Priming-Dependent Modulation of Secretome in Placenta- and Adipose-Derived Mesenchymal Stromal/Stem Cells: Toward Cell-Free Immunoregenerative Therapies for Osteochondral and Degenerative Joint Diseases

Abstract

Age-related joint diseases are driven by structural deterioration, chronic inflammation, and impaired regenerative capacity. Mesenchymal stromal/stem cells (MSCs) have emerged as promising therapeutic candidates due to their immunomodulatory and regenerative effects, largely mediated by their secretome and extracellular vesicles (EVs). However, the therapeutic efficacy of MSC-derived products depends on both the cell source and the conditioning stimuli to which MSCs are exposed. In this study, we characterized secretomes derived from placenta-derived MSCs (PDSCs) and adipose-derived MSCs (ASCs) by performing a comparative proteomic and exosomal microRNA (miRNA) profiling. Moreover, the impact of different priming strategies, including hypoxia, interferon-gamma (IFNγ), and interleukin-1 beta (IL1β), on the paracrine properties of these cells was assessed.

Proteomic analysis identified over 7,000 proteins, with PDSC secretome enriched in pathways related to osteogenesis, chondrogenesis, extracellular matrix organization, angiogenesis, and immune regulation, whereas ASC secretome displayed limited enrichment in these processes. Functional scoring highlighted IL1β priming as the most effective strategy to enhance osteochondral and immunomodulatory protein signatures in PDSCs. Differently, IFNγ priming selectively expanded the repertoire of exosomal miRNAs, with enrichment in signalling networks including Wnt/β-catenin, TGF-β, NF-κB, and T cell receptor pathways, underscoring its role in fine-tuning immune and regenerative functions.

Together, our findings revealed that PDSCs secrete a broader and more functionally relevant spectrum of bioactive molecules for osteochondral applications compared with ASCs. Moreover, distinct priming strategies showed to differentially regulate cell paracrine outputs. In particular, while IL1β primarily enhances protein-driven regenerative and immunomodulatory activity, IFNγ promotes a functionally enriched exosomal miRNA cargo. These results indicate PDSC-derived secretomes as versatile candidates for next-generation, cell-free therapies in joint diseases.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s12015-026-11122-8.

Introduction

Articular cartilage and subchondral bone form a highly integrated functional unit, in which continuous biochemical and mechanical crosstalk ensures joint homeostasis and tissue integrity [1]. Cartilage, a specialized avascular connective tissue, provides low-friction articulation and load distribution, while subchondral bone supports mechanical stress and contributes to the metabolic exchange with overlying cartilage [2, 3]. This osteochondral interface is maintained through a finely regulated interplay between chondrocytes, osteoblasts, and osteoclasts, mediated by cytokines, growth factors, and extracellular matrix (ECM) components [4]. With aging, both cartilage and bone undergo structural and functional changes that compromise this balance. Chondrocytes progressively lose their ability to synthesize ECM and collagen, leading to reduced cellularity, water content, and elasticity, together with increased stiffness, protein aggregation, and accumulation of advanced glycation end-products [2, 3]. Concurrently, trabecular thinning and decreased bone mineral density weaken the underlying bone, further disrupting mechanical coupling with cartilage and contributing to joint degeneration [5]. A key driver of these degenerative processes is chronic, low-grade inflammation, commonly referred to as “inflammaging”, which progressively alters the osteochondral microenvironment [6]. Elevated levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNFα), interleukin-1 beta (IL1β), and interleukin-6 (IL6), inhibit chondrocyte and osteoblast function while promoting osteoclastogenesis and ECM degradation [2, 5, 7]. These inflammatory cues perpetuate tissue catabolism, oxidative stress, and cellular senescence, amplifying the deterioration of both cartilage and bone [3]. Hormonal decline (e.g., estrogen and testosterone deficiency), nutritional imbalances, and reduced mechanical loading further accelerate osteochondral degeneration by impairing bone remodelling and chondrocyte metabolism [8–10]. Moreover, oxidative stress, which rises with age, also promotes the accumulation of reactive oxygen species (ROS) that damage bone cells, stimulate osteoclastogenesis, and suppress osteoblast function [11]. The confluence of these factors leads to a progressive breakdown of the osteochondral unit, underlying the pathogenesis of major degenerative joint disorders such as osteoarthritis, with significant clinical and socioeconomic impact.

To counteract these processes, regenerative medicine and tissue engineering have focused on mesenchymal stromal/stem cells (MSCs) as promising tools for restoring osteochondral homeostasis. MSCs are multipotent cells capable of differentiating into osteoblasts, chondrocytes, and adipocytes, while exerting potent immunomodulatory and trophic effects through paracrine signalling [12–17]. Their secretome, particularly extracellular vesicles (EVs) including exosomes (EXOs), mediates intercellular communication by transferring proteins, lipids, and regulatory RNAs that influence inflammation, angiogenesis, and matrix remodelling [18–22]. Increasing evidence attributes the immunoregulatory and regenerative functions of MSCs to their exosomal microRNA (miRNA) cargo, capable of reprogramming target cells and promoting tissue repair in osteoarticular contexts [23–26]. These discoveries have driven a paradigm shift toward cell-free therapeutic strategies, leveraging MSC-derived secretomes and EVs to overcome the limitations of live-cell therapy.

Among MSC sources, bone marrow-derived MSCs (BM-MSCs) remain the most studied. However, adipose-derived MSCs (ASCs) and placenta-derived MSCs (PDSCs) have gained increasing attention for their accessibility, abundance, and distinct biological profiles [27–30]. PDSCs exhibit an immature phenotype, high proliferative capacity, strong immunomodulatory function, and low immunogenicity, whereas ASCs are easily obtained and widely used, although with more variable immunoregulatory outcomes [31, 32]. Importantly, MSC therapeutic efficacy can be further enhanced through priming strategies, such as exposure to hypoxia or pro-inflammatory cytokines (IFNγ, IL1β), which increase their secretion of immunosuppressive and pro-regenerative factors [33–37]. Priming can increase MSC expression of IDO1, COX-2, TGF-β, and HLA-G, and modulate their exosomal cargo to improve angiogenesis, ECM remodelling, and immune modulation [23, 24, 38–40]. However, the effects of priming are highly context-dependent, varying according to stimulus type and cell source [34]. Understanding how these intrinsic differences affect secretome composition, exosomal cargo, and therapeutic efficacy is essential for optimizing cell-free approaches, particularly for age-related osteochondral disorders where both immunomodulation and tissue regeneration are required.

In this scenario, the present study aimed to compare placenta-derived and adipose-derived MSCs to identify the most suitable cell source for the development of innovative MSC-based cell-free therapeutic strategies for osteochondral and degenerative joint disorders. In addition, we investigated the effects of distinct priming stimuli, including hypoxia, IFNγ, and IL1β, on their immunomodulatory and regenerative properties. This study seeks to elucidate how cell origin and preconditioning modulate the composition and functionality of the MSC-derived secretome, thereby providing mechanistic insights to guide the optimization of cell-free approaches for the treatment of age-related joint diseases.

