pMSCs were isolated from the bone marrow of femurs and tibiae obtained under aseptic conditions from healthy piglets. Initially, primary (P0) pMSCs appeared scattered after adhesion. After 72 h, pMSCs proliferated to form distinct colony clusters. Upon passaging, the cells adopted a homogeneous, elongated, spindle-shaped morphology (Figures 1A,B). The isolated cells were positive for the mesenchymal stem cell markers CD90 and CD44 (both >98% positivity) and negative for the hematopoietic and immune markers CD4, CD14, CD19, CD34, CD45 and CD80, confirming a characteristic pMSC surface phenotype (Figure 1C; Supplementary Figures S1A–C). The multipotent differentiation potential was evaluated by inducing osteogenic, adipogenic, and chondrogenic lineages. After induction, the formation of calcium nodules was confirmed by Alizarin Red staining, lipid droplet accumulation was revealed by Oil Red O staining, and glycosaminoglycan deposition in the cartilage matrix was detected by Alcian Blue staining (Figure 1D). These results verify that pMSCs have the capability to differentiate into osteocytes, adipocytes, and chondrocytes. In summary, through morphological assessment, surface marker analysis, and multilineage differentiation assays, we successfully isolated and expanded highly pure and functional pMSCs.

The efficacy of human cell xenotransplantation into porcine hosts may be limited by interspecies physiological differences, particularly with respect to body temperature and the competition among xenogeneic cells. We first investigated the impact of temperature using CCK-8 assays. The results indicated that the physiological temperature of pigs (39 °C) significantly inhibited the proliferation of hMSCs. In contrast, the proliferation of pMSCs was not significantly affected when comparing 37 °C–39 °C, underscoring their strong adaptability to human body temperature. These findings demonstrate that the porcine thermal environment exerts a substantial inhibitory effect on hMSCs (Figures 2A,B).

We then investigated the interactions between human and porcine cells by four experiments: hMSCs were cultured at 37 °C (L separate culture), hMSCs were cultured at 39 °C (H separate culture), hMSCs were co-cultured with pMSCs at 37 °C (L co-culture), and hMSCs were co-cultured with pMSCs at 39 °C (H co-culture). The proliferation of GFP-labeled hMSCs was evaluated using cell counting and flow cytometry (Figure 2C). Flow cytometric analysis revealed a time-dependent decline in the proportion of GFP-positive hMSCs during co-culture. Compared to the separate culture group, the proliferation rate of hMSCs was significantly reduced under co-culture conditions (Figures 2D,E). These results indicate that pMSCs could impair the survival and proliferation of hMSCs under co-culture conditions at both 37 °C and 39 °C, thereby influencing the overall efficiency of human cell xenotransplantation.

Given that porcine cells significantly outnumber human cells during xenotransplantation of human cells into pigs, we examined the effect of pMSC concentration on hMSC proliferation through five experimental groups: hMSCs cultured alone and hMSCs co-cultured with pMSCs at cell number ratios of 1:1, 1:4, 1:6, and 1:9. The results indicated that higher pMSC ratios led to a stronger inhibition of hMSC proliferation, which was consistently observed at both 37 °C and 39 °C (Figures 3A,B). Notably, the higher temperature (39 °C) and co-culture exerted a synergistic inhibitory effect on hMSC proliferation. To investigate the underlying mechanism, immunofluorescence analysis was performed, and the results revealed a markedly increased expression of the apoptosis-related protein cleaved caspase-3 in GFP-labeled hMSCs under co-culture conditions compared to the control (Figures 3C,D), suggesting that pMSCs may inhibit proliferation by inducing apoptosis in hMSCs through undefined mechanisms.

To elucidate the potential mechanisms underlying the inhibition of hMSC proliferation mediated by pMSCs, we employed a transwell non-contact co-culture assay to investigate whether the soluble factors secreted by pMSCs contribute to the suppression of hMSC growth. Notably, the results demonstrated that pMSCs at different proportions in the non-contact co-culture system did not significantly affect hMSC proliferation (Figures 4A,B), suggesting that the inhibitory effect of pMSCs on hMSCs may be attributed to the direct cell-to-cell contact rather than to the secreted soluble factors.

