Generating Neurons by Reprogramming Human Dermal Fibroblast Cells

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.

Prepare a cellular suspension of 1,320,000 cells in 13.2 milliliters of fibroblast medium in order to achieve 100,000 cells per milliliter of medium. After aspirating the gelatin from the plate, wash the plate with Dulbecco's phosphate-buffered saline twice. Then, add 500 microliters of cell suspension to each of the wells, and incubate at 37 degrees Celsius overnight.

Warm 13.2 milliliters of fibroblast medium to 37 degrees Celsius. Then, thaw the lentiviral vector in the laminar hood at room temperature. Add the required volume of lentivirus necessary to infect the adult human dermal fibroblasts at a multiplicity of infection of 20 to the medium without any transduction enhancers.

Then, replace the medium with 500 microliters of fibroblast medium with the addition of the lentiviral vector. Incubate the plate at 37 degrees Celsius overnight. The following day, replace the medium containing lentivirus with fresh fibroblast medium without the lentiviral vector. On the third day after viral transduction, remove the fibroblast medium and add 500 microliters of early neuronal conversion medium.

Twice or thrice a week, replace 225 microliters of old medium from each well with 250 microliters of fresh, early neuronal conversion medium. On the 18th day after viral transduction, remove the early neuronal conversion medium and replace with 500 microliters of late neuronal conversion medium. Keep changing the medium every two to three days as before until the 25th day or the experimental endpoint.