Materials and Methods

Isolation and Culture of MSCs, Priming, and Conditioned Medium Production

To obtain ASCs, fresh adipose tissue (Biopredic International, France) was washed with PBS containing antibiotics and digested with collagenase type I (0.075%) for 40–90 min at 37 °C under gentle agitation. After digestion, the suspension was filtered (100 μm), centrifuged, and the pellet was resuspended in complete ASC medium (DMEM-HG, 10% FBS, 1% HEPES, 1% NaP, 1% PS, and L-Glutamine). Cells were seeded at 50,000 cells/cm² and cultured at 37 °C, 5% CO₂. At passage 1, cells were cryopreserved in 90% FBS/10% DMSO.

PDSCs were isolated from amniotic membranes of the human term placenta (38–40 weeks) collected within 6 h after birth, after informed consent and ethical approval (IRRB/39/20). After washing and decontamination, fragments were enzymatically digested using dispase (Corning, USA) and collagenase A/DNase (Roche, Germany), filtered (100–70 μm), centrifuged, and plated in Chang medium (Irvine Scientific, USA) for expansion at 37 °C, 5% CO₂.

Three independent batches of human PDSCs and three independent batches of human ASCs were expanded, with medium replacement every 2–3 days. Cell surface markers for PDSCs and ASCs (CD90⁺, CD73⁺, CD14⁻, CD19⁻, CD34⁻, CD45⁻, and HLA-DR⁻) were analyzed by flow cytometry using an MSC Phenotyping Cocktail Kit (Miltenyi Biotec, Germany), according to the manufacturer’s instructions. In addition, the multipotent differentiation potential of PDSCs and ASCs was evaluated in vitro. PDSC multipotency was assessed using the Human Mesenchymal Stem Cell Functional Identification Kit (cat. N. SC006, Bio-Techne, USA) as previously described [27]. Briefly, osteogenic and adipogenic differentiation were induced by culturing the cells for 14 days in α-MEM supplemented with 10% FBS (Thermo Fisher Scientific, USA) and lineage-specific osteogenic or adipogenic differentiation supplements, respectively (Bio-Techne, USA). For chondrogenic differentiation, cells were cultured for 28 days in DMEM/F12 (Thermo Fisher Scientific, USA) supplemented with ITS and a chondrogenic differentiation supplement (Bio-Techne, USA). Lineage commitment was assessed by immunofluorescence using a panel of antibodies including anti-mFABP4, anti-hACAN, and anti-hOC to identify adipogenic, chondrogenic, and osteogenic differentiation, respectively (Bio-Techne, USA). ASC multipotency was evaluated following a previously reported protocol [41]. In particular, the cells were cultured for 14 days in either osteogenic or adipogenic differentiation media. Osteogenic differentiation was induced using DMEM-HG supplemented with 10 mM β-glycerophosphate, 10 nM dexamethasone, 150 µM L-ascorbic acid-2-phosphate, and 10 nM cholecalciferol (Sigma-Aldrich, USA). Adipogenic differentiation was performed using a cyclic induction protocol consisting of 48 h in induction medium (DMEM-HG supplemented with 1 µM dexamethasone, 10 µg/mL insulin, 500 µM IBMX, and 200 µM indomethacin, Sigma-Aldrich, USA), followed by 48 h in maintenance medium containing 10 µg/mL insulin. For chondrogenic differentiation, the cells were pelleted by centrifugation and cultured for 28 days in serum-free chondrogenic medium composed of DMEM-HG supplemented with 10 ng/mL TGF-β3, 100 nM dexamethasone, 50 µg/mL ascorbic acid-2-phosphate, 100 µg/mL sodium pyruvate, 40 µg/mL proline, ITS-plus supplement, and 10 ng/mL BMP-6 (Sigma-Aldrich, USA). After adipogenic, osteogenic, or chondrogenic differentiation, cells were fixed with 10% formalin and stained with 0.3% Oil Red O, 40 mM Alizarin Red S, or 1% Alcian Blue in 0.1 N HCl (Sigma-Aldrich), respectively. Stained cells were then observed under an inverted microscope.

For conditioned medium (CM) collection, PDSCs and ASCs at passage 4 and 80–90% confluence were cultured as monolayers in serum-free DMEM-HG (cat. Number: 11965092, Thermo Fisher Scientific, USA). Cell viability was routinely assessed before and after the serum-free incubation using the trypan blue exclusion assay. The percentage of non-viable cells relative to the total cell number was calculated, and no significant differences in cell viability were observed between pre-starvation and post-starvation conditions. During serum-free culture, for cytokine-primed conditions, the medium was supplemented with or without 10 ng/mL IL1β (Miltenyi Biotec, Germany) or 200 IU/mL IFNγ (Miltenyi Biotec, Germany), and cells were incubated at 37 °C, 20% O₂ and 5% CO₂. For hypoxic conditioning, cells were cultured in serum-free DMEM-HG at 37 °C, 1% O₂ and 5% CO_2, using a Whitley H85 HEPA Hypoxystation (Don Whitley Scientific, UK). After 48 h, the supernatants were collected, centrifuged to remove cell debris, and stored at − 80 °C until use.

Isolation and Characterization of EXOs

EXOs were isolated from both primed and unprimed CM using ultracentrifugation (Optima XE-100, rotor: SW32TI, Beckman Coulter, USA). The CM was first centrifuged at 300 × g for 10 min to remove debris, followed by a 25 min centrifugation at 17,000 × g at 4 °C to eliminate remaining cells and debris. Finally, ultracentrifugation at 120,000 × g for 90 min at 4 °C was performed to pellet EXOs. For characterization, the size distribution and concentration of EXOs were assessed using nanoparticle tracking analysis (NTA) with a NanoSight NS3000 (Malvern Instruments Ltd., Malvern, UK). Three 60-second videos were recorded for each sample using a camera level of 16 and manual temperature monitoring. Data were analyzed using NTA software version 3.3 (Build 3.3.104, Analitik LTD, UK). EXOs were also characterized using the human cytofluorimetric bead-based MACSPlex EV kit (Miltenyi Biotec, Germany) according to the manufacturer’s instructions. Briefly, approximately 1 × 10⁹ EXOs were diluted in MACSPlex buffer (MPB) to a final volume of 120 µL. Subsequently, 15 µL of MACSPlex exosome capture beads, containing a cocktail of 39 exosomal markers, were added to each sample. Samples were then counterstained with 15 µL of an APC-conjugated antibody mix against CD9, CD63, and CD81 and incubated overnight at room temperature in the dark on an orbital shaker at 450 rpm. Following incubation, beads were washed with 1 mL of MPB and centrifuged at 3000 × g for 5 min. An additional washing step was performed by incubating the beads in 1 mL of MPB on an orbital shaker for 15 min. Samples were then centrifuged again at 3000 × g for 5 min, and the supernatant was removed, leaving a residual volume of 150 µL per tube for acquisition. Median fluorescence intensity (MFI) values for all 39 exosomal markers were corrected for medium background and gated based on their respective fluorescence signals. Cytofluorimetric analyses were performed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec, Germany) equipped with MACSQuantify software version 3.1.