To further validate the role of soluble factors, the conditioned medium (CM) from pMSCs was extracted and purified. The impact of the CM on hMSC proliferation was assessed via CCK-8 assays at varying concentrations and treatment times. The results indicated that concentrations of 200–400 μg/ml of pMSC-CM did not exert a significant effect on hMSC proliferation. Treatment with 600 μg/mL of CM for 12 h induced a slight promotion, which disappeared when the treatment time was extended to 24 h (Figures 4C,D). The modest promotion observed at the 12-h time point for pMSC-CM may attribute to the presence of growth factors, cytokines, and other bioactive components. However, many of these factors have relatively shorter half-lives, complicating the maintenance of their activity over extended periods. Furthermore, subtle variations in the concentrations of active components may occur between different batches of pMSC-CM, contributing to the observed fluctuations in the promotional effect at the 12-h time point across multiple independent replicate experiments. These results suggest that the soluble factors secreted by pMSCs may not be the primary inhibitors of hMSC proliferation.

To elucidate the molecular mechanisms underlying the suppression of hMSC proliferation by pMSCs, we isolated GFP-labelled hMSCs from the co-culture system using cell sorting technology and conducted RNA-seq analysis (Figure 5A). Compared to monoculture, hMSCs in co-culture exhibited significant upregulation of 889 genes and downregulation of 270 genes. Notably, several proliferation-related genes, including CYP1B1, SLC7A11, PSAT1, and TFAP2C, were significantly downregulated in the co-culture group, which was further confirmed by qPCR (Figures 5B,C). Gene Ontology (GO) enrichment analysis indicated that the differentially expressed genes were primarily associated with biological processes such as endoplasmic reticulum protein folding, response to misfolded proteins, and amino acid transport, suggesting that co-culture disrupts protein homeostasis and metabolic functions in hMSCs (Figure 5D). Given that direct cell-cell contact could cause an inhibitory effect on proliferation, we next examined the expression alterations of cell junction-related molecules. Notably, significant downregulation of key tight junction molecules, including OCLN, CLDN1, CLDN11, and TJP1, was found in co-cultured hMSCs. Among adhesion junction-associated molecules, the baseline expression level of CDH1 (encoding α-catenin) decreased, whereas CDH2 (encoding β-catenin) showed no significant difference between groups we tested (Figure 5E).

Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed significant enrichment of signaling pathways associated with cellular competition and proliferation regulation, including NF-κB, PI3K-AKT, and JAK-STAT. Among these, alterations in the NF-κB pathway were particularly pronounced (Figure 6A; Supplementary Figures 2A-C). qPCR results confirmed that the mRNA expression of key NF-κB pathway genes, IL1B and TNFAIP3, was significantly upregulated in co-cultured hMSCs (Figure 6B). Employing in vitro stimulation of hMSCs with interleukin-1β (IL-1β), the activation status of the NF-κB pathway and its role in regulating hMSC proliferation was further investigated. Western blot (WB) analysis demonstrated a decrease in IκBα expression alongside elevated levels of p-IκBα and TNFα, confirming NF-κB pathway activation. Importantly, following NF-κB activation, the expression levels of cyclin D1 and proliferating cell nuclear antigen (PCNA) proteins were significantly downregulated, suggesting an impairment in cellular proliferation capacity (Figure 6C). Further immunofluorescence results indicated a significant reduction in the mean fluorescence intensity of the proliferation marker protein Ki67 in the IL-1β-treated group (Figures 6D,E). Moreover, the CCK-8 assay confirmed that the cell proliferation was markedly suppressed following IL-1β treatment (Figure 6F). These findings collectively suggest that pMSCs impair the proliferative capacity of hMSCs by activating the NF-κB pathway.

