Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.
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.
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 vitiligo1,2. 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 transplantation3,4,5. 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 effective2,6. 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 considered14,15. 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.
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
2. Direct reprogramming of fibroblasts to melanocytes (Figure 2A)
3. Optimization for direct reprogramming and identification
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) after infecting HEK-293T cells for 48 h with unconcentrated lentivirus (1x) and concentrated lentivirus (100x). The titer of the concentrated virus was 108 TU/mL or more, which is relatively high (Figure 1C).
Direct reprogramming of fibroblasts to melanocytes is achieved through the infection with transcription factor lentivirus and transformation with the optimized reprogramming medium. The scheme for the generation of iMels from MEFs is shown in Figure 2A. Cell morphology gradually changes during direct reprogramming. Cell synapses become elongated, and cell nuclei enlarge. However, these cells gradually age after ~Day 20 (~5 passages) (Figure 2B).
One transcription factor was removed at a time from the original set of six transcription factors (Mitf, Pax3, Sox10, Sox9, Sox2, and Snai2) to determine the transcription factor with the greatest impact on reprogramming. Removal of Mitf, Pax3, or Sox10 resulted in the silencing of the expression of melanocytic genes Tyr, Tyrp1, and Mlana (Figure 3A), indicating that these three transcription factors had the greatest impact on fibroblast conversion to melanocytes. The expression of melanocytic genes induced by direct reprogramming with three transcription factors was higher than that of all six transcription factors (Figure 3B).
Finally, the characteristics of the iMels obtained by using this optimized system of direct reprogramming were identified. The expression of melanocytic markers (TYR, TYYP1) was detected using immunofluorescent staining (Figure 4A). Melanin-specific staining methods, including DOPA and Masson-Fontana staining, also showed positive results (Figure 4B).
Figure 1: Production of concentrated virus and estimation of titer. (A) Fluorescence intensity of GFP after infecting HEK-293T cells with unconcentrated lentivirus (1x) and concentrated lentivirus (100x) (B) Comparison of the infection rates of unconcentrated lentivirus (1x) and concentrated lentivirus (100x) as detected by flow cytometry. (C) Titer estimation of concentrated lentivirus. Scale bars = 250 µm. Abbreviations: GFP = green fluorescent protein; TU = transducing units. Please click here to view a larger version of this figure.
Figure 2: Generation of iMels from MEFs. (A) Schematic diagram of iMels generation. (B) Changes in cell morphology during conversion from MEFs to iMels in direct reprogramming on day 0, day 3, day 10, and day 20+. Scale bars = 100 µm. Abbreviations: iMels = induced melanocytes; MEFs = mouse embryonic fibroblasts. Please click here to view a larger version of this figure.
Figure 3: Screening for optimized transcription factors. (A) qRT-PCR of Tyr, Tyrp1, and Mlana mRNA levels in cells transduced with five transcription factors (one of the original six transcription factors has been removed). MEF + EV and MEF + 6F are used as negative control and positive control, respectively. The mRNA expression is normalized to Gapdh expression. (B) qRT-PCR of Tyr,Tyrp1, and Mlana mRNA levels in cells transduced with the original six transcription factors and with three transcription factors. MEF, MEF + EV, and MMC are used as a blank, negative control, and positive control, respectively. The mRNA levels are normalized to Gapdh levels. All values are mean ± SD of three independent experiments. The primer sequences are shown in Table 3. Abbreviations: qRT-PCR = quantitative reverse-transcription PCR; MEF = mouse embryonic fibroblasts; MEF+EV = mouse embryonic fibroblasts + empty vector; MMC = mouse melanocyte; 6F = six transcription factors comprising Mitf, Pax3, Sox10, Sox9, Sox2, and Snai2; 3F: three transcription factors comprising Mitf, Pax3, and Sox10. Please click here to view a larger version of this figure.
Figure 4: Functional identification of iMels. (A) Immunostaining of melanocytic markers (TYR, TYYP1) in iMels. Scale bar = 50 µm. See the Table of Materials for the dilution of antibodies used in this study. (B) Masson-Fontana staining and DOPA staining. MEF and the Melan-a cell line are used as negative control and positive control, respectively. Scale bar = 50 µm. Abbreviations: iMels = induced melanocytes; DOPA = 3,4-dihydroxyphenylalanine; MEF = mouse embryonic fibroblast. Please click here to view a larger version of this figure.
Components | Dose (concentration, volume) | Final Concentration | |
Normal Medium (100 mL) | DMEM/HIGH GLUCOSE | 89 mL | |
Heat-inactivated FBS | 10 mL | ||
Antibiotics(Pen/Strep) | 10000 U/mL, 1 mL | 100 U/mL | |
Reprogramming medium (100 mL) | RPMI-1640 | 88 mL | |
Heat-inactivated FBS | 10 mL | ||
Recombinant Human SCF | 200 μg/mL, 50 μL | 100 ng/mL | |
Recombinant Human bFGF | 4 μg/mL, 250 μL | 10 ng/mL | |
Recombinant Human insulin | 10 mg/mL, 50 μL | 5 μg/mL | |
EDN3 human | 100 μM, 100 μL | 0.1 μM | |
Cholera Toxin | 0.3 mg/mL, 0.56 μL | 20 pM | |
Phorbol 12-myristate 13-acetate (TPA) | 1 mM, 20 μL | 200 nM | |
Hydrocortisone | 100 μg/mL, 500 μL | 0.5 μg/mL | |
Adenine | 40 mg/mL, 60 μL | 24 μg/mL |
Table 1: Components of normal and reprogramming media.
