RESUMO
Endothelial-like cells may be obtained from CD133+ mononuclear cells isolated from human umbilical cord blood (hUCB) and expanded using endothelial-inducing medium (E-CD133 cells). Their use in regenerative medicine has been explored by the potential not only to form vessels but also by the secretion of bioactive elements. Extracellular vesicles (EVs) are prominent messengers of this paracrine activity, transporting bioactive molecules that may guide cellular response under different conditions. Using RNA-Seq, we characterized the miRNA content of EVs derived from E-CD133 cells cultivated under normoxia (N-EVs) and hypoxia (H-EVs) and observed that changing the O2 status led to variations in the selective loading of miRNAs in the EVs. In silico analysis showed that among the targets of differentially loaded miRNAs, there are transcripts involved in pathways related to cell growth and survival, such as FoxO and HIF-1 pathways. The data obtained reinforce the pro-regenerative potential of EVs obtained from E-CD133 cells and shows that fine tuning of their properties may be regulated by culture conditions.
Assuntos
Vesículas Extracelulares , MicroRNAs , Proliferação de Células , Células Endoteliais/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Hipóxia/metabolismo , MicroRNAs/metabolismoRESUMO
Tissue engineering is a branch of regenerative medicine, which comprises the combination of biomaterials, cells and other bioactive molecules to regenerate tissues. Biomaterial scaffolds act as substrate and as physical support for cells and they can also reproduce the extracellular matrix cues. Although tissue engineering applications in cellular therapy tend to focus on the use of specialized cells from particular tissues or stem cells, little attention has been paid to endothelial progenitors, an important cell type in tissue regeneration. We combined 3D printed poly(lactic acid) scaffolds comprising two different pore sizes with human adipose-derived stromal cells (hASCs) and expanded CD133+ cells to evaluate how these two cell types respond to the different architectures. hASCs represent an ideal source of cells for tissue engineering applications due to their low immunogenicity, paracrine activity and ability to differentiate. Expanded CD133+ cells were isolated from umbilical cord blood and represent a source of endothelial-like cells with angiogenic potential. Fluorescence microscopy and scanning electron microscopy showed that both cell types were able to adhere to the scaffolds and maintain their characteristic morphologies. The porous PLA scaffolds stimulated cell cycle progression of hASCs but led to an arrest in the G1 phase and reduced proliferation of expanded CD133+ cells. Also, while hASCs maintained their undifferentiated profile after 7 days of culture on the scaffolds, expanded CD133+ cells presented a reduction of the von Willebrand factor (vWF), which affected the cells' angiogenic potential. We did not observe changes in cell behavior for any of the parameters analyzed between the scaffolds with different pore sizes, but the 3D environment created by the scaffolds had different effects on the cell types tested. Unlike the extensively used mesenchymal stem cell types, the 3D PLA scaffolds led to opposite behaviors of the expanded CD133+ cells in terms of cytotoxicity, proliferation and immunophenotype. The results obtained reinforce the importance of studying how different cell types respond to 3D culture systems when considering the scaffold approach for tissue engineering.
RESUMO
BACKGROUND: Previous studies have demonstrated remarkable tropism of mesenchymal stem cells (MSCs) toward malignant gliomas, making these cells a potential vehicle for delivery of therapeutic agents to disseminated glioblastoma (GBM) cells. However, the potential contribution of MSCs to tumor progression is a matter of concern. It has been suggested that CD133+ GBM stem cells secrete a variety of chemokines, including monocytes chemoattractant protein-1 (MCP-1/CCL2) and stromal cell-derived factor-1(SDF-1/CXCL12), which could act in this tropism. However, the role in the modulation of this tropism of the subpopulation of CD133+ cells, which initiate GBM and the mechanisms underlying the tropism of MSCs to CD133+ GBM cells and their effects on tumor development, remains poorly defined. METHODS/RESULTS: We found that isolated and cultured MSCs (human umbilical cord blood MSCs) express CCR2 and CXCR4, the respective receptors for MCP-1/CCL2 and SDF-1/CXCL12, and demonstrated, in vitro, that MCP-1/CCL2 and SDF-1/CXC12, secreted by CD133+ GBM cells from primary cell cultures, induce the migration of MSCs. In addition, we confirmed that after in vivo GBM tumor establishment, by stereotaxic implantation of the CD133+ GBM cells labeled with Qdots (705 nm), MSCs labeled with multimodal iron oxide nanoparticles (MION) conjugated to rhodamine-B (Rh-B) (MION-Rh), infused by caudal vein, were able to cross the blood-brain barrier of the animal and migrate to the tumor region. Evaluation GBM tumors histology showed that groups that received MSC demonstrated tumor development, glial invasiveness, and detection of a high number of cycling cells. CONCLUSIONS: Therefore, in this study, we validated the chemotactic effect of MCP-1/CCL2 and SDF-1/CXCL12 in mediating the migration of MSCs toward CD133+ GBM cells. However, we observed that, after infiltrating the tumor, MSCs promote tumor growth in vivo probably by release of exosomes. Thus, the use of these cells as a therapeutic carrier strategy to target GBM cells must be approached with caution.
