID Protein name Protein ID Protein name [454]P02795 Metallothionein-2 [455]P14618 Pyruvate kinase PKM [456]P62937 Peptidyl-prolyl cis–trans isomerase A [457]P07602 Prosaposin [458]P01033 Metalloproteinase inhibitor 1 [459]P14314 Glucosidase 2 subunit beta [460]P09211 Glutathione S-transferase P [461]P06744 Glucose-6-phosphate isomerase [462]P16035 Metalloproteinase inhibitor 2 [463]P04264 Keratin, type II cytoskeletal 1 [464]P20618 Proteasome subunit beta type-1 [465]P0DMV8 Heat shock 70 kDa protein 1A [466]P78417 Glutathione S-transferase omega-1 [467]P11142 Heat shock cognate 71 kDa protein [468]P63104 14–3-3 protein zeta/delta [469]P11021 Endoplasmic reticulum chaperone BiP [470]P25788 Proteasome subunit alpha type-3 [471]P08253 72 kDa type IV collagenase [472]P28070 Proteasome subunit beta type-4 [473]P09871 Complement C1s subcomponent [474]O00584 Ribonuclease T2 [475]P00736 Complement C1r subcomponent [476]P25786 Proteasome subunit alpha type-1 [477]O00391 Sulfhydryl oxidase 1 [478]P47756 F-actin-capping protein subunit beta [479]P08238 Heat shock protein HSP 90-beta [480]P00491 Purine nucleoside phosphorylase [481]P06396 Gelsolin [482]P37837 Transaldolase [483]P05556 Integrin beta-1 [484]P07858 Cathepsin B [485]P55072 Transitional endoplasmic reticulum ATPase [486]P07355 Annexin A2 [487]P14625 Endoplasmin [488]P04083 Annexin A1 [489]P13639 Elongation factor 2 [490]P04075 Fructose-bisphosphate aldolase A [491]Q14764 Major vault protein [492]P60709 Actin, cytoplasmic 1 [493]P55786 Puromycin-sensitive aminopeptidase [494]P36222 Chitinase-3-like protein 1 [495]P10253 Lysosomal alpha-glucosidase [496]P07339 Cathepsin D [497]P15144 Aminopeptidase N (CD13) [498]Q8NBS9 Thioredoxin domain-containing protein 5 [499]P18206 Vinculin [500]P27797 Calreticulin [501]P08123 Collagen alpha-2(I) chain [502]P68363 Tubulin alpha-1B chain [503]P02461 Collagen alpha-1(III) chain [504]P50395 Rab GDP dissociation inhibitor beta [505]P02452 Collagen alpha-1(I) chain [506]Q01518 Adenylyl cyclase-associated protein 1 [507]P01024 Complement C3 [508]P03956 Interstitial collagenase [509]P46940 Ras GTPase-activating-like protein IQGAP1 [510]P30101 Protein disulfide-isomerase A3 [511]P35579 Myosin-9 [512]P07237 Protein disulfide-isomerase [513]P02751 Fibronectin [514]P21333 Filamin-A [515]O75369 Filamin-B [516]Q13813 Spectrin alpha chain, non-erythrocytic 1 [517]Open in a new tab Another over-represented pathway identified in the hAMSCs secretome is the Hemostasis pathway. This pathway plays a crucial role in the physiological response that ultimately leads to the arrest of bleeding from an injured vessel [[518]37]. A total of 34 proteins resulted involved in this pathway (Table [519]7) and, among these, STRING analysis revealed 13 significantly enriched clusters. These clusters include “RHO GTPases Activate WASPs and WAVEs, and actin filament organization” (CL:17,048), “Mixed, incl. profilin binding, and ef-hand, ca insensitive”(CL:17,130), “RHO GTPases Activate WASPs and WAVEs, and actin filament organization” (CL:17,049), “Collagen formation, and Defective B3GALTL causes Peters-plus syndrome (PpS)”( CL:16,429), “EF-hand, Ca insensitive, and Vinculin”(CL:17,150), “Collagen formation, and Matrix metalloproteinases”(CL:16,430), “Mixed, incl. dissolution of fibrin clot, and negative regulation of metallopeptidase activity”(CL:16,571), “Collagen type i trimer, and lateral cystocele”(CL:16,514), “Mixed, incl. conjunctivochalasis, and metalloproteinase inhibitor 1”(CL:16,575), “Integrin alpha5-beta1 complex, and integrin alphav-beta6 complex”(CL:16,873), “Profilin conserved site, and Baraitser-Winter syndrome”(CL:17,132), “Mixed, incl. annexin a5, and transgelin-2”(CL:17,241), and “Dissolution of Fibrin Clot, and positive regulation of sterol import”(CL:16,595). Table 7. List of the 34 proteins involved in the