Direct Reprogramming of Mouse Fibroblasts into Melanocytes

This work was approved by the Laboratory Animal Management and Use Committee at Jiangsu University (UJS-IACUC-AP–20190305010). The experiments were performed in strict accordance with the standards established by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). There were no experiments involving humans, so this work did not need approval from the human research ethics committee. Refer to the Table of Materials for details about reagents.

1. Construction of a concentrated lentivirus packaging system for transcription factors

1. Production of the concentrated virus (Figure 1A, B)
   1. Plate 1.5 × 10⁶ HEK-293T cells into a 60 mm dish and culture these cells with normal medium (see Table 1) at 37 °C in a humidified incubator with 5% CO₂.
      NOTE: If the virus needs to be packaged in batches, a 100 mm cell culture dish can be used (for details, see Table 2).
   2. After 24 h, ensure that the HEK-293T cells have reached 80-90% confluence on the day of transduction, and replace the medium with 3.5 mL of DMEM (150 µL of DMEM per 1 cm²). Leave the cells at 37 °C in a humidified incubator with 5% CO₂ for 2 h.
      NOTE: The replacement medium must be serum-free, so that the cells can be "starved" for better plasmid transfection.
   3. Prepare the mixture of plasmids (mix A) containing 3 µg of the target plasmids of Mitf, Sox10, Pax3, Sox2, Sox9, or Snai2; 1 µg of the packaging plasmid PMD2.G, and 2 µg of the packaging plasmid PSPAX2. Make up the volume of mix A to 150 µL with serum-free DMEM. Prepare the mix of the transfection reagent (mix B) by adding 12 µL of the transfection reagent (the volume is twice the total mass of all the plasmids), and make up the volume of mix B to 150 µL with serum-free DMEM.
      NOTE: When preparing the mixes, it is important to add liquid slowly to avoid air bubbles.
   4. Combine mix A and mix B after allowing them to stand for 5 min at room temperature. Incubate the mixture at room temperature for 20-30 min to form the transfection complex.
   5. Take the HEK-293T cells out of the incubator, replace the medium with DMEM + 2% FBS, add the mixture from step 1.1.4 dropwise, and mix the liquid gently.
   6. After 8 h, change the medium with 3.5 mL of normal medium. After changing the medium, collect the virus supernatant every 24 h and 48 h.
   7. Mix the virus supernatants collected at two different time points. Centrifuge at 200 × g for 5 min at 4 °C. Pass the supernatant through 0.45 µm filters and collect it in a 50 mL sterile conical tube.
      NOTE: The virus collected at 24 h can be stored at 4 °C and mixed with the virus collected at 48 h.
   8. Concentrate the virus supernatant by centrifuging at 6000 × g at 4 °C overnight (~16 h). Ensure that the virus pellet is visible at the bottom of the conical tube after centrifuging.
   9. Pour out the supernatant slowly. Dissolve the virus pellet in a volume of normal medium that is 1/100^th of the volume of the virus supernatant. Using a P1000 micropipette, pipette up and down gently until a homogeneous mixture is obtained. Divide the concentrated virus into microcentrifuge tubes as needed. Store at −80 °C.
      NOTE: The virus is concentrated 100x with this method. The concentrated virus (100x) can be stored for >1 year at −80 °C. Avoid repeated freezing and thawing of the virus.
2. Detection of concentrated virus titer (Figure 1C)
   1. Plate 1 × 10⁵ HEK-293T cells into one well of a 6-well plate. Remember to add one well as a negative control. Culture these cells with normal medium at 37 °C in a humidified incubator with 5% CO₂.
   2. Add 0.1 µL or 0.2 µL of fluorescent concentrated virus (100x) to each well after 24 h, and add 4 ng/µL of the cationic polymeric transfection reagent to each well. About 8-12 h after infection, replace the medium with normal medium.
      NOTE: To ensure the accuracy and efficiency of the fluorescence detection, the infection rate must be 10-30%. Adding 0.1 µL or 0.2 µL of the fluorescent concentrated virus can maintain the infection efficiency within this range. The concentrated virus titer can reach 1 × 10⁸ transducing units (TU)/mL at least.
   3. Approximately 48 h after infection, wash the dish with 1 mL of sterile phosphate-buffered saline (PBS) to remove dead cells.
   4. Trypsinize these cells using 250 µL of 0.05% trypsin-EDTA per well in a 6-well plate for 1 min at room temperature. Centrifuge at 200 × g for 5 min at 4 °C and then remove the supernatant.
   5. Resuspend the cell pellet in 1 mL of PBS, add the suspension to a 5 mL polystyrene round-bottom tube, and detect the infection efficiency of the virus fluorescence (green fluorescent protein, GFP⁺) using flow cytometry.
   6. Calculate the concentrated virus titer using the following formula: 10⁵ (cell volume) × infection rate (GFP⁺%)/added virus volume (0.1 µL or 0.2 µL).

