Human Pluripotent Stem Cell Differentiation in Neurons Using Engineered Lentiviruses
Transcript
Two days before induction of differentiation, detach ES cells by adding 1 milliliter of cell detachment solution, and incubating at room temperature for 10 minutes. Transfer the cells to a tube. Then, wash the well with 2 milliliters of media and add it to the same tube. Centrifuge the cells at 300 times g for 5 minutes. Then, resuspend the pellet in media and plate the cells onto matrix-coated six-well plates at the seating density of 1 times 10 to the fifth cells per well.
On the next day, add lentiviruses expressing Ngn2 plus PuroR and tetracycline transactivator together with polybrene in fresh ES cell maintenance medium. On day zero, add 2 micrograms per milliliter doxycycline in DMEM/F12 medium supplemented with N2 and without morphogens. On day one, add puromycin in fresh DMEM/F12 plus N2 and doxycycline to the final concentration of 1 microgram per milliliter.
Select the transduced cells in puromycin for at least 24 hours. On day two, detach differentiating neurons with cell detachment solution and replate them on 24-well plates coated with matrix solution. Maintain them in NBA/B27 medium without doxycycline. Culture pure induced neurons on the plates coated with extracellular matrix-based solutions.