Culture Dish | Seeding Density (cells/dish) | Medium (mL) | Plasmid System | Transfection reagent (µL) | ||
Target plasmid (μg) | PMD2.G (μg) | PSPAX2 (μg) | ||||
35 mm | 6 ×105 | 1.5 | 1.5 | 0.5 | 1 | 6 |
60 mm | 1.5 × 106 | 3.5 | 3 | 1 | 2 | 12 |
100 mm | 4 × 106 | 8 | 8 | 3 | 6 | 34 |
Table 2: Details of the lentivirus packaging system.
RT-PCR Primers | |
Target | Primer sets |
Gapdh | Forward: CAACGACCCCTTCATTGACC |
Reverse: CATTCTCGGCCTTGACTGTG | |
Tyr | Forward: GGGCCCAAATTGTACAGAGA |
Reverse: ATGGGTGTTGACCCATTGTT | |
Tyrp1 | Forward: AAGTTCAATGGCCAGGTCAG |
Reverse: TCAGTGAGGAGAGGCTGGTT | |
Mlana | Forward: AGACGCTCCTATGTCACTGCT |
Reverse: TCAAGGTTCTGTATCCACTTCGT |
Table 3: Primer information.
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 to make the concentrated virus must be healthy cells within 10 passages (the titer decreases with higher passages).
Another critical point is the proportion of the transfection reagent (in this case, lipofectamine) used. As cells are damaged after adding the transfection reagent, the ratio of the reagent to plasmids must be checked repeatedly to ensure the optimal condition of the cells and the quality of the virus. The 2:1 ratio of transfection reagent to plasmids is most suitable for HEK-293T cells for packaging the virus in this system. In addition, all steps require gentle handling throughout the process. As virus particles can be absorbed after coating, which affects the virus titer, it is not advisable to coat the dish with any matrix when packaging the virus. Due to the possibility of detachment of HEK-293T, the supernatant must be replaced carefully, especially when fresh medium is added after collecting the virus supernatant after 24 h. Some other considerations include cell density, the length of the transfection time, and the serum content in the medium. These are important issues that affect transfection efficiency. The conditions presented in this protocol are the most suitable, based on results obtained after many repeated experiments.
In the process of direct reprogramming to melanocytes, it is important to consider the state of the original cell MEFs and the density of MEFs used for direct reprogramming. Before starting the direct reprogramming, MEFs must be cultured in non-coated cell culture dishes. Cells in the proliferation phase (40-50% confluence) should be selected for direct reprogramming; the infection efficiency decreases with increasing cell density. The reprogrammed cells need to be plated in gelatin-coated culture dishes.
The length of time needed for the virus to infect cells is also critical; too long an infection time will impair cell survival, whereas too short an infection time will reduce the efficiency. Eight hours were found to be appropriate for the concentrated virus to infect MEFs in this protocol. The last critical point is the correct way of changing the reprogramming medium. MEFs need to adapt to a new medium. The state of the cells must be checked every day, and the medium must be changed carefully according to the cell status. Changing the reprogramming medium too quickly will cause a large number of cells to die.
When cultivating and subculturing iMels, the passage density of the cells is critical. iMels need a relatively high density to maintain growth. Cell synapses should be in contact with each other for the normal proliferation of these cells. If the density is too low, the cells may stop proliferating. Here, the density of 3 × 104/cm2 was found to be a suitable passage density for iMels. After 5 passages, the iMels begin aging (Figure 2B); the melanin is more mature at this time, and the resultant cells can be used for immunofluorescence, DOPA, or Masson-Fontana staining. Earlier passages of cells can be used for RT-PCR because the expression of genes will change at a relatively early stage.
Although direct reprogramming is widely studied, there is little research on the direct reprogramming of fibroblasts to melanocytes. Different studies have used different transcription factors14,15, leading to much confusion. This protocol explains how to produce high-quality concentrated viruses and screen several transcription factors to select the most important ones for direct reprogramming to melanocytes. The medium was supplemented with nutritional factors for the direct reprogramming to melanocytes in this protocol (Table 2). Finally, functional iMels were identified successfully. The clear and optimized melanocyte direct reprogramming system could provide new treatment strategies for depigmentation diseases such as vitiligo.
However, there are still some limitations to the technology presented in this article. First, it is challenging to estimate the efficiency of this protocol. CRISPR-Cas9 gene editing could be combined with this protocol to knock in melanocytic genes, such as Tyr or Tyrp-1, into the initial cells to observe the percentage of induced cells during the reprogramming process. In addition, the lentivirus introduction system used in this protocol may carry the risk of gene recombination and insertion mutation19. In the future, more efficient and safer introduction methods could be used, such as non-viral recombinant protein expression vectors or mRNA vectors. The experiment in this protocol is still at the in vitro stage. The next step is to reprogram the melanocytes directly in vivo, causing non-pigmented skin to become pigmented and to use the direct reprogramming system to design an effective treatment of vitiligo.
The authors have nothing to disclose.
This study was partially supported by grants from the National Natural Science Foundation of China (82070638 and 81770621) and the Natural Science Foundation of Jiangsu Province (BK20180281).
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 |