The overall goal of this protocol is to generate a high yield culture of induced neurons from human adult skin fibroblasts. This method can help answer key questions in the field of neurological disorders as it allows for the degeneration of a patient-based neural system that maintains the age of the cell. The main advantage of this method is that it allows for the generation of induced neurons from people of any age in a very standardized and efficient manner in only 25 days.
It allows for a vast array of biomedical application, including disease modeling, toxicology, diagnostics, as well as drug screening assays on a very large scale. Demonstrating the procedure will be Shelby, a Ph.D.student within the unit where brain train, as well as Karlonia, a in our lab. To begin, thaw the adult human dermal fibroblasts using an automated cell thawing system.
After counting the cell number with an automated cell counter, seed two hundred thousand cells per T-75 flask containing 10 milliliters of fibroblast medium. Maintain at 37 degrees Celsius and 5%carbon dioxide. The next day, replace the old medium with fresh fibroblast medium.
Keep changing the fibroblast medium every three to four days until the cells reach 95%confluency. An hour before plating the adult human dermal fibroblasts from reprogramming, coat each well of a 24-well plate with 250 microliters of 0.1%gelatin. Incubate the plate at 37 degrees Celsius.
Aspirate the fibroblast medium from the flask, and wash with Dulbecco's Phosphate-Buffered Saline once. After washing, lyse the cells with 1.5 milliliters of 0.05%trypsin per T-75 flask at 37 degrees Celsius for three minutes. Then, add three milliliters of fibroblast medium for neutralizing the trypsin solution.
Cool the detached cells in a 15 milliliter tube by flushing out the cells in the flask twice. Then, centrifuge the cells at 400 G for five minutes. Expel the supernatent and re-suspend the cell pellet in one milliliter of fibroblast medium.
Then, 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. Use a 1000 microliter pipette to remove the medium. Then add two 250 microliters of cell dissociating agent to each well and leave the plate in the incubator for 10 to 20 minutes until the cells detach and float as single cells.
In the meantime, prepare the FACS buffer. Triturate with a 1000 microliter pipette. Incubate a little longer if there are cell clumps remaining.
Transfer the single cell suspension obtained into a 1.5 milliliter tube. Flush the wells twice with late neuronal conversion medium and transfer to the same 1.5 milliliter tube. Then, centrifuge at 400 G for five minutes and discard the supernatent.
Next, re-suspend the cell pellet in 200 microliters of FACS buffer. Again, spin down the cells at 400 G for five minutes and discard the supernatent. Re-suspend the cells in FACS buffer and centrifuge.
Repeat two more times. Then, re-suspend the cell pellet in 50 microliters of FACS buffer containing human CD56 antibody at a one to 50 concentration for 15 minutes on ice, away from the light. Centrifuge the cells at 400 G for five minutes and dissolve the cell pellet in 200 microliters of FACS buffer to wash the cells.
Again, centrifuge the cells at 400 G for five minutes. Wash with FACS buffer twice followed with centrifugation. Finally, re-suspend in 200 microliters of FACS buffer containing 10 micrograms per milliliter of propidium iodide.
Sort the NCAM positive and propidium iodide negative cells using gating on the basis of fluorescence intensity level of control samples that were not stained with the NCAM antibody or propidium iodide. Use an automated cell counter to count the cell number and seed 50, 000 cells per centimeter square on a Poly-L-ornithine fibronectin and laminin-coated plate with late neuronal conversion medium. Keep changing half of the medium two to three times per week with late neuronal conversion medium until the experimental endpoint.
Representative phase-contrast images show the morphological changes in the cells during the conversion period. A significant transformation in cellular morphology is visible from day five. By the 22nd day, approximately half of the cells have converted into neurons.
Representative amino fluorescence images show the presence of standard neuronal markers in the cells. By the 25th day, cells obtain neuronal morphology and express neuronal markers such as MAP2 and TAU. The nuclei are stained with DAPI.
After watching this video, you should have a good understanding of how to generate induced neurons from adult human fibroblasts. And this includes plating the cells for reprogramming, viral transduction, cell maintenance, as well as FACS sorting for purification. The development of this technique paves the way for researchers in the field of neurological disorders to study different features associated with the disease as well as potential therapies.
And that can be done in a system that is not only a human neural system but that can be both patient-and disease-specific.
