Direct reprogramming to generate melanocytes has been reported. However, various transcription factors are used in different studies. In this protocol, we validated and reduced transcription factor number still with higher reprogramming efficiency.
We established a direct reprogramming system to generate melanocytes using only three transcription factors, Mitf, Sox10 and PAX3, and modified the culture system to make it more stable and robust. How to regenerate functional melanocytes is a significant question that may help address therapy limitations for depigmentation diseases like vitiligo. The right reprogramming provides a prospective Our technique has the characteristics of convenience, strong operability, and stability, which is easier to promote and repeat.
To begin the production of the concentrated lentivirus, plate 1.5 times 10 to the 6 HEK-293T cells into a 60-millimeter dish, and culture the cells with normal medium at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide. After 24 hours of the incubation, when the HEK-293T cells have reached 80 to 90%confluency, replace the medium with 3.5 mL of DMEM, then incubate the cells at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for two hours. After removing the cells from the incubator, replace the medium with DMEM containing 2%FBS, and add the freshly prepared transfection complex mixture to the cells in a drop-wise manner.
Mix the liquid mixture gently. At the completion of eight hours, change the cell medium with 3.5 mL of normal medium. Collect the virus supernatant at 24 and 48 hours.
Next, mix the virus supernatants collected at two different time points and centrifuge the mixture at 200 xg for five minutes at four degrees Celsius. Then pass the collected supernatant through 0.45 micron filters to collect the filtrate in a 50-mL sterile conical tube. Concentrate the filtered virus supernatant by centrifugation at 6, 000 xg at four degrees Celsius overnight.
Once done, ensure that the virus pellet is visible at the bottom of the conical tube. Pour out the supernatant slowly. For dissolving the virus pellet in the normal medium with one by 100th*volume of the virus supernatant.
Use a P1000 micropipette to pipette up and down gently until a homogenous 100x concentrated virus mixture is obtained. Divide the concentrated virus into the microcentrifuge tubes to store at 80 degrees Celsius. Plate 1 times 10 to the 5th HEK-293T cells into one well of a six-well plate.
Culture these cells with the normal medium at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide. After 24 hours, add 0.1 to 0.2 microliters of the 100x fluorescent concentrated virus to each well. Followed by the addition of four nanograms per microliters of the cationic polymeric transfection reagent to each well.
About eight to twelve hours after infection with the virus, replace the medium with the normal medium. At forty-eight hours post-infection, wash the dish with 1 mL of sterile PBS to remove the dead cells. Then trypsinize the cells using 250 microliters of 0.05%trypsin-EDTA solution per well of a six-well plate at room temperature.
Centrifuge the plate at 200 xg for five minutes at four degrees Celsius. After removing the supernatant, re-suspend the cell pellet in one mL of PBS and add the cell suspension to a five mL polystyrene round-bottom tube. Then place the tube into the flow cytometer to analyze the sample.
For direct reprogramming of fibroblasts, coat one well of a six-well cell culture plate with 1 mL of 0.1%gelatin solution at room temperature. After 15 to 30 minutes, when the well is completely covered with the gelatin, aspirate 0.1%gelatin solution. Next, plate 5 times 10 to the 4th mouse embryonic fibroblasts, or MEFs, into one well of a six-well plate coated with 0.1%gelatin, and culture the cells overnight with the normal medium at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide.
After 24 hours, when the MEFs have reached 40 to 50%confluency, replace the medium with the normal medium. Next, melt the frozen, concentrated virus on ice. Calculate the volume of the virus to be added using equation and then add the concentrated virus for the six transcription factors to each well, according to the calculated volume.
Add four micrograms per mL of the cationic polymeric transfection agent to the well. Consider it as day zero of the lentivirus infection. On day one, after 8 to 12 hours of infection, replace the medium containing the virus with the fresh normal medium, while adding 0.5 micrograms per mL puromycin to screen stable infected cell lines.
On day two, 48 hours after infection, replace the supernatant medium gradually with the reprogramming medium. Change one-fourth of the total medium volume before adding three micromolar CHIR99021. From day three to day seven, depending on the condition of the cells, change the medium by gradually replacing it with a higher proportion of reprogramming medium to switch to the complete reprogramming medium within five days.
Next, detach the cells with 500 microliters of 0.05%trypsin-EDTA digestive enzyme for three minutes at room temperature. When approximately sixty percent of the cells have floated up, stop the digestion by adding the normal medium, two times the volume of the digestive enzyme. Collect the cell suspension in a 15-mL sterile conical tube to centrifuge at 200 xg for five minutes at four degree Celsius.
Remove the supernatant before re-suspending the cell pellet with the reprogramming medium. When done, plate the re-suspended cells in a 60-millimeter sterile dish. Culture the cells at 37 degrees Celsius in a humidified 5%carbon dioxide incubator.
After forty-eight hours of lentivirus infection in HEK-293T cells, the success of concentrated lentivirus production was evaluated by observing the fluorescence intensity of GFP or by flow cytometry. The titer of the concentrated virus was found to be high at 10 to the 8th TU/mL. During the direct reprogramming of the fibroblasts, the cell morphology changed gradually to elongated cell synapse and enlarged cell nuclei.
However, the cells progressively aged after day twenty. Removal of the transcription factors, Mitf, Pax3 or Sox10, resulted in the silencing of the expression of melanocytic genes, Tyr, Tyrp1, and Mlana, indicating the greatest impact on fibroblast conversion to melanocytes. Hence, the expression of melanocytic genes induced by the direct programming with three transcription factors was higher when compared with six transcription factors.
To identify MEF-induced melanocytes, or iMels, the expression of melanocytic markers, TYRP1 and DCT was detected using immunofluorescent staining. Moreover, the melanin-specific staining methods such as DOPA and Masson-Fontana staining showed positive results. The 293T cells should be distributed evenly.
Also, the addition of transfection mixture to cells must be done gently to prevent the cells from floating. The protocol is stable and practical and it can be used to reprogram other types of cells. It expects to provide a reference and a strategy for in vivo direct reprogramming in melanocytes regeneration in future medicine.