Human fetal bone marrow MSCs were purchased from Cyagen Biosciences (HUXMF-01001, China). hMSCs and pMSCs were cultured in low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (OriCell, China), 1% non-essential amino acids (Gibco, USA), 1% GlutaMAX (Gibco, USA), and 1% penicillin-streptomycin (Transgen, China) at 95% humidity and 5% CO₂, with temperatures set at either 37 °C or 39 °C. HEK293FT cells were maintained in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

Healthy piglets within 7 days postnatally were selected. Under aseptic conditions, the femurs and tibiae were dissected, disinfected twice with 75% ethanol, and rinsed three times with phosphate-buffered saline (PBS) containing penicillin-streptomycin. Bone marrow was flushed out using complete medium and cultured at 37 °C with 5% CO₂ under 95% humidity. Half of the medium was replaced every 12 h. After 72 h, the culture dish was washed twice with PBS to remove non-adherent cells, and complete medium was replenished for continued culture. Thereafter, the medium was changed every 3 days. Upon reaching 80%–90% confluence, the cells were either cryopreserved or passaged for subsequent experiments.

pMSCs (1 × 10⁶) were washed with FACS buffer and resuspended in 100 μL of FACS buffer. The cells were incubated at 4 °C for 30 min in the dark with fluorochrome-conjugated primary antibodies: anti-CD90 APC (559869, BD), anti-CD44 BV785 (103041, BD), anti-CD45 BV480 (566115, BD), anti-CD14 BV650 (563420, BD), and anti-CD34 FITC (555821, BD). After washing, the cells were analyzed using A5 flow cytometry, and the data were processed using FlowJo software.

pMSCs were seeded into a 12-well plate at a density of 2 × 10⁴ cells per well. Once the cell density reached 70%, the medium was replaced with osteogenic induction medium (OriCell, China), which was changed every 3 days during the osteogenic differentiation process. On day 9, when calcium nodule formation was observed, the cells were fixed with 4% paraformaldehyde (PFA) for 30 min. Subsequently, the cells were stained with alizarin red for 5–10 min. The results were then observed and photographed under microscope.

Cells were seeded into a 12-well plate at a density of 3 × 10⁴ cells per well. Once the cell density reached 100%, the medium was replaced with adipogenic induction medium A (OriCell, China). After 3 days of culture, medium A was substituted with induction medium B, and the two media were alternated thereafter. Approximately 2 weeks later, when lipid droplet formation was observed, the cells were fixed with 4% PFA for 30 min. Subsequently, the cells were stained with Oil Red O for 30 min. The results were then observed and photographed under a microscope.

pMSCs were cultured at a density of 4 × 10⁵ cells in 15 mL tubes and centrifuged at 250 g for 5 min. The cell pellets were resuspended in 0.5 mL of chondrogenic media (OriCell, China) and subjected to a second centrifugation at 150 g for 5 min. The chondrogenic media was replaced every 2 days until chondrospheres were observed. Subsequently, the chondrospheres were fixed in 4% PFA for 30 min. Following fixation, the chondrospheres were embedded in optimal cutting temperature compound and stored at −80 °C. Continuous frozen sections were prepared, and Alcian blue staining was performed. The results were then observed and photographed under a microscope.

Cell proliferation assays were conducted using a CCK-8 reagent (Beyotime, China). Cells were seeded in 96-well plates at a density of 1 × 10³ cells per well in 100 μL of complete medium. At the indicated time points, 10 μL of CCK-8 reagent was added to each well and incubated at 37 °C for 2 h. The absorbance was measured at 450 nm. All samples were tested in three independent experiments.

