Human Pluripotent Stem Cell Differentiation in Neurons Using Engineered Lentiviruses
Transcription
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.
