Role Of Fasudil, TRPM8 And MicroRNA-124a In Induction Of MSCs To Differentiate Into Nerve Cells

BackgroundMesenchymal stem cells (mesenchymal stem cells, MSCs) are mesoderm derived multipotent stem cells with ability of differentiation to the bone cells, cartilage cells, tendon cells, muscle cells, stem cells, fat cells and hematopoietic cells and self-renewal capacity. In vivo and in vitro, MSCs can differentiate into neurons and glial cells under certain conditions. Recently, a number of animal studies reported therapy of a variety of neurodegenerative diseases, stroke and central nervous system injury by transplantation of bone marrow mesenchymal stem cells, and achieved certain results.Rho/Rho kinase (ROCK) signaling pathway is an important signaling pathway in vivo, including three components:Rho protein, Rho kinase, Rho effector kinase (ROCK), mainly involved in regulation of cytoskeleton formation, cell migration, gene transcription, cell proliferation, apoptosis and other biological behaviors and functions, which regulate intracellular actin cytoskeleton polymerization state through the small G protein GDP-GTP transition and plays a "molecular switch" role, involved in the regulation of cytoskeletal proteins synthesis, degradation, movement and shrinkage of cell division, contraction, adhesion, migration, secretion and other activities. We observed the role of the ROCK inhibitor fasudil in vitro induced MSCs differentiation into neuron-like cells. TRP (transient receptor potential) channels are a class of six transmembrane non-selective cation channels. They are highly conserved in evolution, widely expressed in mammals, involved in many important physiological functions, such as temperature, pain, auditory perception, and fertilization.TRPM8 is a TRP channel subfamily, express abundant in the human central nervous system, and its main function is to sense temperature. Surprisingly, this study found that when inducing MSCs to differentiate into neural cells, first detected the expression of TRPM8.microRNA-124a is most abundant expression in brain tissue of a class of microRNA, approximately 25%-48% of the total microRNA in brain tissue. Lim, whose studies have shown that the microRNA-124a infected HeLa cells and led to a series of expression of non-neuronal transcripts was inhibited, gene expression patterns of HeLa cells transform into the neuron. In the mouse brain, microRNA-124 is limited to expressed in already differentiated and mature neurons and very little in neural progenitor cells. This study was to construct the rat microRNA-124a lentiviral vector containing green fluorescent protein (green fluorescence protein, GFP) reporter gene, infection to the MSCs and passage, observed differentiation of bone marrow mesenchymal stem cells into nerve cells.Part 1 fasudil in vitro induce bone marrow mesenchymal stem cells into nerve cellsObjectiveResearch the feasiblity of Rho/Rho kinase (ROCK) inhibitor, fasudil in vitro induce rat bone marrow mesenchymal stem cells(MSCs) into neuron-like cell.MethodsRat MSCs were isolated from Wistar rats and cultured to 15-18 passages.We used fasudil (200?mol/L) as inducers to differentiate MSCs respectively, and then observed morphologic changes of differentiated cells,stained with AO/EB mixed fluorescent dye to estimate the apoptosis of MSCs, identified them by detecting NF200, NSE and GFAP by immunofluorescence.ResultsAO-EB staining identified cell survival after inductionCytoplasm of surviving cells after the induction of green, nuclear is bright green, nuclear shape rules for the round or oval-shaped, dead cells showed red cytoplasmic, nuclear was bright red, nuclear shrinkage or fragmentation. With the induction of prolonged increase in the number of dead cells. By AO-EB staining identified fasudil induced by 30 min,60min,90min,120min and 180min after the cell survival rates were 96.7±2.2%,95.3±1.9%,93.8±1.8%,92.5±2.1% and 90.1±1.3%.Immunofluorescence after inductionFasudil induced group, with the induction of prolonged, NSE, NF200 was significantly increased, GFAP expression less. After the induction of 60,90,120 and 180 min, identified by immunofluorescence, NSE-positive rates (respectively 66.5±1.9%,88.1±3.2%,93.6±1.9%,93.5±5.4%) and NF200-positive rates (70.1±2.9%, 89.5±1.3%,98.1±1.6%,98.3±1.9%) increased, while GFAP expression in each group were less than 5%.Part 2 in vitro Expression of TRPM8 of bone marrow mesenchymal stem cells differentiation into nerve cellsObjectivesThe transient receptor potential (TRP) channels are a large family of proteins have been involved in a wide range of processes ranging from sensing of thermal and chemical signals to reloading intracellular stores after responding to an extracellular stimulus. TRPM8 is conventionally reported as a cold- and menthol- sensing cation channel implicated in thermosensation. Here we surprisingly showed that expression of TRPM8 might incease greatly in neural cells derived from rat bone marrow mesenchymal stem cells (MSCs) in vitro.MethodsIn this study, we firstly characterized in vitro properties of neural cells derived from MSCs by immunocytochemistry, Western blot.ResultsBefore the induction of rat MSCs did not express TRPM8; induced 30min; MSCs began to express TRPM8 (45.3%±1.58%); induced 60min, possible increase in the expression (57.50%±2.45%); induced 90min, TRPM8 expression of the highest (89.56%±12.24%); induction of 120 min, TRPM8 expression decreased (59.25%±9.15%), Western Blot has a similar trend.Part 3 Construct the microRNA-124a lentiviral vectors and infection of rat bone marrow mesenchymal stem cells ObjectiveTo investigate the role of microRNA-124a in bone marrow mesenchymal stem cells differentiating into neurons.MethodsConstruction of preparations containing green fluorescent protein (green fluorescence protein, GFP) reporter gene in the rat lentiviral microRNA-124a, its infections and rat bone marrow mesenchymal stem cells (mesenchymal stem cells, MSCs), and passaged. Fasudil induced MSCs to differentiate into neuron. After infection fluorescence expression of MSCs was observed under fluorescence microscope; Expression of neuron-specific enolase (NSE), neurofilament protein (NF200) and glial fibrillary acidic protein (GFAP) was detected by western blot and immunocytochemistry. MTT was used to detect cell survival.ResultsImmunocytochemical staining1h after induction of infection, NSE, NF200 expression rates were 83.2±2.0%, 79.6±0.4%, significantly higher than the other two groups (P<0.05). GFAP expression in each group were lower than 5%, no significant difference.Western Blot ResultsThree groups were, respectively, after 1h inducing the expression of NSE, NF200, no infection and no positive control group statistically significant differences in the expression of the infected group NSE, NF200 was significantly higher than the other two groups, a significant difference. Conclusions1 Rho-kinase inhibitor fasudil can rapidly and efficiently induce rat bone marrow derived MSCs differentiating into neuron-like cells.2 These findings indicate that TRPM8 may play some important roles in MSCs differentiation into neural cells.3 microRNA-124a can promote bone marrow mesenchymal stem cells to differentiate into neural cells.