Direct Reprogramming of Mouse Fibroblasts into Melanocytes

요약

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

초록

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming.

Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

서문

Vitiligo is a skin disease that seriously affects the physical and mental health of the affected individuals. For various reasons, including metabolic abnormalities, oxidative stress, generation of inflammatory mediators, cell detachment, and autoimmune response, the functional melanocytes are lost, and the secretion of melanin is stopped, leading to the development of vitiligo^1,2. This condition occurs widely and is particularly problematic on the face. The main treatment is the systemic use of corticosteroids and immunomodulators. Phototherapy can be used for systemic or local diseases, and there are surgical treatments, such as perforated skin transplantation and autologous melanocyte transplantation^3,4,5. However, patients who use drug therapy and phototherapy are prone to relapse, and these treatments have poor long-term therapeutic effects. Surgical treatment is traumatic and only moderately effective^2,6. Therefore, a new and effective therapeutic strategy is needed for vitiligo.

The reprogramming of induced pluripotent stem cells (iPSCs) reverses these cells from their terminal state to a pluripotent state, a process mediated by the transcription factors, Oct4, Sox2, Klf4, and c-Myc⁷. However, due to the possibility of tumorigenicity and the long production time, this technology has been met with skepticism when applied to clinical settings⁸. Direct reprogramming is a technology that makes one type of a terminal cell transform into another type of a terminal cell⁹. This process is achieved by suitable transcription factors. Various cells have already been directly reprogrammed successfully, including cardiomyocytes¹⁰, neurons¹¹, and cochlear hair cells¹². Some researchers have even reprogrammed skin tissue directly in situ, which can be used for wound repair¹³. The advantages of direct reprogramming include reduced wait times and costs, lower risk of cancer, fewer ethical problems, and a better understanding of the mechanism underlying cell fate determination⁹.

Although the direct reprogramming method is widely used, there is currently no definite method for the direct reprogramming of skin cells into melanocytes, especially because of the numerous transcription factors to be considered^14,15. The transcription factors, Mitf, Sox10, and Pax3, have been used for direct reprogramming of skin cells into melanocytes¹⁴. In contrast, the combination of MITF, PAX3, SOX2, and SOX9 has also been used for direct reprogramming of skin cells into human melanocytes in another study¹⁵. In this protocol, despite the use of a different screening method, the same result was obtained with the combination of Mitf, Sox10, and Pax3 for direct reprogramming of skin cells into melanocytes as described previously¹⁴. Developing a system to generate melanocytes from other skin cells can provide a scheme for transforming other skin cells of vitiligo patients into melanocytes. Hence, it is crucial to construct a simple and efficient method for this direct reprogramming to generate melanocytes successfully.

프로토콜

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.

결과

This article includes the protocols of a concentrated lentivirus packaging system to produce lentivirus of transcription factors for direct reprogramming of fibroblasts to melanocytes and protocols for screening for transcription factors and direct reprogramming of melanocytes from MEFs.

The success of concentrated lentivirus production was evaluated by observing the fluorescence intensity of GFP (Figure 1A) or by flow cytometry (Figure 1B

토론

The quality of the virus is crucial for the success of direct reprogramming to melanocytes in this protocol. The method of packaging and concentrating viruses in this protocol is simple and easy to repeat and does not rely on any other auxiliary concentrated reagent. This protocol can be followed successfully in most laboratories. To ensure the quality of the concentrated virus, the following points need special attention. One is the cell status of HEK-293T. Although HEK-293T cells are immortalized cells, the cells used ...

자료

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA	Gibco	25300-062	Stored at -20 °C
0.45 μM filter	Millipore	SLHVR33RB
5 mL polystyrene round bottom tube	Falcon	352052
95%/100% ethanol	LANBAO	210106	Stored at RT
Adenine	Sigma	A2786	Stock concentration 40 mg/mL Final concentration 24 µg/mL
Alexa Fluor 555 Goat anti-Mouse IgG2a	Invitrogen	A21137	Dilution of 1:500 to use
Antibiotics(Pen/Strep)	Gibco	15140-122	Stored at -20 °C
Anti-TRP1/TYRP1 Antibody	Millipore	MABC592	Host/Isotype: Mouse IgG2a Species reactivity: Mouse/Human Dilution of 1:200 to use
Anti-TRP2/DCT Antibody	Abcam	ab74073	Host/Isotype: Rabbit IgG Species reactivity: Mouse/Human Dilution of 1:200 to use
CHIR99021	Stemgent	04-0004	Stock concentration 10 mM Final concentration 3 μM
Cholera toxin	Sigma	C8052	Stock concentration 0.3 mg/mL Final concentration 20 pM
Cy3 Goat anti-Rabbit IgG (H+L)	Jackson Immunoresearch	111-165-144	Dilution of 1:500 to use
DMEM (High glucose)	HyClone	SH30243.01	Stored at 4 °C
DMSO	Sigma	D2650	Stored at RT
FBS	Gibco	10270-106	Stored at -20 °C Heat-inactivated before use
Gelatin	Sigma	G9391	Stored at RT
GFP-PURO plasmids (Mitf, Sox10, Pax3, Sox2, Sox9 and Snai2)	Hanheng Biological Technology Co., Ltd.	pHBLPm003198 pHBLPm001143 pHBLPm002968 pHBLPm002981 pHBLPm004348 pHBLPm000325	Stored at -20 °C
Hematoxylin	Abcam	ab220365	Stored at RT
Human EDN3	American-Peptide	88-5-10A	Stock concentration 100 μM Final concentration 0.1 μM
Hydrocortisone	Sigma	H0888	Stock concentration 100 µg/mL Final concentration 0.5 µg/mL
L-DOPA	Sigma	D9628	Stored at RT
Lipofectamine 2000	Invitrogen	11668-019	Transfection reagent, stored at 4 °C
Masson-Fontana staining kit	Solarbio	G2032	Stored at 4 °C
Neutral balsam	Solarbio	G8590	Stored at 4 °C
Paraformaldehyde	Sigma	P6148	Stored at RT
PBS (-)	Gibco	C10010500BT	Stored at RT
Phorbol 12-myristate 13-acetate (TPA)	Sigma	P8139	Stock concentration 1 mM Final concentration 200 nM
Polybrene	Sigma	H9268	cationic polymeric transfection reagent; Stock concentration 8 μg/µL Final concentration 4 ng/µL
Puromycin	Gibco	A11138-03	Stored at -20 °C
Recombinant human bFGF	Invitrogen	13256-029	Stock concentration 4 μg/mL Final concentration 10 ng/mL
Recombinant human insulin	Sigma	I3536	Stock concentration 10 mg/mL Final concentration 5 µg/mL
Recombinant human SCF	R&D	255-SC-010	Stock concentration 200 μg/mL Final concentration 100 ng/mL
RPMI-1640	Gibco	11875-093	Stored at 4 °C
Xylene	Sigma	1330-20-7	Stored at RT