This article describes a protocol for expanding induced neural progenitor cells derived from adult human fibroblasts. The process involves culturing these cells in a laminin-coated environment and utilizing specific growth factors and inhibitors to promote cell proliferation and prevent apoptosis.
Begin with an adherent culture of induced neural progenitor cell colonies derived from adult human fibroblasts in a laminin-coated multi-well plate.
Wash the colonies with a buffer to remove non-adherent cells, then add fresh buffer.
Scrape around individual colonies to remove surrounding cells.
Transfer the colonies to fresh laminin-coated wells containing neuroinduction medium.
Mechanically disaggregate the colonies to obtain a single-cell suspension.
Laminin, an extracellular matrix protein, interacts with the surface proteins of the progenitors, facilitating cell adhesion to the well surface.
Add a small molecule inhibitor that enters the cells and blocks specific protein kinases, preventing cellular apoptosis.
Growth factors and small molecules present in the neuroinduction medium facilitate the proliferation of the progenitors.
Periodically replace the medium to replenish nutrients, facilitating continued progenitor proliferation.
A confluent culture, or a fully covered cell layer, indicates the successful expansion of induced neural progenitor cells.
One day later, aspirate laminin from the plates, and add 200 microliters of neural induction medium to the wells. Wash the six-well plates containing the colonies to be picked, once with DPBS, before adding 2 milliliters of neural induction medium per well of a six-well plate.
Mechanically pick the cells by scraping around the colonies using a thin needle to get rid of surrounding cells, and picking the colonies using pipette tips mounted on a 200-microliter pipette man set on 50 microliters.
Transfer each colony into one well of a laminin-coated 48-well plate and generate a single-cell suspension mechanically by pipetting up and down 10 times. Add Rho-kinase inhibitor in a final concentration of 10 micromolar to the cells. Grow the cells on the 48-well plate at 37 degrees Celsius, 5% CO2 for two days. Change the medium every second day until the cells reach 80% to 90% confluency.
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