Project description:Cervical cancer (CC) remains a global health challenge, with chemotherapy resistance and tumor recurrence limiting treatment success. Our study investigated the effects of mesenchymal stem cell-derived exosome (MSC-Exos) pretreatment on chemotherapy sensitivity in 3D spheroids generated from HeLa and SiHa CC cell lines. Proteomic profiling of MSC-Exos revealed key proteins, including ANXA1, ANXA2, EF2, LGALS1, and PKM2, with potential roles in tumor regeneration and chemosensitization. Our findings highlight the context-dependent nature of MSC-Exo activity: while they may promote chemoresistance via drug efflux, metabolic reprogramming, and stress adaptation, they can also enhance chemosensitivity by modulating apoptosis, DNA damage response, and integrin-mediated signaling pathways. Functionally, spheroids pretreated with MSC-Exos exhibited altered responses to paclitaxel in combination with either cisplatin or carboplatin, underscoring the potential of MSC-Exos as modulators of chemotherapy response in CC. In HeLa spheroids, pretreatment with MSC-Exo significantly enhanced chemotherapy-induced cytotoxicity, evidenced by decreased cell viability, increased caspase activity, and upregulation of pro-apoptotic markers such as Bax, suggesting sensitization to apoptosis via chemotherapy. Conversely, SiHa spheroids exhibited variable responses. Although MSC-Exo pretreatment did not sensitize SiHa spheroids to paclitaxel-cisplatin, it improved responsiveness to paclitaxel–carboplatin, particularly in the spheroid core. Molecular analysis revealed upregulated SOX2 expression in SiHa spheroids, indicating partial activation of stemness pathways without full acquisition of a cancer stem cell phenotype. Overall, our findings reveal that MSC-Exo pretreatment exerts cell type- and drug-specific effects in CC spheroids. They enhance chemotherapeutic efficacy in HeLa spheroids and selectively alter drug sensitivity in more resistant SiHa spheroids. The results, therefore, represent promising candidates for engineered exosome-based adjuvant therapies in CC.
Project description:Exosomes derived from mesenchymal stem cells (MSCs) have shown to have effective application prospects in the medical field, but exosome yield is very low. The production of exosome mimetic vesicles (EMVs) by continuous cell extrusion leads to more EMVs than exosomes, but whether the protein compositions of MSC-derived EMVs (MSC-EMVs) and exosomes (MSC-exosomes) are substantially different remains unknown. The purpose of this study was to conduct a comprehensive proteomic analysis of MSC-EMVs and MSC-exosomes and to simply explore the effects of exosomes and EMVs on wound healing ability. This study provides a theoretical basis for the application of EMVs and exosomes.In this study, EMVs from human umbilical cord MSCs (hUC MSCs) were isolated by continuous extrusion, and exosomes were identified after hUC MSC ultracentrifugation. A proteomic analysis was performed, and 2,315 proteins were identified. The effects of EMVs and exosomes on the proliferation, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by cell counting kit-8, scratch wound, Transwell and tubule formation assays. A mouse mode was used to evaluate the effects of EMVs and exosomes on wound healing . Bioinformatics analyses revealed that 1,669 proteins in both hUC MSC-EMVs and hUC MSC-exosomes play roles in retrograde vesicle-mediated transport and vesicle budding from the membrane. The 382 proteins unique to exosomes participate in extracellular matrix organization and extracellular structural organization, and the 264 proteins unique to EMVs target the cell membrane. EMVs and exosomes can promote wound healing and angiogenesis in mice and promote the proliferation, migration and angiogenesis of HUVECs.
Project description:Mesenchymal stromal cells (MSCs) hold great promise in regenerative medicine due to their potential to promote tissue repair and regeneration. However, applying MSC-based cell therapy in actual clinical settings remains challenging due to issues such as immunocompatibility and cell stability. Recent shifts in therapeutic strategies have highlighted MSC-derived exosomes, small vesicles carrying various bioactive molecules, as a promising cell-free therapy to promote tissue regeneration. However, it remains largely unknown regarding the ability to customize the content of MSC-derived exosomes, how alterations in the MSC microenvironment influence exosome content, and the effects of such alterations on healing efficiency and mechanical properties in tissue repair when utilized as a therapy. In this study, we used an in vitro system of human MSC-derived exosomes and an in vivo rat ligament injury model to address these questions. We found a context-dependent correlation between exosomal and parent cell RNA content. Under native conditions, the correlation was moderate but heightened with microenvironmental changes. In vivo rat ligament injury model showed that MSC-derived exosomes increased ligament max load and stiffness. We also found that changes in the MSCs' microenvironment significantly influence the mechanical properties driven by exosome treatment. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.
Project description:Compared to conventional monolayer cell culture, three-dimensional (or spheroid) cultures are more reflective of the in vivo environment and represent a better means of representing normal tissue. Therefore, understanding the biology of spheroid-derived cells is important for a more complete appreciation of in vivo tissue function. Although it has been shown that culture conditions such as nutrients, oxygen, cell passage etc. alter gene expression patterns of the cells, the gene expression profile of spheroid as compared to conventional monolayer culture in primary cells has not been studied. To investigate the effects of spheroid culture system on gene expression of primary cells, single cell RNA sequencing (scRNAseq) was performed on mouse dermal fibroblasts (tail/ear fibroblasts, TEFs) cultured as monolayers on cell culture dishes or grown as spheroids. After quality control, a total of 11,515 cells (8,022 spheroid cells and 3,493 cells of monolayer fibroblasts) were analyzed using Uniform Manifold Approximation and Projection (UMAP) which identified 2 largely separated libraries. These libraries contained cells with 2,491 and 8722 detected genes (nFeature_RNA) for monolayer and spheroid cells respectively. UMAP clustering of the integrated datasets identified 8 significant cell clusters (resolution=0.3) in which clusters 0, 1, 2, 4 and 7 had a higher number of cells in the spheroid library. Differential gene expression analyses revealed that each cluster was characterized by a specific transcriptional profile. Among cell clusters, cluster 4 exhibited significantly increased expression of collagen family genes, including Col1a2, Col3a1, Col1a1, Col5a2, Col4a, Col4a2, Col5a3, Col5a1, Col6a1, Col12a1 and Col15a1. Furthermore, expression of mesenchymal stem cell genes [Sca-1 (Ly6a), CD29 (Itgb1), CD44, CD90.1 (Thy1)] as well as a group of genes important for stem cell self-renewal including Notch2, Sox4, Sox9 Klf2, and Foxp1 were significantly increased in cluster 4. Immunofluorescence (IF) staining was performed to validate the protein expression of selected genes. Our work provides a significant advance in characterizing the global gene expression profile of spheroid culture of mouse TEFs compared to monolayer culture. We reached a high resolution at single-cell level, which enabled us to identify specific clusters associated with mesenchymal stem-like cells.