Mass Spectrometry Analysis of Conditioned Medium

Sample Processing

Samples were processed using the Filter-Aided Sample Preparation (FASP) method with Vivacon Spin filters (10 kDa MWCO; Sartorius, Germany) [42]. Briefly, 2 mL of conditioned media were first reduced with 20 mM dithiothreitol (DTT; Thermo Fisher Scientific, USA) in 100 mM Tris/HCl containing 8 M urea (pH 8.5) at 37 °C for 30 min. Subsequently, proteins were alkylated with 50 mM iodoacetamide (Thermo Fisher Scientific, USA) in the dark at room temperature for 5 min. After two washes with 100 mM Tris/HCl, 8 M urea (pH 8.0), proteins were digested overnight with 0.2 µg LysC (Promega, USA) in 25 mM Tris/HCl, 2 M urea (pH 8.0), followed by a 4-hour digestion with 0.1 µg trypsin (Promega, USA) in 50 mM ammonium bicarbonate. The resulting peptides were desalted using StageTips packed with reverse-phase C18 material (Supelco Analytical Products, Sigma-Aldrich, USA), and eluted in 40 µL of 60% acetonitrile containing 0.1% formic acid [43]. The eluate was dried using a SpeedVac (Thermo Fisher Scientific, USA) and reconstituted in 20 µL of 0.1% formic acid for further analysis.

LC-MS/MS and Data Analysis

An amount of 350 nanograms per sample was injected into a Vanquish Neo UHPLC nanoLC system, which was coupled online to an Orbitrap Astral mass spectrometer (Thermo Fisher Scientific, USA), using a 75 μm × 150 mm DNV PEPMap Neo analytical column (Thermo Fisher Scientific, USA). Data-independent acquisition (DIA) was performed using a full MS1 scan (380–980 m/z), followed by 299 sequential DIA windows of 2 m/z each, without overlap, with window placement optimization enabled. Full MS1 scans were acquired at a resolution of 240,000, with an automatic gain control (AGC) target of 5 × 10⁶ and a maximum injection time of 5 ms. The 299 DIA windows were acquired at a fixed Astral detector resolution of 80,000, with an AGC target of 5 × 10⁴ and a maximum injection time of 3 ms. The scan range was set to 100–1000 m/z, and higher-energy collisional dissociation (HCD) was applied at a collision energy of 25%. Data analysis was performed using DIA-NN software (version 2.0.2) [44], using a predicted spectral library generated from an in silico-digested Homo sapiens UniProt reference proteome (UP000005640_9606), downloaded on 04/03/2025. Digestion was set to cleavage at lysine (K) and arginine (R) residues, allowing up to two missed cleavages and a minimum peptide length of six amino acids. The maximum number of variable modifications per peptide was set to one. N-terminal methionine excision and carbamidomethylation of cysteines were set as fixed modifications, while methionine oxidation and N-terminal acetylation were set as variable modifications. The final spectral library contained 20,397 protein isoforms, 32,701 protein groups, and 5,495,479 precursors in 2,932,475 elution groups. The false discovery rate (FDR) for both peptide and protein identification was controlled at 0.01%.

Data Analysis

Label-free quantification (LFQ) was used for protein quantification and analysed using Perseus software (version 1.6.15.0) [45]. LFQ intensity values were log₂-transformed prior to statistical analysis. Protein groups were included in the statistical analysis only if they were quantified in at least 70% of the samples within at least one study group.

Cluster, PCA and Gene Ontology (GO) Analysis of Proteins

Hierarchical cluster analysis of protein abundance, normalized as z-scores, was performed to group biological and technical replicates exhibiting similar expression patterns. Clustering was conducted using Perseus software (version 2.0.11). Principal component analysis (PCA) was performed to reduce dimensionality and explore variance in the log2-transformed proteomic expression matrix. Initial PCA was conducted in Perseus using Euclidean distance and average linkage for hierarchical clustering and dimensionality reduction. To further quantify the contribution of individual proteins to the second principal component (PC2), the same dataset was processed in Python using the scikit-learn library. Data were first standardized using z-score normalization, and PCA was computed via the PCA function with n components = 2. Protein contributions to PC2 were extracted from the component loading matrix, representing the coefficients of each protein in the linear combination defining PC2. Proteins were ranked by the absolute value of their PC2 loadings to identify those most relevant to the separation between ASCs and PDSCs. The top contributors were visualized using hierarchical cluster analysis. Protein-protein interaction (PPI), gene ontology (GO) and reactome enrichment analyses were subsequently carried out using the STRING web tool [46], based on the identified protein dataset.

Quantitative Profiling of Osteochondral and Immunomodulatory Protein Expression Across Priming Conditions

To assess the phenotypic shifts induced by different priming conditions, we evaluated the expression profiles of key osteogenic and immunomodulatory markers across priming treatments. Protein intensities were first log2-transformed and then normalized using z-score normalization. The osteogenic and immunomodulatory scores were calculated for each condition by averaging the normalized expression values of the respective marker sets. These combined scores were plotted in a two-dimensional scatter plot to enable comparative visualization of the osteogenic versus immunomodulatory potential of each priming strategy.

RNA Extraction and Real-time PCR Analysis of miRNAs

The expression of miRNAs was analyzed using the TaqMan Array Human MicroRNA Panels A and B v3.0, following the manufacturer’s protocol (Thermo Fisher Scientific, USA). Total RNA was extracted using the miRNeasy Mini Kit and treated with DNase I (Qiagen, Germany) to remove genomic DNA contamination. Subsequently, 300 ng of total RNA were reverse transcribed into single-stranded cDNA using the High Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, USA). Quantitative real-time PCR (qRT-PCR) was performed for 754 human miRNAs using the QuantStudio 7 Pro Real-Time PCR System (Thermo Fisher Scientific, USA). Relative miRNA expression levels were calculated using the 2^^−ΔΔCT method, with U6 small nuclear RNA serving as the endogenous control. Hierarchical clustering analysis was employed to identify groups of samples with similar miRNA expression profiles. Euclidean distance and average linkage methods were applied to group samples according to their expression profiles, allowing the identification of clusters based on similarity patterns across the dataset. Clustering was performed using the Cluster 3.0 software, and the resulting heatmap was visualized with Java TreeView.

miRNA Target Gene Prediction

Functional enrichment analysis of differentially expressed miRNAs was performed using the online tool miRNA Enrichment Analysis and Annotation Tool 2.0 (miEAA 2.0) (https://ccb-compute2.cs.uni-saarland.de/mieaa/) [47], focusing on Gene Ontology (GO) terms related to biological processes. Predicted target genes of these miRNAs were further analyzed using the miRNet platform (https://www.mirnet.ca/faces/home.xhtml) [48] and the STRING database to construct PPI networks, thereby elucidating the potential biological roles of the identified miRNAs.

