ABSTRACT
We recently reported that transplantation of mesenchymal stem cells (MSCs) significantly reduced bacterial growth and brain injury in neonatal meningitis induced by Escherichia coli (E. coli) infection in newborn rats. As a next step, to verify whether the MSCs protect against brain injury in a paracrine manner, this study was designed to estimate the efficacy of MSC-derived extracellular vesicles (MSC-EVs) in E. coli meningitis in newborn rats. E. coli meningitis was induced without concomitant bacteremia by the intra-cerebroventricular injection of 5 × 102 colony-forming units of K1 (-) E. coli in rats, at postnatal day 11. MSC-EVs were intra-cerebroventricularly transplanted 6 h after the induction of meningitis, and antibiotics were administered for three consecutive days starting at 24 h after the induction of meningitis. The increase in bacterial growth in the cerebrospinal fluid measured at 24 h after the meningitis induction was not significantly reduced following MSC-EV transplantation. However, an increase in brain cell death, reactive gliosis, and inflammation following meningitis were significantly attenuated after MSC-EV transplantation. Taken together, our results indicate that MSCs show anti-apoptotic, anti-gliosis, and anti-inflammatory, but not antibacterial effects, in an EV-mediated paracrine manner in E. coli-induced neonatal meningitis.
ABSTRACT
Co-culture system, in which two or more distinct cell types are cultured together, is advantageous in that it can mimic the environment of the in vivo niche of the cells. In this study, we presented a strategy to analyze the secretome of a specific cell type under the co-culture condition in serum-supplemented media. For the cell-specific secretome analysis, we expressed the mouse mutant methionyl-tRNA synthetase for the incorporation of the non-canonical amino acid, azidonorleucine into the newly synthesized proteins in cells of which the secretome is targeted. The azidonorleucine-tagged secretome could be enriched, based on click chemistry, and distinguished from any other contaminating proteins, either from the cell culture media or the other cells co-cultured with the cells of interest. In order to have more reliable true-positive identifications of cell-specific secretory bodies, we established criteria to exclude any identified human peptide matched to bovine proteins. As a result, we identified a maximum of 719 secreted proteins in the secretome analysis under this co-culture condition. Last, we applied this platform to profile the secretome of mesenchymal stem cells and predicted its therapeutic potential on osteoarthritis based on secretome analysis.
Subject(s)
Methionine-tRNA Ligase , Animals , Cattle , Click Chemistry , Coculture Techniques , Methionine-tRNA Ligase/genetics , Mice , Proteins , SecretomeABSTRACT
Neonatal stroke leads to mortality and severe morbidity, but there is no effective treatment currently available. Erythropoietin (EPO) has been shown to promote cytoprotection and neurogenesis and decrease subventricular zone morphologic changes following brain injury. The long-term cellular response to EPO has not been defined, and local changes in cell fate decision may play a role in functional improvement. We performed middle cerebral artery occlusion in P10 rats. EPO treatment (5 U/g i.p.) significantly preserved hemispheric brain volume 6 weeks after injury. Furthermore, EPO increased the percentage of newly generated neurons while decreasing newly generated astrocytes following brain injury, without demonstrating long-term differences in the subventricular zone. These results suggest that EPO may neuroprotect and direct cell fate toward neurogenesis and away from gliogenesis in neonatal stroke.
