Generating Neurons by Reprogramming Human Dermal Fibroblast Cells
Transcrição
Introduce adult human dermal fibroblasts or aHDFs, suspended in a fibroblast medium, into an extracellular matrix protein-coated microplate. Incubate to allow cell adherence.
Introduce a transgenic lentiviral vector. Upon internalization into the host cell, the viral RNA is reverse-transcribed into DNA, which integrates into the host genome.
The viral DNA expresses transcription factors and short hairpin RNAs, or shRNAs. Cellular nucleases process the shRNAs into small interfering RNAs or siRNAs.
The generated siRNAs degrade the mRNA encoding transcriptional repressors, thereby inhibiting their expression and enabling the activation of neuronal differentiation genes.
The transcription factors activate the genes, promoting neuronal differentiation of the aHDFs.
Remove the medium containing the non-internalized vectors.
Add an early neuronal conversion medium containing growth factors and small molecules that induce differentiation into neuron-like cells.
Replace the medium with a late neuronal conversion medium containing neurotrophic factors for maturation into neurons.
The mature neurons are ready for downstream assays.