Statistical Analysis

For multiple hypothesis testing in proteomic comparisons, p-values were adjusted using a false discovery rate (FDR) correction based on the Benjamini–Hochberg procedure, as implemented in Perseus. Statistical differences in protein and miRNA expression between groups were evaluated using either the parametric Student’s t-test or the non-parametric Mann–Whitney U test, depending on data distribution. Paired or unpaired t-tests were applied as appropriate for the comparison of fold-change values. For comparisons involving more than two groups, one-way ANOVA followed by appropriate post hoc tests was performed using GraphPad Prism 6.0 (USA). A p-value < 0.05 was considered statistically significant. Hierarchical clustering was conducted using the Euclidean distance metric to group samples or features based on expression similarity.

Results

Phenotypic Characterization of PDSCs and ASCs

After the initial phase of cell expansion, phase-contrast microscopy of PDSCs and ASCs revealed highly spindle-shaped, fibroblastic cell morphology, which is consistent with the well-established phenotypic characteristics of MSCs (Fig. 1A). A cell surface marker panel also confirmed that both PDSCs and ASCs were positive for the classical MSC-associated markers CD73 and CD90, while lacking expression of CD14, CD19, CD34, CD45, and HLA-DR (Fig. 1B). The multipotent differentiation potential of both cell types was further confirmed by trilineage differentiation assays. Under lineage-specific induction conditions, ASCs showed adipogenic differentiation evidenced by oil red o-positive lipid droplets, osteogenic differentiation by alizarin red staining of mineralized matrix, and chondrogenic differentiation by alcian blue staining of glycosaminoglycan-rich extracellular matrix (supplementary Fig. S1). Similarly, PDSCs demonstrated adipogenic differentiation marked by FABP4 expression, osteogenic differentiation indicated by osteocalcin (OC) staining, and chondrogenic differentiation confirmed by aggrecan (ACAN) immunofluorescence (supplementary Fig. S2).

Fig. 1.

Characterization of ASCs and PDSCs. A Representative images of ASC and PDSC morphology grown as monolayers. B Surface marker analysis of ASCs and PDSCs (passage 4) for both positive (CD90, CD73) and negative surface markers (CD14/CD19/CD34/CD45 and HLA-DR)

Proteomic Profiling Reveals Functional Differences in PDSC and ASC Secretomes

To gain insights into the paracrine profiles of PDSCs and ASCs, we performed a mass spectrometry-based proteomic analysis of their secretomes, identifying a total of 7,049 proteins. Hierarchical clustering of the whole dataset (Fig. 2A) revealed consistent protein expression patterns among technical replicates, indicating high experimental reproducibility. However, variability was observed among the three biological replicates, as well as between the two MSC sources, suggesting intrinsic donor-specific and tissue-specific differences in secretome composition. PCA of the same dataset (Fig. 2B) stratified the samples into two clearly separated clusters along component 2 (accounting for 32.3% of the variance), effectively discriminating PDSCs from ASCs based on their proteomic profiles. To further characterize the molecular drivers of this separation, we identified the top 100 proteins contributing most significantly to component 2 and visualized their expression patterns through hierarchical clustering (Fig. 2C). These differentially expressed proteins strongly distinguished the two MSC populations, underscoring the existence of distinct paracrine signatures with potential functional relevance.

Fig. 2.

Proteomic analysis of secretome derived from PDSCs and ASCs. A Hierarchical clustering heatmap of 7,049 proteins identified by mass spectrometry in the secretomes of both PDSCs and ASCs. B Principal component analysis (PCA) of the proteomic dataset, showing sample separation based on protein expression. C Heatmap of the top 100 proteins contributing most to the separation along PCA component 2

We performed a PPI analysis on the 100 proteins upregulated in the PDSC secretome and contributed most significantly to the cluster separation between PDSCs and ASCs in the PCA. As shown in Fig. 3A, the resulting PPI network revealed a high degree of connectivity among the selected proteins, indicating extensive physical and functional interactions. Functional annotations based on GO and reactome enrichment analysis (Fig. 3B) showed that these proteins are significantly associated with key biological processes related to both immunomodulation (e.g., innate and adaptive immune system regulation, cytokine signaling) and osteochondral tissue regeneration (e.g., osteoblast differentiation, chondrogenesis, extracellular matrix organization). The node colors in the PPI network reflect the functional categories identified in the enrichment analysis. To further delineate the biological roles of individual proteins within these pathways, we examined their specific associations with the 15 most significantly enriched processes (Fig. 3C). This analysis highlighted distinct protein subsets involved in critical functions such as chondroitin sulfate metabolism, WNT signaling, immune system regulation, and positive regulation of endothelial cell migration, emphasizing the multifaceted regenerative and immunoregulatory potential of the PDSC secretome. In parallel, a functional enrichment analysis was also performed on the top 100 proteins upregulated in ASCs that also contributed to the separation along PCA component 2. However, no significant enrichment was found for pathways related to immunomodulation, osteogenesis, or chondrogenesis in this set (supplementary Table 1).

Fig. 3.

Functional interaction and enrichment analysis of the top 100 PDSC-enriched proteins driving PCA component 2 separation. A Protein-protein interaction (PPI) network generated using the top 100 proteins upregulated in placenta-derived MSCs (PDSCs) that most strongly contribute to PCA component 2. Nodes represent individual proteins, and edges indicate predicted physical or functional interactions. Node colors correspond to biological processes identified by gene ontology and reactome enrichment analyses. B Bubble plot showing a subset of significantly enriched biological processes (GO/reactome terms) associated with the PDSC-upregulated proteins. Dot size reflects the number of proteins per pathway; color indicates false discovery rate (FDR). C Matrix heatmap showing the association between the 15 most significantly enriched biological processes and the individual proteins among the top 100 PDSC-upregulated candidates. Green intensity indicates the strength of association for each protein-process pair

Priming Strategies Modulate the PDSC Secretome Toward Enhanced Immunomodulatory and Osteochondral Regenerative Potential

Given that PDSCs secrete a broader repertoire of bioactive molecules associated with pathways related to osteochondral regeneration and immune regulation compared with ASCs, we next explored whether specific priming strategies could further potentiate the therapeutic potential of the PDSC secretome. To this end, we applied three priming conditions, such as treatment with hypoxia (HYP), IFNγ, and IL1β, and analyzed the resulting changes in secretome composition relative to non-primed PDSCs. Volcano plots (Figs. 4A-4C) highlight differentially expressed proteins under each condition compared with control, with a considerable number of upregulated proteins identified mainly in IFNγ and IL1β priming. The overlap and distribution of upregulated proteins across conditions are summarized in the Venn diagram in Fig. 4D. We then investigated the biological significance of these secretome alterations, performing GO enrichment analysis. In contrast to hypoxia priming (data not shown), as displayed in Fig. 4E and 4F, both IFNγ and IL1β priming were associated with significant enrichment in pathways related to immune system regulation (e.g., activation of immune effector processes, cytokine-mediated signaling, T cell modulation) and osteochondral tissue repair (e.g., osteoblast differentiation, ossification, and regulation of chondrogenesis). These results indicate that IFNγ and IL1β priming may selectively enhance the functional capacity of the PDSC secretome toward applications in inflammatory and osteochondral disorders.

