Protocol for Directed Induced Differentiation of Mouse Embryonic Stem Cells to Neural Stem Cells

• The generation of neurons or glia that can be used to replace lost or damaged tissue is a key step in stem cell-based therapies designed to treat neurodegenerative disease and neurological disorders.
• Several different candidate sources have been proposed for producing these cells. Based upon their proliferative and pluripotential properties, embryonic stem cells (ESCs) are an attractive alternative to fetal or adult-derived central nervous system (CNS) tissue. The derivation of specific neuronal or glial cell types from ESCs invariably includes the production of neural stem cells (NSCs), the self-renewing, pluripotential stem cells of the nervous system, capable of differentiating into neurons, astrocytes, and oligodendrocytes.

• The 35 mm Petri dishes are coated with 0.1% gelatin 1 h in advance, and the gelatin is discarded after 1 h of incubation.
• The pluripotent stem cells cultured on the feeder layer were digested with 0.05% Trypsin for 2 min, and the digestion was terminated by adding an equal volume of cell basal medium. Centrifugation is performed at 1000 rpm for 5 min, the supernatant is discarded, and the cells are resuspended with a neural stem cell culture medium. Then, the cells are added to the gelatin-coated 35 mm dishes at a cell density of 2×10⁵ cells per dish, and the cells are left to stand for 30 min at 37°C in a 5% CO₂ incubator. The supernatant is aspirated to remove the trophoblastic layer attached to the gelatin.
• The supernatant is centrifuged at 1000 rpm for 5 min, the supernatant is discarded and the cells are resuspended with EB medium, and the cells are inoculated into 100 mm dishes at a density of 1×10⁶ cells per dish, and then incubated at 37°C with 5% CO₂ for 48 h. In the following, 3 mL of EB medium was added to each 100 mm dish every day.
• On day 5 of differentiation, 500 μL of 20 μg/mL fibronectin is added to each well of a 12-well plate and left at room temperature overnight.
• On day 6 of differentiation, the fibronectin in the 12-well plate is discarded and 1 mL of EB medium is added to each well. Pipette EB spheres from 100 mm Petri dishes into 12-well plates, and place about 20 EB spheres per well.
• On the 2^nd day after EB inoculation for wall apposition, EBM is discarded and 2 mL of N2B27 culture medium is added to each well. The fresh N2B27 culture medium is replaced half daily thereafter.
• On day 6 after EB inoculation, a large number of nerve cells and occasionally neurons can be seen crawling out of the wall-adherent balls at this time.
• The cells are digested with 0.05% Trypsin for 2 min and terminated with an equal volume of 10% FBS. 1000 rpm centrifugation is performed for 5 min, and the cells are resuspended with NSCM planted in 35 mm dishes at a density of 5×10⁵ cells per dish, and cultured at 37°C in a 5% CO₂ incubator.
• When the cell density reaches 80%, the cells are passaged, usually once in 3-4 d. After several passages, the neural stem cells grew as floating neuro-spheres.

Creative Bioarray Relevant Recommendations

• Creative Bioarray provides embryonic stem cells from both humans and mice. Embryonic stem cells (ES cells) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage preimplantation embryo.
• We also provide our customers with a large and unique collection of high-purity, low-passage human and animal neural stem cells, including but not limited to Strain C57BL/6 Mouse Neural Stem Cells, Sprague-Dawley (SD) Rat Neural Stem Cells, Human Neural Stem Cells-cortex region, Human iPSC-Derived Neural Stem Cells, Human Neural Stem Cells-ventral mesencephalon region, Mouse Spinal Cord Neural Stem Cells, Rat Hippocampal Neural Stem Cells, and others.

• After inoculation of the petri dish, the cell suspension should be shaken well before putting it into the incubator. Otherwise, the EBs tend to stick together.
• Aspirate and add liquid in 12-well plates gently, with the tip of the gun pressed against the wall of the dish to avoid blowing up the EB balls.

Embryonic Stem Cells

For research use only. Not for any other purpose.