ABSTRACT
Obtaining oligodendroglial cells from dispensable tissues would be of great interest for autologous or immunocompatible cell replacement therapy in demyelinating diseases, as well as for studying myelin-related pathologies or testing therapeutic approaches in culture. We evaluated the feasibility of generating oligodendrocyte precursor cells (OPCs) from adult rat adipose tissue by expressing genes encoding transcription factors involved in oligodendroglial development. Adipose-derived mesenchymal cells were lentivirally transduced with tetracycline-inducible Sox10, Olig2, Zfp536, and/or Nkx6.1 transgenes. Immunostaining with the OPC-specific O4 monoclonal antibody was used to mark oligodendroglial induction. O4- and myelin-associated glycoprotein (MAG)-positive cells emerged after 3 weeks when using the Sox10 + Olig2 + Zfp536 combination, followed in the ensuing weeks by GFAP-, O1 antigen-, p75NTR (low-affinity NGF receptor)-, and myelin proteins-positive cells. The O4+ cell population progressively expanded, eventually constituting more than 70% of cells in culture by 5 months. Sox10 transgene expression was essential for generating O4+ cells but was insufficient for inducing a full oligodendroglial phenotype. Converted cells required continuous transgene expression to maintain their glial phenotype. Some vestigial characteristics of mesenchymal cells were maintained after conversion. Growth factor withdrawal and triiodothyronine (T3) supplementation generated mature oligodendroglial phenotypes, while FBS supplementation produced GFAP+- and p75NTR+-rich cultures. Converted cells also showed functional characteristics of neural-derived OPCs, such as the expression of AMPA, NMDA, kainate, and dopaminergic receptors, as well as similar metabolic responses to differentiation-inducing drugs. When co-cultured with rat dorsal root ganglion neurons, the converted cells differentiated and ensheathed multiple axons. We propose that functional oligodendroglia can be efficiently generated from adult rat mesenchymal cells by direct phenotypic conversion.
ABSTRACT
BACKGROUND: Insulin receptor substrate 4 (IRS-4) is an adaptor protein for which new evidence suggests plays a role in tumour promotion. METHODS: We described nuclear IRS-4 in RKO colon cancer cell lines in biopsies of patients with colorectal cancer (CRC) (n = 20) and in matched adjacent normal colorectal (MANC) tissue (n = 20). RESULTS: Treatment with physiological doses of IGF-1 promoted nuclear influx of IRS-4 from cellular cytosol in RKO cells. When exogenous IRS-4 was overexpressed in RKO cells, there was an increase in cyclin D1, cyclin E, E2F1, pRB Ser 809/811 and pRB Ser 705 levels compared with the empty vector-transfected cells. Some of these changes returned to control values after wortmannin treatment. Subcellular fractionation showed an overexpression of IRS-4 in the cytoplasm, membrane, and nuclei of tumour samples, whereas the levels of the protein were barely detectable in the three compartments of normal samples. Immunohistochemical studies showed positive nuclear IRS-4 staining in over 74% of the tumour cells. IRS-4 was strongly overexpressed in tumoural tissues from CRC patients compared to MANC tissues. The up-regulation of IRS-4 in CRC samples correlated significantly with the increase of several G1 checkpoint proteins including cyclin D1 (r = 0.6662), Rb (r = 0.7779), pRb Serine 809/811 (r = 0.6864), pRb serine 705 (r = 0.6261) and E2F1 (r = 0.8702). CONCLUSIONS: Taken together, our findings suggest that IRS-4 promotes retinoblastoma-cyclin-dependent kinase activation and it may serve as a pharmacological target since its expression is very low in normal tissue, including colonic epithelium.
Subject(s)
Adenocarcinoma/metabolism , Adenoma/metabolism , Carcinoma, Hepatocellular/metabolism , Colorectal Neoplasms/metabolism , Cyclin D1/metabolism , E2F1 Transcription Factor/metabolism , Insulin Receptor Substrate Proteins/metabolism , Liver Neoplasms/metabolism , Retinoblastoma Protein/metabolism , Aged , Aged, 80 and over , Cell Line, Tumor , Cell Membrane/metabolism , Cell Nucleus/metabolism , Cell Proliferation/genetics , Colon/metabolism , Colorectal Neoplasms/pathology , Cytoplasm/metabolism , Female , Humans , Insulin Receptor Substrate Proteins/genetics , Insulin-Like Growth Factor I/pharmacology , Male , Middle Aged , Neoplasm Grading , Neoplasm Staging , Proliferating Cell Nuclear Antigen/metabolism , Protein Transport/drug effects , Rectum/metabolism , Signal Transduction , Up-RegulationABSTRACT
Extracellular vesicles (exosomes and shedding vesicles) released by mesenchymal stem cells (MSCs) are regarded as a storable, cell-free alternative with comparable therapeutic potential to their parent cells. Shedding vesicles originate as bulges on the cell surface but little is known about their turnover or how their formation can be stimulated. We have used atomic force microscopy (AFM) to follow the formation dynamics of bulges in living adipose tissue-derived MSCs. AFM images showed that, in general, MSCs present hundreds of nanosized protrusions on their surface with life spans of 10-20 min. Scanning electron microscopy confirmed those images and showed that bulges are also formed on filamentous processes. Extracellular vesicles deposited on the culture surface have comparable sizes to those of bulges showing up on the cell surface. The amount of protrusions on cells treated with progesterone or PDGF-BB, two treatments that stimulate the secretion of extracellular vesicles in MSCs, was evaluated by AFM. Measurements of the cross-area at 50 nm over the cell surface provided estimates of the amount of protrusions and showed that these values increased with the stimulating treatments. Our study suggests that shedding vesicles constitute a large population of the extracellular vesicle pool.