Hemostasis pathway Protein ID Protein name Protein ID Protein name [520]P62328 Thymosin beta-4 [521]P60709 Actin, cytoplasmic 1 [522]P62937 Peptidyl-prolyl cis–trans isomerase A [523]P07093 Glia-derived nexin [524]P55145 Mesencephalic astrocyte-derived neurotrophic factor [525]P05121 Plasminogen activator inhibitor 1 [526]P37802 Transgelin-2 [527]P68363 Tubulin alpha-1B chain [528]P01033 Metalloproteinase inhibitor 1 [529]Q01518 Adenylyl cyclase-associated protein 1 [530]P63104 14–3-3 protein zeta/delta [531]P03956 Interstitial collagenase [532]P47756 F-actin-capping protein subunit beta [533]P07602 Prosaposin [534]P09486 SPARC [535]Q08380 Galectin-3-binding protein [536]P08758 Annexin A5 [537]P02768 Albumin [538]O43852 Calumenin [539]P11021 Endoplasmic reticulum chaperone BiP [540]P07355 Annexin A2 [541]P02787 Serotransferrin [542]P04075 Fructose-bisphosphate aldolase A [543]O00391 Sulfhydryl oxidase 1 [544]P07996 Thrombospondin-1 [545]P05556 Integrin beta-1 [546]P02452 Collagen alpha-1(I) chain [547]P12814 Alpha-actinin-1 [548]Q9Y490 Talin-1 [549]O43707 Alpha-actinin-4 [550]P02751 Fibronectin [551]P18206 Vinculin [552]P21333 Filamin-A [553]P08123 Collagen alpha-2(I) chain [554]Open in a new tab The pathway of Developmental biology includes several developmental processes such as the transcriptional regulation of pluripotent stem cells, gastrulation, and the activation of HOX genes during differentiation. A total of 31 proteins resulted to be involved in this important pathway (Table [555]8) and, among these, STRING analysis revealed16 significantly enriched clusters. The clusters included “Collagen formation, and Matrix metalloproteinases”, “Collagen biosynthesis and modifying enzymes”, “Proteasome”, “Keratin type II head”, “Proteasome subunit, and proteasome regulatory particle”, “Myosin ii complex, and rho gtpases activate rocks”, “Collagen biosynthesis and modifying enzymes”, “Bethlem myopathy, and NCAM1 interactions”, “EF-hand domain, and myosin II filament”, “Keratin”, “Mixed, incl. laminin-10 complex, and sprouting of injured axon”, “Protein complex involved in cell adhesion, and met activates ptk2”, “signalling”, “Fibrillar collagen, C-terminal, and Lateral cystocele”, “Mixed, incl. cystadenoma, and intrahepatic cholangiocarcinoma”, “Proteasome regulatory particle, and proteasome subunit”, and “Mixed, incl. profilin binding, and ef-hand, ca insensitive”. Some of these clusters are shared with the enriched clusters of proteins involved in the Hemostasis pathway. Table 8. List of the 31 proteins involved in the Developmental biology pathway Protein ID Protein name [556]P05387 60S acidic ribosomal protein P2 [557]P60660 Myosin light polypeptide 6 [558]P19105 Myosin regulatory light chain 12A [559]P62906 60S ribosomal protein L10a [560]P20618 Proteasome subunit beta type-1 [561]P25788 Proteasome subunit alpha type-3 [562]P28070 Proteasome subunit beta type-4 [563]P25786 Proteasome subunit alpha type-1 [564]P60709 Actin, cytoplasmic 1 [565]P08727 Keratin, type I cytoskeletal 19 [566]P68363 Tubulin alpha-1B chain [567]Q01518 Adenylyl cyclase-associated protein 1 [568]P05787 Keratin, type II cytoskeletal 8 [569]P13645 Keratin, type I cytoskeletal 10 [570]P35527 Keratin, type I cytoskeletal 9 [571]P35908 Keratin, type II cytoskeletal 2 epidermal [572]P04264 Keratin, type II cytoskeletal 1 [573]P11142 Heat shock cognate 71 kDa protein [574]P08253 72 kDa type IV collagenase [575]P08238 Heat shock protein HSP 90-beta [576]P05556 Integrin beta-1 [577]P12109 Collagen alpha-1(VI) chain [578]P02461 Collagen alpha-1(III) chain [579]P08572 Collagen alpha-2(IV) chain [580]P11047 Laminin subunit gamma-1 [581]P20908 Collagen alpha-1(V) chain [582]P07942 Laminin subunit beta-1 [583]P35579 Myosin-9 [584]Q9Y490 Talin-1 [585]Q13813 Spectrin alpha chain, non-erythrocytic 1 [586]P12111 Collagen alpha-3(VI) chain [587]Q13813 Spectrin alpha chain, non-erythrocytic 1 [588]Open in a new tab The pathway of Extracellular matrix organization is involved in the regulation of cell differentiation processes, such as the establishment and maintenance of stem cell niches, branching morphogenesis, angiogenesis, bone remodeling, and wound repair [[589]38]. A total of 37 proteins were found to be involved in this pathway (Table [590]9) and, among these, STRING analysis revealed the enrichment of clusters mostly related to collagen biosynthesis and degradation (Table [591]9). Among these, collagen is most abundant fibrous protein in the ECM that constitutes up to 30% of total proteins in multicellular animals. It provides tensile strength and it is associated with elastic fibers composed of elastin and fibrillin microfibrils, which give tissues the ability to recover after stretching. Other ECM proteins, such as fibronectin, laminins, and matricellular proteins, participate as connectors or linking proteins [[592]38], (Table [593]9). Table 9. List of the 37 proteins involved in the Extracellular matrix organization Protein ID Protein name [594]P01033 Metalloproteinase inhibitor 1 [595]P23284 Peptidyl-prolyl cis–trans isomerase B [596]P16035 Metalloproteinase inhibitor 2 [597]P09486 SPARC [598]P07858 Cathepsin B [599]P07339 Cathepsin D [600]P05121 Plasminogen activator inhibitor 1 [601]P50454 Serpin H1 [602]Q15113 Procollagen C-endopeptidase enhancer 1 [603]O95967 EGF-containing fibulin-like extracellular matrix protein 2 [604]P03956 Interstitial collagenase [605]P09238 Stromelysin-2 [606]P07237 Protein disulfide-isomerase [607]P08253 72 kDa type IV collagenase [608]O00469 Procollagen-lysine,2-oxoglutarate 5-dioxygenase 2 [609]Q9Y4K0 Lysyl oxidase homolog 2 [610]P05556 Integrin beta-1 [611]P12814 Alpha-actinin-1 [612]Q9Y6C2 EMILIN-1 [613]P12109 Collagen alpha-1(VI) chain [614]P08123 Collagen alpha-2(I) chain [615]P07996 Thrombospondin-1 [616]P02461 Collagen alpha-1(III) chain [617]P02452 Collagen alpha-1(I) chain [618]Q14112 Nidogen-2 [619]P08572 Collagen alpha-2(IV) chain [620]P11047 Laminin subunit gamma-1 [621]P20908 Collagen alpha-1(V) chain [622]Q14766 Latent-transforming growth factor beta-binding protein 1 [623]Q14767 Latent-transforming growth factor beta-binding protein 2 [624]P07942 Laminin subunit beta-1 [625]P02751 Fibronectin [626]P35555 Fibrillin-1 [627]Q99715 Collagen alpha-1(XII) chain [628]P12111 Collagen alpha-3(VI) chain [629]P13611 Versican core protein [630]P98160 Basement membrane-specific heparan sulfate proteoglycan core protein [631]Open in a new tab The pathway of Cellular responses to stimuli is essential for normal development, maintenance of homeostasis in mature tissues, and effective defensive responses to potentially noxious agents [[632]39]. A total of 30 proteins were found to be involved in this pathway (Table [633]10) and, among these, STRING analysis revealed 8 significantly enriched clusters. These clusters include “Photodynamic therapy-induced unfolded protein response, and protein disulfide isomerase activity”, “Photodynamic therapy-induced unfolded protein response, and Inhibition of PKR”, “Protein processing in endoplasmic reticulum, and Insertion of tail-anchored proteins into the endoplasmic reticulum membrane”, “Sequestering of calcium ion, and disulfide isomerase”, “Proteasome”, “Proteasome subunit, and proteasome regulatory particle”, “Chaperone complex, and chaperone cofactor-dependent protein refolding”, and “Fructose 1,6-bisphosphate metabolic process, and xylulose biosynthetic process”. Table 10. List of the 30 proteins involved in the pathway of Cellular responses to stimuli Protein ID Protein name Protein ID Protein name [634]P05387 60S acidic ribosomal protein P2 [635]Q15084 Protein disulfide-isomerase A6 [636]P99999 Cytochrome c [637]P27797 Calreticulin [638]P02795 Metallothionein-2 [639]P68363 Tubulin alpha-1B chain [640]P21291 Cysteine and glycine-rich protein 1 [641]P07237 Protein disulfide-isomerase [642]Q06830 Peroxiredoxin-1 [643]P29401 Transketolase [644]P09211 Glutathione S-transferase P [645]P02768 Albumin [646]P62906 60S ribosomal protein L10a [647]P0DMV8 Heat shock 70 kDa protein 1A [648]P20618 Proteasome subunit beta type-1 [649]P11142 Heat shock cognate 71 kDa protein [650]P25788 Proteasome subunit alpha type-3 [651]Q16881 Thioredoxin reductase 1, cytoplasmic [652]Q16270 Insulin-like growth factor-binding protein 7 [653]P11021 Endoplasmic reticulum chaperone BiP [654]P62258 14–3-3 protein epsilon [655]P02545 Prelamin-A/C [656]P28070 Proteasome subunit beta type-4 [657]P08238 Heat shock protein HSP 90-beta [658]P25786 Proteasome subunit alpha type-1 [659]P55072 Transitional endoplasmic reticulum ATPase [660]P47756 F-actin-capping protein subunit beta [661]P14625 Endoplasmin [662]P37837 Transaldolase [663]Q9Y490 Talin-1 [664]Open in a new tab Evaluation of the most abundant proteins in the hAMSCs secretome Finally, we identified the most abundant proteins among the 157 commonly-identified proteins in the hAMSCs secretome. We thus applied a filter to the protein area data obtained from the Proteome Discoverer analysis of the LC–MS raw files, specifically considering protein area values ≥ 5 × 10^7. This resulted in a total of 33 proteins that showed protein area values in the range 5 × 10^7–1 × 10^9 (Additional file [665]5: Table S5A and B). These proteins collectively constitute the most abundant proteins of the hAMSCs secretome. Figure [666]8 illustrates the label-free relative quantitation graph for these proteins across the four hAMSCs secretome pools. This quantitation is based on the average protein area values obtained from LC–MS analysis performed in triplicate. These data provide valuable insights into the proteins that significantly contribute to the overall protein profile of the hAMSC secretome. Fig. 8. [667]Fig. 8 [668]Open in a new tab Histogram of the relative quantitation of the most abundant proteins identified inside the group of 157 commonly-identified proteins in the hAMSCs secretome. The average protein area values and the stabndard deviation as resulting from analytical triplicate analysis are reported on the y-axis This group of abundant proteins was further analysed using STRING and Cytoscape bioinformatic tools ([669]https://cytoscape.org) to discover specific functional relationships. Figure [670]9 illustrates the Cytoscape-STRING network, which represents interactions among the 33 most abundant proteins in the hAMSCs secretome. Additionally, it highlights the top 6 enriched terms, which are annotated in different colours at the nodes. The results demonstrate that each protein appears to be involved in multiple categories. Interestingly, a large number of the abundant proteins are classified as components of the Extracellular region, Extracellular space, matrix, and exosomes and vescicle categories. Fig. 9. [671]Fig. 9 [672]Open in a new tab Functional interaction network and biological pathways of the most abundant proteins of the hAMSC secretome Discussion To enhance our understanding of the therapeutic mechanisms of mesenchymal stromal cells (MSC) in regenerative medicine and immune-related disorders, we conducted an in-depth analysis of the proteins secreted by human amniotic membrane-derived MSCs (hAMSCs). In the hAMSCs secretome, we identified 157 highly abundant proteins, 79 of which are reported as secreted in the Human Protein Atlas (HPA) database, and the remaining 78 have not been previously categorized as secreted proteins. Reactome analysis unveiled several pathways prominently represented in the hAMSCs secretome, including Immune system, Signal transduction, Gene expression (transcription), Hemostasis, Developmental biology, DNA repair, Disease, Extracellular matrix organization, Cellular responses to stimuli, and Nephrin family interactions. Some of these pathways, such as Extracellular matrix organization, Hemostasis, and Immune system, directly relate to processes like tissue remodeling, particularly evident in wound healing [[673]40–[674]42]. The significant presence of elements associated with the Developmental biology pathway may stem from the fetal origin of hAMSCs [[675]43, [676]44]. Among the proteins in the Immune system cluster, we found those with immunoregulatory action such as metallothionein-2 (MT2), glutathione S-transferase Omega 1, elongation factor 2 (eEF-2 K), various immunoproteasome-related proteins, and complement proteins. Metallothioneins (MT) have been reported to play an immunoregulatory role in autoimmune diseases, infections, and inflammatory bowel diseases [[677]45, [678]46]. Among the MT, MT2 showed better outcomes in a mouse model of autoimmune encephalomyelitis, preserving myelin and reducing neuroinflammation compared to MT1 [[679]47]. In an acute lung injury model, MT knockout (-/-) mice displayed increased edema, proinflammatory molecules, and NF-κB nuclear localization compared to wildtype mice [[680]48]. MT also protected against inflammatory organ damage in a study with MT knockout mice, reducing prothrombin, C-reactive protein, and fibrinogen production after LPS exposure [[681]49]. Furthermore, in vitro studies evidenced that MT can inhibit the proliferation of cytotoxic T lymphocytes, as also previously reported for macrophages and lymphocytes [[682]50, [683]51], possibly by interfering with cell–cell interactions, resulting in immature T lymphocytes and reduced differentiation to effector CTLs [[684]52]. eEF-2 K is another protein with immune-regulatory action identified in the hAMSCs secretome. CD8 lymphocytes from eEF-2 K KO mice show increased proliferation but reduced post-activation survival, likely due to premature induction of senescence via hyperactivation of the Akt-mTOR-S6K pathway [[685]53]. In addition, several proteins in hAMSCs secretome identified in the Immune cluster have been shown to be crucial for the activation of immune responses. For example, some immunoproteasome-related proteins (Proteasome subunit beta type-1, Proteasome subunit alpha type-3, Proteasome subunit beta type-4, Proteasome subunit alpha type-1) which have been reported to be crucial for inflammatory T helper lymphocyte differentiation and implicated in autoimmune disease pathogenesis [[686]54, [687]55]. The enzyme glutathione S-transferase omega 1 (GSTO1-1), which has been reported to play a pro-inflammatory role in response to LPS [[688]56]. Furthermore, knockdown of GSTO1-1 in macrophage-like cells was shown to block NADPH oxidase 1 expression and ROS generation after LPS stimulation. Paradoxically, GSTO1-1-deficient mice exhibited a more severe inflammatory response and increased bacterial escape in a model of inflammatory bowel disease [[689]57]. Other proteins identified in the hAMSCs secretome that were reported to be crucial for immune responses are complement protein C3 and their proteolytic products C3a, which has chemotactic properties, and C3b which stimulates the innate immune response by opsonizing pathogens. C3 combined with collagen type 1 has been shown to boost inflammation, collagen deposition, and wound healing [[690]58]. Among the other proteins represented in the Immune cluster, are tissue inhibitor of metalloproteinases 1 and 2 (TIMP1 and TIMP2), which are reported to exert an immunoregulatory action and also play a major role in matrix remodeling. During wound healing, keratinocytes produce TIMP1 and TIMP2 to promote tissue remodeling and homeostasis, but dysregulated expression can lead to fibrosis [[691]59]. Increased