The pLenti-CMV-GFP cloning vector (Addgene 17448), psPAX2, and pMD2.G were co-transfected into HEK293FT cells using Lipofectamine 2000 reagent (Invitrogen, USA) according to the manufacturer’s instructions. The culture supernatant containing lentivirus was collected and concentrated using ultrafiltration centrifuge tubes (Millipore, USA). MSCs were seeded in a 12-well plate at a density of 3 × 10⁴ cells per well and cultured until reaching 30%–40% confluence. The cells were infected with the concentrated lentivirus and 5 μg/mL Polybrene (Sigma, USA) to enhance transduction efficiency. After 12 h of infection, the virus-containing medium was removed, and fresh complete medium was added for continued cultivation. About 7 days later, GFP-positive cells were sorted using a BD flow cytometer (BD, USA) after digestion with 0.25% trypsin and filtration through a 70 μm sterile cell strainer. The sorted cells were cultured for 1 week to establish stable cell lines. The transduction efficiency of the lentivirus was confirmed through qPCR and WB analysis.

The experiment included five groups: hMSCs cultured alone, and hMSCs co-cultured with pMSCs at ratios of 1:1, 1:4, 1:6, and 1:9. The initial total cell number was kept postnatally across all groups by adjusting the seeding density of each cell type. On days 4 and 8 after seeding, the cells were digested and collected. The total cell number was obtained using a cell counter, and the proportion of GFP-positive hMSCs was determined by flow cytometry. The absolute number of hMSCs in each group was calculated by multiplying the total cell count by the percentage of GFP-positive cells. Data were processed using the formula: normalized growth curve = log_e(final cell number/initial cell number)/log_e2, and the normalized growth curve of hMSCs was plotted accordingly.

The indicated cells (1 × 10⁴) were seeded on glass slides in 24-well plates. The cells were washed with PBS and fixed in 4% PFA for 20 min at room temperature. Fixed cells were permeabilized with 1% Triton X-100 for 10 min and blocked with 1% bovine serum albumin supplemented with 22.52 mg/mL glycine for 30 min at room temperature. Subsequently, the cells were incubated overnight at 4 °C with primary antibodies: c-caspase3 (Cell Signaling Technology, USA) or Ki67 (Abcam, USA). The cells were then washed three times with PBST (PBS +0.1% Tween 20) and incubated at room temperature for 1 h with secondary antibodies: goat anti-rabbit IgG H&L (Alexa Fluor® 555) (Abcam, USA) or goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam, USA). The cell nuclei were stained with DAPI (Beyotime, China) for 15 min at room temperature in the dark. Finally, images were captured using a Zeiss LSM880 confocal microscope.

Human and porcine MSCs were indirectly co-cultured in a Transwell system with 0.4 μm pores. The upper chamber was seeded with hMSCs (1 × 10⁴), while the lower chamber contained either hMSCs (separate culture) or pMSCs (co-culture) at varying seeding ratios. After 4 days of culture, the proliferation of hMSCs in the upper chamber was assessed using the CCK-8 kit.

The pMSCs with 80%–90% confluence were washed three times with PBS and cultured in serum-free medium for 24–48 h. The supernatant was collected and centrifuged at 300 g for 10 min at 4 °C to remove residual cells, followed by a second centrifugation at 2000 g for 10 min at 4 °C to pellet cellular debris. The solution was then filtered through a 0.22 μm membrane and concentrated using a 3 kDa Millipore ultrafiltration device by centrifugation at 4,000 g for 40 min at 4 °C. The protein concentration of the CM was quantified using the Pierce™ BCA Protein Detection Kit (Thermo, USA) and stored at −80 °C for future use.

A total of 16 × 10⁴ GFP-labeled hMSCs (single-culture group) or 2.28 × 10⁴ GFP-labeled hMSCs combined with 13.72 × 10⁴ pMSCs (co-culture group) were seeded into 10 cm culture dishes, with three replicates for each group. Once the cells reached confluence, they were digested, centrifuged at 1,000 rpm for 5 min, and resuspended in 800 μL of FACS buffer. The suspension was then filtered through a 70 μm sterile cell strainer. GFP-expressing hMSCs (5 × 10⁴ cells) were isolated using cell sorting, washed with PBS, and centrifuged again at 1,000 rpm for 5 min. The resulting cell pellet was resuspended in 400 μL of TRIzol and stored at −80 °C.

