Human Pluripotent Stem Cell Differentiation in Neurons Using Engineered Lentiviruses

Treat the adhered human pluripotent stem cells with engineered lentiviruses in a transfection reagent.

The virus carries genes for antibiotic resistance and the neurogenic transcription factor, controlled by the tetracycline-responsive and regulatory elements.

The positively charged transfection reagent enhances viral fusion and its content release.

Viral RNA reverse-transcribes into DNA and integrates into the stem cell DNA, enabling the expression of regulatory elements that produce activator proteins.

Add a basal medium with tetracycline derivative, which forms a complex with the activator proteins.

This complex binds to the tetracycline-responsive element, promoting gene expression and producing the neurogenic transcription factors that induce stem cell differentiation into neurons.

Add a basal medium with an antibiotic drug to eliminate the untransfected cells.

Treat the cells with a detachment solution containing proteolytic enzymes that degrade the extracellular matrix or ECM and release the cells.

Remove the enzymes and re-plate the cells onto ECM-coated wells with a maturation medium, promoting neuronal maturation.

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.