Fig. 4.

Secretome modulation of PDSCs by priming with hypoxia (HYP), interferon-gamma (IFNγ), and interleukin 1β (IL1β). A–C Volcano plots showing differentially expressed protein in the secretome of PDSCs after priming with HYP (A), IFNγ (B), or IL1β (C), compared with non-primed controls. Red and blue dots indicate significantly upregulated and downregulated proteins, respectively (cutoff: log2 fold change > 1.5, p < 0.05). D Venn diagram representing the number of upregulated proteins unique or shared among the three priming conditions. E–F Gene ontology enrichment analysis of upregulated proteins in IFNγ-primed (E) and IL1β-primed (F) secretomes. Selected enriched biological processes include pathways involved in immune regulation, osteogenic and chondrogenic regeneration. Dot size reflects the number of proteins per pathway; color indicates false discovery rate (FDR)

Comparative Scoring Analysis Identifies IL1β as the Most Effective Priming Strategy for Enhancing the Dual Therapeutic Potential of PDSC Secretome

After identification of IFNγ and IL1β as the two most promising priming conditions for enhancing the immunomodulatory and osteochondral regenerative potential of the PDSC secretome, we sought to determine which of the two more effectively supports combined therapeutic applications. We identified two protein sets representing key mediators of osteochondral tissue homeostasis and inflammatory regulation, respectively, and calculated z-score–based expression indices for each priming condition. Figure 5A displays the osteochondral score across all conditions, highlighting that IL1β priming yielded the highest upregulation of proteins related to osteochondral regeneration. Similarly, the immunomodulatory score (Fig. 5B) demonstrated a marked upregulation of immune-related mediators with IFNγ treatment. To better visualize the combined therapeutic profile of each priming strategy, we plotted the osteochondral and immunomodulatory scores in a two-dimensional space (Fig. 5C). This analysis clearly positioned IL1β as the priming condition that most strongly enhances both osteochondral and immunoregulatory components of the PDSC secretome. IFNγ showed intermediate activity, while the non-primed (CTR) and hypoxia-treated conditions exhibited reduced therapeutic signatures. These results suggest that IL1β priming is associated with an increased abundance of proteins involved in biological processes related to osteochondral regulation and immune modulation within the PDSC secretome.

Fig. 5.

Comparative scoring of osteochondral and immunomodulatory protein expression reveals IL1β as the most effective priming strategy for enhancing the therapeutic potential of PDSC secretome. A Heatmap showing the osteochondral protein expression score across four conditions: hypoxia (HYP), interferon-gamma (IFNγ), interleukin-1β (IL1β), and non-primed control (CTR). The score was calculated using z-scores of key proteins involved in osteochondral regeneration. B Heatmap displaying the immunomodulatory protein expression score for the same conditions, based on z-scores of protein related to immune regulation. C Two-dimensional scatter plot summarizing each condition's combined osteochondral and immunomodulatory potential

Exosomal miRNA Profiling Reveals Tissue-specific Signatures and Functional Enrichment in Placental MSCs

To further investigate the paracrine properties of both PDSCs and ASCs in the context of osteochondral disorders, we performed a comparative profiling of 754 microRNAs (miRNAs) in exosomes isolated from their secretomes. NTA revealed that EVs released by both PDSCs and ASCs displayed a size distribution consistent with small EV populations (Fig. 6A-B). The modal particle size was 143.5 nm for PDSC-derived EVs and 147.5 nm for ASC-derived EVs, indicating a comparable vesicle size profile between the two MSC sources. Surface marker profiling performed using the MACSPlex EV Kit confirmed the presence of canonical EV tetraspanins (CD9, CD63, CD81) together with MSC-associated markers (CD29, CD105, CD44) in vesicles derived from both cell types (Fig. 6C-D). In contrast, hematopoietic markers such as CD45 and CD14 showed negligible fluorescence signal, supporting the MSC origin and purity of the isolated EV preparations.

Fig. 6.

Characterization of extracellular vesicles released by PDSCs and ASCs. A–B Nanoparticle tracking analysis (NTA) showing the size distribution and concentration of extracellular vesicles isolated from conditioned media of PDSCs (A) and ASCs (B). Representative particle images obtained during NTA acquisition are shown in the insets. C–D Surface marker profiling of extracellular vesicles derived from PDSCs (C) and ASCs (D) using the MACSPlex EV Kit. Median APC fluorescence intensity values indicate the presence of EV-associated markers (CD9, CD63, CD81) and MSC-related markers (CD29, CD105, CD44), while hematopoietic markers (CD45, CD14) showed negligible signal. Data are presented as median fluorescence intensity ± SD

Hierarchical clustering analysis (Fig. 7A) revealed distinct miRNA expression patterns between the two MSC sources. Principal component analysis (Fig. 7B) further confirmed this separation, with samples clustering according to tissue of origin along principal component 1 (62% of total variance). This separation reflects the significant contribution of the cellular source to the EV-associated miRNA profiles, indicating the presence of distinct source-specific exosomal signatures between PDSCs and ASCs. Differential expression analysis identified 82 miRNAs significantly upregulated in PDSC-derived exosomes and 22 in ASC-derived exosomes (Fig. 7C). To gain functional insights into the biological relevance of the exosomal miRNAs enriched in PDSCs, we performed GO enrichment analysis (Fig. 7D). This revealed that upregulated miRNAs from PDSCs are potentially involved in key biological processes associated with osteochondral tissue regeneration, such as osteoblast differentiation, Wnt signaling, collagen binding, and SMAD signaling, as well as immunoregulatory pathways, including T cell activation, immune response modulation, and monocyte differentiation. In contrast, GO enrichment analysis of the 22 miRNAs upregulated in ASC-derived exosomes did not reveal any significant associations with biological pathways related to osteochondral regeneration or immune system regulation (supplementary Table 2). This further supports the interpretation that PDSC-derived exosomes contain a more functionally relevant miRNA cargo for therapeutic applications targeting osteoimmunological and regenerative processes.

Fig. 7.