Assuntos
Antígeno AC133/metabolismo , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Neoplásicas/patologia , Tropismo , Animais , Neoplasias Encefálicas/ultraestrutura , Carcinogênese/metabolismo , Carcinogênese/patologia , Ensaios de Migração Celular , Proliferação de Células , Separação Celular , Quimiocinas/metabolismo , Glioblastoma/ultraestrutura , Humanos , Imunofenotipagem , Masculino , Células-Tronco Mesenquimais/ultraestrutura , Modelos Biológicos , Células-Tronco Neoplásicas/ultraestrutura , Pontos Quânticos/metabolismo , Ratos Wistar , Receptores de Quimiocinas/metabolismo , Esferoides Celulares/patologia , Células Tumorais CultivadasRESUMO
Background Previous studies have demonstrated remarkable tropism of mesenchymal stem cells (MSCs) toward malignant gliomas, making these cells a potential vehicle for delivery of therapeutic agents to disseminated glioblastoma (GBM) cells. However, the potential contribution of MSCs to tumor progression is a matter of concern. It has been suggested that CD133+ GBM stem cells secrete a variety of chemokines, including monocytes chemoattractant protein-1 (MCP-1/CCL2) and stromal cell-derived factor-1(SDF-1/CXCL12), which could act in this tropism. However, the role in the modulation of this tropism of the subpopulation of CD133+ cells, which initiate GBM and the mechanisms underlying the tropism of MSCs to CD133+ GBM cells and their effects on tumor development, remains poorly defined. Methods/results We found that isolated and cultured MSCs (human umbilical cord blood MSCs) express CCR2 and CXCR4, the respective receptors for MCP-1/CCL2 and SDF-1/CXCL12, and demonstrated, in vitro, that MCP-1/CCL2 and SDF-1/CXC12, secreted by CD133+ GBM cells from primary cell cultures, induce the migration of MSCs. In addition, we confirmed that after in vivo GBM tumor establishment, by stereotaxic implantation of the CD133+ GBM cells labeled with Qdots (705 nm), MSCs labeled with multimodal iron oxide nanoparticles (MION) conjugated to rhodamine-B (Rh-B) (MION-Rh), infused by caudal vein, were able to cross the blood-brain barrier of the animal and migrate to the tumor region. Evaluation GBM tumors histology showed that groups that received MSC demonstrated tumor development, glial invasiveness, and detection of a high number of cycling cells. Conclusions Therefore, in this study, we validated the chemotactic effect of MCP-1/CCL2 and SDF-1/CXCL12 in mediating the migration of MSCs toward CD133+ GBM cells. However, we observed that, after infiltrating the tumor, MSCs promote tumor growth in vivo probably by release of exosomes. Thus, the use of these cells as a therapeutic carrier strategy to target GBM cells must be approached with caution.
RESUMO
Adult stem cells have beneficial effects when exposed to damaged tissue due, at least in part, to their paracrine activity, which includes soluble factors and extracellular vesicles (EVs). Given the multiplicity of signals carried by these vesicles through the horizontal transfer of functional molecules, human mesenchymal stem cell (hMSCs) and CD133+ cell-derived EVs have been tested in various disease models and shown to recover damaged tissues. In this study, we profiled the protein content of EVs derived from expanded human CD133+ cells and bone marrow-derived hMSCs with the intention of better understanding the functions performed by these vesicles/cells and delineating the most appropriate use of each EV in future therapeutic procedures. Using LC-MS/MS analysis, we identified 623 proteins for expanded CD133+-EVs and 797 proteins for hMSCs-EVs. Although the EVs from both origins were qualitatively similar, when protein abundance was considered, hMSCs-EVs and CD133+-EVs were different. Gene Ontology (GO) enrichment analysis in CD133+-EVs revealed proteins involved in a variety of angiogenesis-related functions as well proteins related to the cytoskeleton and highly implicated in cell motility and cellular activation. In contrast, when overrepresented proteins in hMSCs-EVs were analyzed, a GO cluster of immune response-related genes involved with immune response-regulating factors acting on phagocytosis and innate immunity was identified. Together our data demonstrate that from the point of view of protein content, expanded CD133+-EVs and hMSCs-EVs are in part similar but also sufficiently different to reflect the main beneficial paracrine effects widely reported in pre-clinical studies using expanded CD133+ cells and/or hBM-MSCs.