TIMP1 levels, for example, enhance wound healing and have an antiapoptotic effect in diabetic patients [[692]60]. On the other hand, TIMP levels can sometimes decrease in diseased tendons [[693]61], and TIMP1 can inhibit ECM degradation through MMP2 [[694]62]. Interestingly, a key protein known to be involved in the immunomodulatory actions of MSCs, namely indoleamine 2,3 dioxygenase (IDO), which is known to inhibit lymphocyte responses, was not detected in our analysis. This absence of detection could be attributed to the stringent limitations of the analytical method we applied, which exceeded the protein's limit of detection. Other highly represented proteins in the hAMSC secretome were clustered in the Hemostasis pathway and have also been reported to possess immunomodulatory properties. These proteins include transgelin-2, thymosin beta 4, thrombospondin 1, talin-1, filamin A (FlnA), and Galectin 3 binding protein. Transgelin-2 stabilizes cytoskeletal actin, facilitating T-cell synapse interactions with antigen-presenting cells [[695]71]. Thrombospondin-1 (TSP-1) is transiently released in large quantities by neutrophils during the initial stages of acute inflammation and can exert a strong chemotactic action [[696]63]. Its action is mainly carried out by inducing a strong inflammatory action that accelerates the repair process by facilitating the phagocytosis of damaged cells and the generation of T regulatory cells [[697]64, [698]65]. Thus, TSP-1 could represent a compensatory mechanism for controlling the immune response and protecting tissues from excessive damage. Talin-1 has been shown to maintain T regulatory cell homeostasis since its absence in CD4 lymphocytes led to spontaneous activation due to decreased T regulatory cell levels [[699]66]. FlnA, has been shown to affect T lymphocyte adhesion and infiltration into tissues, indirectly impacting their function [[700]67]. Galectin 3 plays a multifaceted role in regulating the inflammatory response by influencing macrophage polarization [[701]68], angiogenesis [[702]69], and fibroblast-to-myofibroblast conversion [[703]70], making it pivotal in wound healing processes [[704]71]. Thymosin beta 4 participates in recruiting stem and progenitor cells, promoting cardiac repair after myocardial infarction [[705]72, [706]73]. It also interferes with TNF-α-mediated NF-κB activation and IL-8 gene transcription, contributing to immunomodulation [[707]74]. Other proteins identified in the hemostasis cluster, but without immunomodulatory actions, are mesencephalic astrocyte-derived neurotrophic factor (MANF) and secreted protein acidic and rich in cysteine (SPARC). MANF is a neurotrophic factor reported to exert protective actions in various central nervous system diseases [[708]75]. For example, pretreatment with MANF before middle cerebral artery occlusion in rats has been shown to improve locomotor abilities and reduced neurological deficits [[709]76]. MANF has also shown benefit in Parkinson's and Alzheimer's disease by protecting dopaminergic neurons and reducing intracellular α-synuclein aggregates in Parkinson's disease [[710]77] and mitigating Aβ1-42-induced neuronal cell death in Alzheimer's disease [[711]78]. SPARC, a matrix glycoprotein, with diverse functions which is highly expressed during tissue damage, inflammation, and in tumors [[712]79], and was shown to inhibit angiogenesis by interfering with the binding of pro-angiogenic factors such as VEGF, PDGF, and bFGF to their receptors, thus countering blood vessel formation [[713]80]. In regenerative medicine, exogenous SPARC was shown to accelerate the proliferation of limbal epithelial stem cells and promote their migration, expediting corneal wound healing in in vivo corneal damage models [[714]81]. Furthermore, proteins found in the Extracellular matrix organization