Exosomal miRNA profiling in PDSCs and ASCs reveals tissue-specific signatures and functional enrichment in PDSC-derived exosomes. A Heatmap of 754 miRNAs profiled by real-time PCR in exosomes derived from PDSCs and ASCs. B Principal component analysis (PCA) of miRNA expression data. C Volcano plot displaying differentially expressed exosomal miRNAs between PDSCs and ASCs. Red dots indicate miRNAs significantly upregulated in ASCs and blue dots those upregulated in PDSCs (cutoff: log2 fold change > 1.5 and p < 0.05). D Gene ontology (GO) enrichment analysis of miRNAs upregulated in PDSC-derived exosomes, highlighting pathways related to osteochondral regeneration and immune system regulation

To further investigate the regulatory landscape underlying the therapeutic potential of PDSC-derived exosomes, we focused on miRNAs previously identified through GO enrichment analysis as being involved in osteoimmunological and regenerative pathways (Fig. 7D). A PPI network was constructed to map the physical and functional relationships between these miRNAs (blue squares) and their experimentally validated or predicted target proteins (pink circles) (Fig. 8A). The resulting network revealed a highly interconnected structure among miRNAs and their target proteins. However, this connectivity should be interpreted with caution, as network density may partly reflect database bias toward well-characterized hub genes (e.g., STAT3, JUN). Therefore, these interactions should be considered indicative of potential regulatory nodes within the enriched pathways rather than definitive evidence of coordinated biological regulation. To quantify the relative regulatory potential of each miRNA, we assessed the number of unique protein targets per miRNA (Fig. 8B). Among the top regulators, miR-15b-5p, miR-29a-3p, and miR-29c-3p exhibited the highest target gene counts, suggesting broader involvement in osteochondral-regenerative networks. We then evaluated the association of these 16 miRNAs with a panel of biological processes identified by GO analysis and directly implicated in osteochondral regeneration (Fig. 8C). The heatmap analysis demonstrated that several miRNAs, most notably miR-107, miR-15b, miR-29 family members, and miR-30c, showed strong correlations with key regulatory pathways, including regulation of TGF-β/BMP signaling, modulation of Wnt/β-catenin signaling, and activation/inhibition of both osteoblasts and osteoclasts.

Fig. 8.

Network and functional association analysis of osteochondral regeneration-related (A-C) and immune system-related (D-F) miRNAs identified in PDSC-derived exosomes. A Protein-protein interaction (PPI) network showing functional and physical interactions between 16 miRNAs (blue squares) and their validated or predicted protein targets (pink circles), based on miRNet databases. The network focuses on miRNAs previously identified as enriched in PDSC exosomes and involved in both osteochondral tissue regeneration and immunoregulatory pathways (Fig. 7D). B Bar-colored matrix displaying the number of target proteins associated with each miRNAs. Color intensity reflects the number of targets, with red indicating higher targeting capacity and green lower targeting capacity. C Heatmap showing the association between the miRNAs and the most significantly enriched biological processes (with a key regulatory protein) related to osteochondral regeneration. D Protein-protein interaction (PPI) network showing functional and physical interactions between 16 miRNAs (blue squares) and their validated or predicted protein targets (pink circles), based on miRNet databases. The network focuses on miRNAs previously identified as enriched in PDSC exosomes and involved in both osteochondral tissue regeneration and immunoregulatory pathways (Fig. 7D). E Bar-colored matrix indicating the number of unique protein targets regulated by each miRNA. Color intensity reflects the number of targets, with red indicating higher targeting capacity and green lower targeting capacity. F Heatmap representing the association between individual miRNAs and the immune system regulation process (with key regulatory proteins)

To elucidate the immunomodulatory potential of PDSC-derived exosomes, also in this case we focused on miRNAs previously identified through GO enrichment analysis as being significantly associated with osteoimmunological and regenerative pathways (Fig. 7D). The PPI network highlighted the central role of multiple immunoregulatory nodes and revealed high connectivity between miRNAs (blue squares) and their predicted or validated protein targets (pink circles), supporting the hypothesis of coordinated regulation of immune signaling cascades by exosomal miRNAs (Fig. 8D). Quantification of miRNA regulatory breadth, based on the number of unique targets per miRNA, is shown in Fig. 8E. We also mapped their predicted or known interactions with a panel of core immune system regulators (Fig. 8F), including FOS, IFNG, IL1A, IL6, JAK1, JUN, REL, and STAT3. The heatmap illustrates strong associations between several miRNAs and these key immune effectors.

To visualize the molecular interactions underlying the therapeutic potential of exosomal miRNAs from PDSCs, we constructed an integrated regulatory diagram (Fig. 9) summarizing the results of the network and functional analyses presented in Fig. 8. The schematic is divided into two panels: osteochondral regeneration (left) and immune system regulation (right). In the osteochondral panel, several miRNAs, including miR-30c-5p, miR-143-3p, miR-199a-3p, miR-100-5p, miR-655-3p, and miR-99a-5p, are shown to negatively regulate key inhibitors of osteochondral regeneration such as SMAD7, FZD3/5/6, WNT5A, and AKT1, thereby promoting a favorable environment for osteoblast/osteoclast activity and matrix formation. These interactions primarily converge on the regulation of WNT/β-catenin and TGF-β signaling, two pathways strongly associated with enhanced osteochondral repair. In the immune regulation panel, a distinct but partially overlapping set of miRNAs, such as miR-15b-5p, miR-29a/c-3p, miR-181a-5p, miR-655-3p, miR-30c-5p, miR-143-3p, and miR-146a-5p, are implicated in the modulation of inflammatory pathways through direct targeting of transcriptional regulators (STAT3, JAK1, FOS, REL, JUN) and pro-inflammatory cytokines (IL1α, IFNγ, IL6). These interactions collectively contribute to the attenuation of inflammatory responses and point to the role of PDSC-derived exosomal miRNAs in immune homeostasis.

Fig. 9.

Integrated schematic of miRNA-mediated regulatory networks involved in osteochondral regeneration and immune system modulation. The diagrams summarize the key molecular interactions identified through protein-protein interaction (PPI) and functional association analyses of PDSC-derived exosomal miRNAs (see Figs. 6 and 7). Left panel: miRNAs involved in osteochondral regeneration target key components of the WNT/β-catenin and TGF-β signaling pathways. Right panel: miRNAs associated with immune system regulation modulate transcription factors (STAT3, REL, FOS, JUN), cytokines (IL1α, IL6, IFNγ), and signaling mediators (JAK1), shaping inflammatory responses. Arrows indicate positive regulatory interactions; bars denote inhibitory effects. Oval shapes represent proteins; rectangles represent miRNAs

Priming-induced Upregulation of Functional Exosomal miRNAs Reveals Selective Enhancement Under IFNγ Stimulation

Following previous analyses highlighting the superior paracrine potential of PDSC-derived exosomal miRNAs, we next investigated whether specific priming strategies could selectively enhance the expression of functionally relevant miRNAs. Thus, PDSCs were subjected to HYP, IFNγ, or IL1β priming, and the resulting exosomal miRNA profiles were analyzed. Figure 10A presents a Venn diagram showing the distinct and overlapping sets of upregulated miRNAs in response to each priming condition. IFNγ priming induced the most diverse set of unique miRNAs, including miR-323b-5p, miR-218-2-3p, miR-1256, miR-9-3p, miR-1178-3p, miR-1245a, miR-63, and miR-549a, while IL1β and hypoxia resulted in more limited and partially overlapping upregulation. To assess the biological relevance of these miRNA expression changes, we performed GO enrichment analysis. Notably, only the IFNγ-associated miRNAs showed significant enrichment in pathways relevant to osteochondral regeneration and immune regulation (Fig. 10B). These included terms such as osteoblast differentiation, positive regulation of chondrocyte differentiation, positive regulation of endothelial cell migration, and cartilage development. In contrast, GO analysis of miRNAs upregulated by hypoxia or IL1β failed to identify enrichment in pathways of therapeutic interest (data not shown). These findings suggest that IFNγ priming appears to be the most effective strategy for enhancing the regenerative and immunomodulatory properties of the PDSC secretome via selective upregulation of functionally active exosomal miRNAs.