2. Direct reprogramming of fibroblasts to melanocytes (Figure 2A)

1. Coat one well of a 6-well cell culture plate with 1 mL of 0.1% gelatin solution at room temperature for 15-30 min. Ensure that the well is completely covered with the 0.1% gelatin solution. Aspirate the 0.1% gelatin solution after coating.
   NOTE: Prepare 0.1% gelatin solution (100 mL) as follows: 0.1 g of gelatin powder is dissolved in 100 mL of ultrapure water in an autoclaved glass bottle and then stored at 4 °C for no more than 2 months.
2. Plate 5 × 10⁴ MEFs into one well of a 6-well plate coated with 0.1% gelatin (as in step 2.1), and culture these cells with normal medium at 37 °C in a humidified incubator with 5% CO₂ overnight.
3. After 24 h, confirm that the MEFs have reached 40-50% confluence. Replace the medium with normal medium.
4. On Day 0, take out the concentrated virus from the freezer and melt the virus on ice. Calculate the volume of the virus to be added by using eq. (1). Add the concentrated virus for the six transcription factors, Mitf, Pax3, Sox10, Sox9, Sox2, and Snai2 (see Table of Materials) to each well according to the calculated volume, and then add 4 µg/mL of the cationic polymeric transfection agent.
   Cell number (5 × 10⁴) × 30 (multiplicity of infection, MOI)/virus titer (1)
5. On Day 1, 8-12 h after infection, remove the medium containing the virus and replace it with fresh normal medium while adding 0.5 µg/mL puromycin to screen stable infected cell lines.
6. On Day 2, 48 h after infection, replace the supernatant medium gradually with the reprogramming medium. First, change 1/4^th of the total medium volume, and add 3 µM CHIR99021.
7. From Day 3 to Day 7, depending on the condition of the cells, change the medium by replacing with a higher proportion of reprogramming medium (see Table 1) gradually, and switch to complete reprogramming medium within 5 days.
   NOTE: During this period, puromycin and CHIR99021 must be used every day. Many dead cells will appear on the first and second day of changing the reprogramming medium. This is normal as the cells gradually adapt to the transformation. Therefore, the medium needs to be changed gradually to ensure the healthy proliferation of the cells.
8. To passage the cells, add 500 µL of 0.05% trypsin-EDTA to digest the cells for 3 min at room temperature. When ~60% of the cells have floated up, stop the digestion by adding normal medium 2x the volume of the digestive enzyme. Collect the cell suspension in a 15 mL sterile conical tube, centrifuge at 200 × g for 5 min at 4 °C, remove the supernatant, resuspend the cell pellet with the reprogramming medium, and plate the cells in a 60 mm sterile dish at a density of 3 × 10⁴/cm². Culture these cells at 37 °C in a humidified 5% CO₂ incubator.
   ​NOTE: From Day 8 to Day 21, these cells are sub-cultured every 3-5 days and cultured in 60 mm sterile dishes to expand. They can be cultured to reach at least passage 5.