cluster, such as laminin, collagen, and fibronectin, play crucial roles in regeneration processes. Laminin can facilitate cell interactions with other extracellular matrix components, such as collagen and heparin sulfate [[715]82]. It can serve as a substrate for the migration of epithelial keratinocytes during re-epithelialization [[716]83] and contribute to the formation and maturation of blood vessels, particularly in processes like neoangiogenesis [[717]84]. Collagen, a fundamental extracellular matrix protein, is essential for wound healing [[718]85]. It undergoes accelerated turnover during skin remodeling and healing. However, alterations in collagen can lead to functional changes in repairing tissue, potentially resulting in fibrosis [[719]86]. Fibronectin can play a significant role in mediating hemostasis and the migration and recruitment of cell progenitors during wound healing processes [[720]87–[721]89]. Conclusions In conclusion, our analysis has revealed specific proteins within the hAMSCs secretome that fall into clusters with significantly enriched interactions. Nevertheless, it's important to acknowledge the existence of many other proteins whose functional roles in regenerative medicine remain undefined. What emerges from our study is the remarkable complexity of the hAMSCs secretome, whereby we observe proteins with distinct but also paradoxical actions. Yet, it's worth noting that this coexistence of proteins with seemingly contradictory roles may be essential to maintain a dynamic balance between inflammatory and immunoregulatory responses which, in turn, could play a pivotal role in promoting a pro-regenerative environment. On another front, proteins identified in clusters related to hemostasis and extracellular matrix organization hold critical importance in processes like endothelial cell recruitment and matrix remodeling during wound healing. These processes influence the matrix-cell interactions and recruit cell progenitors involved in re-epithelialization and angiogenesis. This holistic view, particularly focused on the protein phenotypes, offers valuable insights for understanding the functional impact of the hAMSCs secretome in regenerative medicine. It paves the way for potential breakthroughs in harnessing the therapeutic potential of hAMSC. Supplementary Information [722]13287_2023_3557_MOESM1_ESM.xlsx^ (2.3MB, xlsx) Additional file 1: Table S1. Protein identification data for hAMSCs secretome pools 1-4 [723]13287_2023_3557_MOESM2_ESM.xlsx^ (39.2KB, xlsx) Additional file 2: Table S2. Proteins identification data for DMEMF12 samples (CTRL 1-3) analyzed against Homo sapiens and Bos Taurus databanks (A-F), proteins common to all samples (G) and summary of relative albumin fragments identification data (H) [724]13287_2023_3557_MOESM3_ESM.xlsx^ (51KB, xlsx) Additional file 3: Table S3. Out of the 157 proteins common to secretome pools 1-4, these proteins have been classified as secreted proteins based on the grouping analysis against the Human Protein Atlas secretome database (B) and Sub-categories distribution of the proteins classified as secreted (C) [725]13287_2023_3557_MOESM4_ESM.xlsx^ (135.1KB, xlsx) Additional file 4: Table S4. Classification of the 157 common proteins was carried out using the Human Protein Atlas placenta proteome database as a reference. The relative distribution is based on tissue expression. Furthermore, the grouping analysis shows that the proteins within this group are also classified as secreted proteins [726]13287_2023_3557_MOESM5_ESM.xlsx^ (47.5KB, xlsx) Additional file 5: Table S5. Protein average area values of the 157 proteins common to the hAMSCs secretome pools 1-4 (A), and the list of the 33 most abundant proteins in the hAMSCs secretome (average area in the range1xE9-5xE7 in decreasing order) (B). Acknowledgements