Fig. 10.

Effects of different priming strategies on exosomal miRNA expression in PDSCs. A Venn diagram showing the overlap and specificity of upregulated exosomal miRNAs following priming of PDSCs with hypoxia (HYP), interferon-gamma (IFNγ), or interleukin 1β (IL1β). B Gene ontology (GO) enrichment analysis of miRNAs upregulated in response to IFNγ priming. C Protein-protein interaction (PPI) network generated using miRNet showing the functional interactions between five IFNγ-induced miRNAs (miR-361-3p, let-7f-1-3p, miR-1245a, miR-208a-3p, miR-9-3p; blue squares) and their predicted or validated target proteins (pink circles). D STRING-based PPI network of the same target proteins, color-coded according to their involvement in key biological pathways identified by gene ontology enrichment

To further explore the biological significance of miRNAs upregulated in PDSC-derived exosomes following IFNγ priming, we conducted a PPI analysis of their predicted target genes. Figure 10C showed the network generated using miRNet, illustrating the relationships between five IFNγ-induced miRNAs (miR-361-3p, let-7f-1-3p, miR-1245a, miR-208a-3p, miR-9-3p) and their associated protein targets. The network reveals a high degree of connectivity, suggesting coordinated regulation of proteins involved in multiple functional systems, including immune response and tissue remodelling. To gain additional insight into the signaling context of these interactions, we further mapped the protein targets using STRING database analysis, assigning each protein to its respective biological pathway (Fig. 10D). Proteins are color-coded according to their involvement in specific signaling cascades identified via gene ontology enrichment analysis. Notably, the targets of IFNγ-induced miRNAs are significantly enriched in pathways such as Wnt signaling (11 proteins), T cell receptor signaling (9 proteins), chemokine signaling (8 proteins), osteoclast differentiation (7 proteins), B cell receptor signaling (6 proteins), Th1/Th2 cell differentiation (5 proteins), TGFβ signaling (5 proteins), and NFκB signaling (2 proteins). This analysis further supports that IFNγ priming selectively enhances exosomal miRNAs that converge on signaling networks central to both osteochondral tissue regeneration and immunomodulation.

Discussion

The therapeutic application of MSCs in regenerative medicine has gained considerable importance over the past two decades, particularly in the context of osteochondral disorders where both structural repair and immune modulation are required [49, 50]. Usually, BM-MSCs have been considered the reference source [51], however, increasing evidence points to other sources, such as adipose tissue and placenta, because of their unique biological properties, ease of procurement, and enhanced paracrine activity [31, 52]. The present study contributes to this growing body of evidence by providing a comprehensive proteomic and miRNA-based profiling of the secretomes derived from PDSCs and ASCs, thereby shedding light on the distinctive therapeutic potential of PDSCs for osteochondral regeneration.

The increasing interest in cell-free regenerative therapies represents a paradigm shift in the field of regenerative medicine [53]. While traditional MSC-based transplantation has shown promising results, many concerns regarding tumorigenicity, immunogenicity, and heterogeneity have limited its broad clinical application [54]. In this context, the use of CM and EVs, including EXOs, has emerged as a safer and more controllable strategy that preserves the beneficial paracrine functions of MSCs while avoiding risks associated to live-cell administration [18, 55, 56]. Several studies have demonstrated that MSC-derived secretome contains a plethora of trophic factors, cytokines, and miRNAs capable of modulating inflammation, stimulating angiogenesis, and promoting osteochondral regeneration [57, 58]. EXOs, in particular, act as natural carriers of proteins, lipids, and nucleic acids, ensuring targeted delivery and stability of therapeutic signals in recipient tissues [18]. This has positioned secretome- and EXO-based products as attractive next-generation therapeutic tools for cell-free immunoregenerative therapies, especially in degenerative joint disorders where both tissue repair and immune modulation are critical. However, secretome profile depends on the source of MSCs and the specific stimuli to which they are exposed [23, 34, 38, 53, 59]. Although the functional properties of MSC-derived products may share common regenerative features, for complex and dynamic conditions such as osteochondral disorders, it is crucial that therapeutic approaches be as targeted and disease-specific as possible. Tailoring the secretome composition to the pathological microenvironment can improve therapeutic efficacy, promote tissue-specific regeneration, and reduce off-target effects.

Within the context of degenerative joint disorders, our findings indicate that PDSCs release a broader repertoire of bioactive molecules linked to biological pathways governing osteochondral regeneration and immune regulation compared with ASCs. Proteomic analyses identified over 7,000 proteins in the MSC-derived secretomes, with PCA and clustering consistently separating PDSCs from ASCs (Fig. 2), underscoring tissue-specific differences in their paracrine landscapes. Importantly, proteins enriched in the PDSC secretome were strongly associated with biological pathways central to osteogenesis, chondrogenesis, extracellular matrix remodelling, angiogenesis, and immune regulation (Fig. 3). By contrast, ASC-derived proteins failed to display comparable enrichment in regenerative or immunomodulatory pathways. However, the absence of enrichment does not necessarily imply the absence of biological activity. ASCs remain a valuable source also due to their accessibility. One of the most striking observations of our work concerns the impact of priming strategies on PDSC functionality. It is increasingly recognized that the microenvironment critically shapes MSC behavior, and preconditioning approaches have been developed to mimic or enhance these context-specific stimuli [23, 34, 36, 38, 60, 61]. In this study, cytokine-based priming with IL1β and IFNγ, as well as hypoxic conditioning, was applied to PDSCs to evaluate changes in their secretome composition. Among these, IL1β priming was associated with the strongest enrichment of proteins linked to osteochondral-related biological pathways (Figs. 4 and 5). In particular, IL1β stimulation markedly upregulated proteins involved in bone maturation, ossification, and regulation of chondrogenesis, while simultaneously boosting mediators of immune responses (Fig. 4F). These findings are consistent with earlier evidence that IL1β, despite being classically categorized as a pro-inflammatory cytokine, can trigger adaptive MSC responses, leading to the release of immunoregulatory and trophic factors that facilitate tissue repair [62]. In fact, in our combined scoring analysis, IL1β priming conferred the highest osteochondral and immunomodulatory indices, reinforcing its value as a useful conditioning stimulus (Fig. 5). IFNγ priming, on the other hand, exerted a more pronounced functional impact on the exosomal miRNA repertoire. Even under basal conditions, PDSC-derived exosomes exhibited a unique miRNA cargo enriched in regulators of osteochondral and immune pathways, including members of the miR-29 family, miR-30c, and miR-15b (Fig. 7D). These miRNAs targeted key regulatory proteins governing processes such as osteoclast and osteoblast activation, Wnt/β-catenin and TGF-β signaling, and immune modulation (Figs. 7 and 8) [63–66]. Following IFNγ stimulation, PDSC-derived exosomes showed a selective upregulation of additional functionally relevant miRNAs, which were significantly enriched in pathways related to osteochondral regeneration and immune regulation, including Wnt signaling, T cell receptor signaling, chemokine signaling, osteoclast differentiation, B cell receptor signaling, Th1/Th2 polarization, TGF-β signaling, and NF-κB signaling (Fig. 10). These results suggest that IFNγ priming selectively enhances the exosomal component of the PDSC secretome, broadening its regulatory capacity beyond what is achievable through protein secretion alone. Interestingly, IL1β priming was most effective at enhancing therapeutic protein secretion, whereas IFNγ induced a broader and more functionally enriched exosomal miRNA profile. One possible explanation for this difference may lie in the distinct intracellular signaling pathways activated by these cytokines. IL1β is known to primarily signal through NF-kB [67], promoting the transcription and secretion of inflammatory and regulatory proteins, whereas IFNγ mainly activates the JAK/STAT pathway [68], which can modulate gene expression programs including miRNA transcription. In addition, scientific evidence suggests that specific signaling pathways may influence endosomal sorting mechanisms involved in the selective loading of miRNAs into extracellular vesicles [69]. Although these mechanisms were not directly investigated in the present study, this differential secretome modulation suggests that distinct priming stimuli may selectively shape protein- versus RNA-mediated components of MSC paracrine signaling.