Total RNA was extracted from MSCs using TRIzol reagent (Invitrogen, USA) in accordance with the manufacturer’s protocol. For single library preparation, the total RNA requirement was set at ≥1 μg, with a concentration of ≥35 ng/μL, an OD260/280 ratio of ≥1.8, and an OD260/230 ratio of ≥1.0. RNA concentration and purity were assessed using the ND-2000 spectrophotometer (NanoDrop, USA). RNA purification, reverse transcription, library preparation, and sequencing were performed following the operational guidelines (Illumina, USA). Sequence libraries underwent 2 × 150 bp paired-end sequencing on the Illumina NovaSeq 6,000 platform, facilitated by Cosmos Wisdom Biotech Co., Ltd. (Hangzhou, China). Raw data were filtered using fastp software to remove adapter-containing reads, trim terminal N bases, and discard low-quality reads (reads where bases with Q_phred ≤ 20 constitute over 50% of the total read length). Alignment against the human reference genome (Homo sapiens GRCh38 release-109) was conducted using Hisat2 software. Based on the alignment results, differentially expressed genes (DEGs) were analyzed using the DESeq2 R package. Genes were identified as differentially expressed if they met the criteria of a P-value ≤0.05 and an absolute fold change ≥2. GO and KEGG enrichment analyses of DEGs were conducted using the cluster Profiler R package, with enrichment significance assessed using a corrected P-value ≤ 0.05 threshold. Finally, ggplot2 was utilized to visualize the enrichment results.

Total RNA was extracted from the MSCs using TRIzol (Invitrogen, USA). The purity and concentration of the RNA were determined using a NanoDrop ND-2000 spectrophotometer (Thermo, USA), with an A260/A280 ratio of 1.8–2.0 considered acceptable. The RNA was reverse-transcribed into cDNA using RevertAid Master Mix (Thermo, USA). The validated or designed primer sets utilized are listed in Table 1 qPCR was performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, USA). Mean cycle threshold values from triplicate measurements were used to calculate gene expression using the 2^−ΔΔCt method, normalizing to GAPDH as the internal control.

When hMSCs reached 70% confluence, 10, 20, or 40 ng/mL of IL-1β (Peprotech, USA) was added into the culture medium to activate the NF-κB pathway. The cells were harvested for subsequent Western blot analyses at the indicated time points.

Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40), supplemented with protease inhibitors (Complete Mini, Roche) and phosphatase inhibitors (phosphatase inhibitor cocktail sets A and B, Biomake). Cell lysates were centrifuged at 12,000 × g for 15 min at 4 °C, and the supernatant was collected. Protein concentrations were determined using the Pierce™ BCA Protein Detection Kit (Thermo, USA). Approximately 30 μg of protein was analyzed via 10% SDS-polyacrylamide gel electrophoresis. The resolved proteins were transferred onto polyvinylidene difluoride membranes, which were blocked with 5% skim milk for 1 h at room temperature. The membranes were incubated for 12 h with primary antibodies: anti-TNFα (Proteintech, China), anti-p-IKBα (Selleck, USA), anti-cyclinD1 and anti-PCNA (Immunoway, China) and anti-IKBα (Abcam, USA) (Su et al., 2014; Aierken et al., 2022; Qian et al., 2024; Zhao et al., 2025; Zhou et al., 2025). To avoid issues of target band overlap and interference from non-specific bands, we selected the anti-Vinculin antibody (Sigma, USA) as our internal control (Wenzel et al., 2024). The membranes were then washed three times with TBST (20 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.1% Tween 20) and incubated with the corresponding secondary antibodies: goat anti-rabbit IgG H&L or goat anti-mouse IgG H&L (Abcam, USA) at room temperature for 1 h. Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Thermo, USA) with an image analyzer.

All experiments were performed with at least three independent biological replicates. Values were represented as mean ± standard error of the mean (SEM). Data analysis was performed GraphPad Prism 9 statistical software. Comparisons between two groups were analyzed using t-test. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.