siRNA-Mediated Gene Silencing in Neural Stem Cells

1. Manipulation of O9-1 cells

1. Performing siRNA knockdown in O9-1 cells
   1. Thaw and coat a 24-well plate with a basement membrane matrix before starting the experiment. Recover and seed O9-1 cells, allowing them to grow to 60% to 80% confluency.
   2. Dilute liposomes in serum-free media according to the appropriate well volume. Dilute siRNA in serum-free media to the final. Add diluted siRNA to diluted liposomes in a ratio recommended by the manufacturer. Mix well by pipetting and then incubate.
      NOTE: Follow the manufacturer's guide on the volume used, time, and temperature of incubation.
   3. Add an appropriate volume of siRNA-lipid complex to cells according to the manufacturer's recommendations.
   4. Incubate cells for 24 h in a standard cell culture incubator (37 °C, 5% CO₂). Perform downstream steps as appropriate (extract RNA, extract proteins, staining, etc.)
      NOTE: siRNA knockdown times and concentrations can vary depending on the individual experiment.

2. O9-1 cell differentiation

1. Differentiation of O9-1 cells into osteoblasts
   1. To prepare osteogenic differentiation media, dilute the following in alpha-MEM (final concentrations are indicated): 0.1 μM dexamethasone, 100 ng/mL bone morphogenetic protein 2 (BMP2), 50 µg/mL ascorbic acid, 10 mM b-glycerophosphate, 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.
   2. Detect the osteoblast marker, osteocalcin, in differentiated osteoblasts by immunostaining, as shown in Figure 1.
      ​NOTE: Osteogenic differentiation can also be evaluated by using other markers of osteoblasts or with Alizarin red staining or alkaline phosphatase staining.
2. Differentiation of O9-1 cells into smooth muscle cells
   1. To prepare smooth muscle cell differentiation media, dilute the following in DMEM (final concentrations are indicated): 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS.
   2. Asses smooth muscle cell differentiation with immunofluorescence staining by using antibodies against markers of smooth muscle cells, such as smooth muscle actin (SMA), shown as an example in Figure 2.