Together, these results suggest that distinct priming strategies can tailor PDSC secretomes toward complementary therapeutic profiles: IL1β enhances protein-driven osteochondral and immunomodulatory pathways, whereas IFNγ boosts the regulatory potential mediated by exosomal miRNAs. The convergence of these findings supports the concept of a modular approach to MSC priming, where specific stimuli can be leveraged to design secretome formulations optimized for particular pathological contexts. In degenerative joint disorders, where chronic inflammation and impaired structural repair coexist [70, 71], the integration of both IL1β-primed proteins and IFNγ-induced miRNAs may offer synergistic benefits, simultaneously promoting matrix regeneration and resolving inflammatory processes. Moreover, given their robust anti-inflammatory and immunoregenerative properties, these primed secretomes may not only represent a promising therapeutic strategy for osteochondral pathologies, but also hold promise for other chronic degenerative illnesses, such as liver, heart, kidney, and lung diseases, in which inflammation and tissue regeneration are key determinants of disease progression and recovery [72].

Beyond their paracrine composition, PDSCs offer intrinsic biological advantages that further justify their use over other adult-tissue-derived MSCs. Perinatal cells are relatively younger in terms of developmental stage, which translates into higher proliferative capacity, genomic stability, and extended telomere length compared with other adult MSCs [73]. This makes PDSCs an appealing source for off-the-shelf therapies, reducing the variability and delays associated with cell preparation.

From a translational perspective, our findings reinforce the shift toward cell-free MSC-based therapies. While cell transplantation carries risks such as uncontrolled differentiation, tumorigenicity, and immune rejection, secretome- or EXO-based approaches offer a safer and more controllable alternative [74, 75]. Several preclinical studies have demonstrated that MSC-derived secretomes can promote cartilage repair, reduce synovial inflammation, and improve subchondral bone quality in models of osteoarthritis and rheumatoid arthritis [76–78]. Moreover, exosomal miRNAs are increasingly recognized as critical effectors of MSC paracrine activity, capable of fine-tuning the inflammatory microenvironment, as well as Wnt and TGF-β signaling, two pathways central to osteochondral homeostasis [18, 63–65]. Interestingly, these observations are in line with our findings (Figs. 4, 5, 8, and 9).

Despite these promising results, several challenges remain before clinical translation. First, the context-specific nature of priming responses highlights the need for precise tailoring to disease settings. What enhances therapeutic efficacy in osteoarthritis may differ from what is optimal in traumatic cartilage defects. Second, while in vitro analyses provide critical mechanistic insights, in vivo validation is essential to determine the stability and functional persistence of primed PDSC-derived products. A limitation of the present study is that the biological functions suggested by the omics analyses were not directly validated through functional assays tests. Therefore, the results should be interpreted as molecular indications of potential biological activity that require further experimental validation. Finally, regulatory considerations must be addressed, particularly regarding the classification of exosome-based therapeutics, their quality control, and their large-scale manufacturing.

Conclusion

In conclusion, the present study underscores the therapeutic potential of placenta-derived MSCs in osteochondral regeneration, particularly when their secretome is modulated through targeted priming strategies. IL1β priming enhances the protein fraction of the PDSC secretome, potentially driving osteogenic, chondrogenic, and immunomodulatory processes, while IFNγ selectively enriches the exosomal miRNA repertoire, extending regulatory influence across critical signaling pathways. Together, these results highlight PDSC-derived products as versatile and powerful candidates for next-generation cell-free therapies, capable of addressing both the structural and immunological hallmarks of degenerative joint diseases. By leveraging the unique properties of PDSCs and strategically applying priming stimuli, it may be possible to design highly tailored therapeutic products that move beyond the limitations of current MSC-based interventions, ultimately paving the way toward more effective and personalized regenerative strategies.

Supplementary Information

Below is the link to the electronic supplementary material.

Authors’ Contributions

M.E.L., E.S., M.L.P., R.V., O.L.R., C.P., G.A., V.I.: Data curation, Investigation, Writing–review and editing. D.S.S., A.G., N.C., S.L., A.C., A.M., N.R.: Data curation, Formal Analysis, Software, Writing–review and editing. V.A., P.G.C.: Critical revision, Writing–review and editing. V.M.: Conceptualization, Data curation, Formal Analysis, Methodology, Writing–original draft, Writing–review and editing. M.M.: Conceptualization, Data curation, Methodology, Project administration, Resources, Supervision, Writing–review and editing. M.B.: Conceptualization, Data curation, Methodology, Project administration, Resources, Supervision, Writing–original draft, Writing–review and editing.

Funding

This work was funded by the European Union - Next Generation EU - NRRP M6C2 - Investment 2.1 Enhancement and strengthening of biomedical research in the NHS (Project title; “Spine Unit modelling coupled with hIgh Throughput analysis (SUIT): targeting degeneration with cell secretome”, PNRR-MAD-2022-12376354, CUP I73C22000560001.

Data Availability

Proteomic data presented in the study are deposited in the ProteomeXchange repository with the dataset identifier PXD068866. Other original data presented in the study are openly available at the following link: https://osf.io/r7cjp/overview?view\_only=a0c5c05112a74887a8df9326e5e0903f.

Declarations

Ethics Approval and Consent to Participate

The studies involving humans were approved by the IRCCS ISMETT’s Institutional Research Review Board (IRRB/39/20). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

Competing Interests

The authors declare no competing interests.