3. Optimization for direct reprogramming and identification

1. Screening for the optimized transcription factors
   1. Repeat steps 2.1-2.7, reducing one of the six transcription factors each time. Infect the MEFs with the virus with combinations of five transcription factors.
   2. Seven days after infection, extract the RNA of these cells¹⁶, and analyze the expression levels of their melanocytic genes using reverse-transcription PCR (RT-PCR)¹⁷ to screen for the transcription factors with the greatest impact on conversion to melanocytes by removing them one by one (Figure 3A).
   3. Use the top three transcription factors impacting the conversion to melanocytes to infect the MEFs. Repeat steps 2.1-2.7.
      NOTE: Seven days after infection, the melanocytic genes should be detectable in these transformed cells (Figure 3B). Primer information for iMels characterization is included in Table 3.
2. Identification of induced melanocytes (iMels)
   1. Use immunofluorescence staining¹⁸ to verify that the iMels express melanocytic proteins, including TYRP-1 and DCT (Figure 4A).
   2. Melanin-specific 3,4-dihydroxyphenylalanine (DOPA) staining (Figure 4B)
      1. Prepare 4% paraformaldehyde (10 mL) as follows: dissolve 0.4 g of paraformaldehyde powder in 10 mL PBS. Place the solution in an oven at 56 °C for 2 h to promote dissolution.
         NOTE: The solution can be kept at 4 °C for no more than one month.
      2. Culture the iMels in a 30 mm dish and wash the dish twice with pre-warmed PBS. Add 1 mL of 4% paraformaldehyde to fix the cells for 20 min, and wash the dish 3 times with PBS.
      3. Prepare 0.1% DOPA stain solution (10 mL) just before use by dissolving 0.01 g of L-DOPA powder in 10 mL of PBS. Place the solution in a water bath at 37 °C for 30 min; shake it several times to promote dissolution.
      4. Add 1 mL of freshly prepared 0.1% DOPA staining solution. Incubate in an oven at 37 °C for 2-5 h. If there are no brown-black particles, continue to incubate at 37 °C for another 2 h but not for >5 h. Check the samples every 30 min.
      5. Wash the dish 3 times with PBS for 1 min each time. Stain the nucleus with 1 mL of hematoxylin staining solution for 2 min.
      6. For long-term storage, dehydrate the samples using 95% ethanol for 3 min and then 100% ethanol for 5 min. Seal the dish with xylene and neutral balsam.
   3. Melanin-specific Masson-Fontana staining (Figure 4B)
      1. Plate iMels in a 30 mm dish and fix the cells with 4% paraformaldehyde for 20 min; wash the dish 3 times with PBS.
      2. Add 1 mL of solution A (ammonia silver solution) from the Masson-Fontana staining kit (see Table of Materials), place the dish in a dark box, and place the dark box in an oven at 56 °C for 15-40 min.
         NOTE: If brown-black particles are not visible after 15 min in the oven, return the samples to the 56 °C oven to continue incubating but not for >40 min. Some water can be added to the dark box to prevent drying up.
      3. Aspirate solution A and wash the dish 5-6 times with distilled water, 1-2 min each time.
      4. Add 1 mL solution B (hypo solution) from the Masson-Fontana staining kit, and leave the dish at room temperature for 3-5 min.
      5. Aspirate solution B and wash the dish with tap water 3 times, 1 min each time.
      6. Add 1 mL of solution C (neutral red dye) from the Masson-Fontana staining kit, and leave the dish at room temperature for 3-5 min.
      7. Aspirate solution C and wash the dish 3 times with distilled water, 1 min each time.
      8. Add 1 mL of 100% ethanol for rapid dehydration, and aspirate the ethanol after 3 min.
         NOTE: Stained samples can be stored for a long time after sealing with xylene and neutral balsam.