This article discusses the process of neuronal reprogramming from genetically altered astrocytes. The method involves using a neuronal differentiation medium to facilitate the transformation of astrocytes into neurons.
Begin with a multi-well plate containing genetically altered astrocytes. These cells are adhered to a coverslip placed in an astrocyte medium.
These genetically modified cells express transformation factors that force their transition into neurons. This process is called neuronal reprogramming.
Take a neuronal differentiation medium containing specific molecules and the B27 supplement. Together, these components provide essential nutrients and factors for differentiation.
Replace the astrocyte medium with the neuronal differentiation medium.
Incubate the astrocytes for a required duration.
During incubation, the transformation factors expressed by the astrocytes trigger a sequence of molecular events that enable the conversion of an astrocyte into a neuron.
Converted neurons have smaller cell bodies and elongated processes, similar to typical neurons.
Prepare neuronal differentiation medium by adding one milliliter B27 supplement to 49 milliliters of basic culture medium. After 24 to 48 hours, depending on whether cells have been transduced or transfected, replace astrocyte medium with one milliliter neuronal differentiation medium per well. Culture the cells at 37 degrees Celsius with 9% carbon dioxide.
To increase the reprogramming efficiency, supplement the neuronal differentiation medium with Forskolin and Dorsomorphin to a final concentration of 30 micromolar and one micromolar respectively when replacing the astrocyte medium with the differentiation medium. When opting to treat cells with Forskolin and Dorsomorphin, provide a second dose of Dorsomorphin two days after the initial treatment. Add this second treatment directly to the culture medium.
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