This article describes a method for direct reprogramming of genetically altered hematopoietic progenitor cells into neural stem cells. The process involves using a neural progenitor medium and an extracellular matrix to facilitate cell growth and transformation.
Begin with a cell suspension of genetically altered hematopoietic progenitor cells.
These cells express transcription factors required for their reprogramming into neural stem cells. This process is called direct reprogramming.
Transfer the cells to an extracellular matrix-coated multiwell plate with a neural progenitor medium, and incubate.
The extracellular matrix provides a three-dimensional scaffold for cell attachment and growth.
During incubation, transcription factors expressed by the hematopoietic progenitor cell initiate a sequence of molecular events that allow its transformation into a neural stem cell.
The neural progenitor medium contains factors that favor the survival of induced neural stem cells.
After incubation, replace this medium with a fresh neural progenitor medium to promote cell multiplication.
Detach the cells using a cell scraper.
Collect this suspension and transfer the cells to a well containing neural stem cell medium.
Incubate for maintenance of induced neural stem cells.
When the cells are 30 to 50% confluent, aspirate the supernatant and reserve, and then add 1 milliliter of neural progenitor medium to each well. To make a backup plate using the spheres in the supernatant, centrifuge the reserve supernatant at 170 times g for 10 minutes.
Then, aspirate the supernatant and resuspend the pellet in neural progenitor medium. Plate in a matrigel-coated 24-well plate; change the medium in each plate of cells every other day until the cells reach 60% to 80% confluence, usually after one week.
Once the cells reach the required confluence, aspirate the supernatant, and add 1 milliliter of neural stem cell medium to each well. Then, dissociate the cells by using a cell scraper followed by very gentle pipetting.
Next, transfer the cells from one well of the 24-well plate into one well of a 6-well plate. Add another milliliter of medium to the well, and after repeating the procedure for all wells, place the 6-well plate into the 37 degrees Celsius 5% carbon dioxide incubator.
Change the full volume of medium every other day until the cells reach 60% confluence. After each time, dissociate and replate the cells at a 1-to-3 ratio as just shown.
繁體中文
