Name: Direct Reprogramming of Mouse Fibroblasts into Melanocytes (Video) | JoVE Journal
Uploaded: 2021-08-27T14:30:00.000+00:00
Duration: 9 min 38 s
Description: Discover direct reprogramming of mouse fibroblasts into melanocytes using Mitf, Sox10, and PAX3 transcription factors. This optimized method enhances efficiency and stability for regenerative medicine applications.

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5 minutes

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).

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
<ol>
	<li>Production of the concentrated virus (Figure 1A, B)
	<ol>
		<li>Plate 1.5 &#215; 106 HEK-293T cells into a 60 mm dish and culture these cells with normal medium (see Table 1) at 37 &#176;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).</li>
		<li>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 &#181;L of DMEM per 1 cm2). Leave the cells at 37 &#176;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 &#34;starved&#34; for better plasmid transfection.</li>
		<li>Prepare the mixture of plasmids (mix A) containing 3 &#181;g of the target plasmids of Mitf, Sox10, Pax3, Sox2, Sox9, or Snai2; 1 &#181;g of the packaging plasmid PMD2.G, and 2 &#181;g of the packaging plasmid PSPAX2. Make up the volume of mix A to 150 &#181;L with serum-free DMEM. Prepare the mix of the transfection reagent (mix B) by adding 12 &#181;L 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 &#181;L with serum-free DMEM. 
		NOTE: When preparing the mixes, it is important to add liquid slowly to avoid air bubbles.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Mix the virus supernatants collected at two different time points. Centrifuge at 200 &#215; g for 5 min at 4 &#176;C. Pass the supernatant through 0.45 &#181;m filters and collect it in a 50 mL sterile conical tube. 
		NOTE: The virus collected at 24 h can be stored at 4 &#176;C and mixed with the virus collected at 48 h.</li>
		<li>Concentrate the virus supernatant by centrifuging at 6000 &#215; g at 4 &#176;C overnight (~16 h). Ensure that the virus pellet is visible at the bottom of the conical tube after centrifuging.</li>
		<li>Pour out the supernatant slowly. Dissolve the virus pellet in a volume of normal medium that is 1/100th 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 &#8722;80 &#176;C. 
		NOTE: The virus is concentrated 100x with this method. The concentrated virus (100x) can be stored for &#62;1 year at &#8722;80 &#176;C. Avoid repeated freezing and thawing of the virus.</li>
	</ol>
	</li>
	<li>Detection of concentrated virus titer (Figure 1C)
	<ol>
		<li>Plate 1 &#215; 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 &#176;C in a humidified incubator with 5% CO2.</li>
		<li>Add 0.1 &#181;L or 0.2 &#181;L of fluorescent concentrated virus (100x) to each well after 24 h, and add 4 ng/&#181;L 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 &#181;L or 0.2 &#181;L of the fluorescent concentrated virus can maintain the infection efficiency within this range. The concentrated virus titer can reach 1 &#215; 108 transducing units (TU)/mL at least.</li>
		<li>Approximately 48 h after infection, wash the dish with 1 mL of sterile phosphate-buffered saline (PBS) to remove dead cells.</li>
		<li>Trypsinize these cells using 250 &#181;L of 0.05% trypsin-EDTA per well in a 6-well plate for 1 min at room temperature. Centrifuge at 200 &#215; g for 5 min at 4 &#176;C and then remove the supernatant.</li>
		<li>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.</li>
		<li>Calculate the concentrated virus titer using the following formula: 105 (cell volume) &#215; infection rate (GFP+%)/added virus volume (0.1 &#181;L or 0.2 &#181;L).</li>
	</ol>
	</li>
</ol>
2. Direct reprogramming of fibroblasts to melanocytes (Figure 2A)
<ol>
	<li>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 &#176;C for no more than 2 months.</li>
	<li>Plate 5 &#215; 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 &#176;C in a humidified incubator with 5% CO2 overnight.</li>
	<li>After 24 h, confirm that the MEFs have reached 40-50% confluence. Replace the medium with normal medium.</li>
	<li>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 &#181;g/mL of the cationic polymeric transfection agent. 
	Cell number (5 &#215; 104) &#215; 30 (multiplicity of infection, MOI)/virus titer (1)</li>
	<li>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 &#181;g/mL puromycin to screen stable infected cell lines.</li>
	<li>On Day 2, 48 h after infection, replace the supernatant medium gradually with the reprogramming medium. First, change 1/4th of the total medium volume, and add 3 &#181;M CHIR99021.</li>
	<li>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.</li>
	<li>To passage the cells, add 500 &#181;L 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 &#215; g for 5 min at 4 &#176;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 &#215; 104/cm2. Culture these cells at 37 &#176;C in a humidified 5% CO2 incubator. 
	&#8203;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.</li>
</ol>
3. Optimization for direct reprogramming and identification
<ol>
	<li>Screening for the optimized transcription factors
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Identification of induced melanocytes (iMels)
	<ol>
		<li>Use immunofluorescence staining18 to verify that the iMels express melanocytic proteins, including TYRP-1 and DCT (Figure 4A).</li>
		<li>Melanin-specific 3,4-dihydroxyphenylalanine (DOPA) staining (Figure 4B)
		<ol>
			<li>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 &#176;C for 2 h to promote dissolution. 
			NOTE: The solution can be kept at 4 &#176;C for no more than one month.</li>
			<li>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.</li>
			<li>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 &#176;C for 30 min; shake it several times to promote dissolution.</li>
			<li>Add 1 mL of freshly prepared 0.1% DOPA staining solution. Incubate in an oven at 37 &#176;C for 2-5 h. If there are no brown-black particles, continue to incubate at 37 &#176;C for another 2 h but not for &#62;5 h. Check the samples every 30 min.</li>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
		<li>Melanin-specific Masson-Fontana staining (Figure 4B)
		<ol>
			<li>Plate iMels in a 30 mm dish and fix the cells with 4% paraformaldehyde for 20 min; wash the dish 3 times with PBS.</li>
			<li>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 &#176;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 &#176;C oven to continue incubating but not for &#62;40 min. Some water can be added to the dark box to prevent drying up.</li>
			<li>Aspirate solution A and wash the dish 5-6 times with distilled water, 1-2 min each time.</li>
			<li>Add 1 mL solution B (hypo solution) from the Masson-Fontana staining kit, and leave the dish at room temperature for 3-5 min.</li>
			<li>Aspirate solution B and wash the dish with tap water 3 times, 1 min each time.</li>
			<li>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.</li>
			<li>Aspirate solution C and wash the dish 3 times with distilled water, 1 min each time.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol><li>Ezzedine, K., Eleftheriadou, V., Whitton, M., van Geel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vitiligo%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Vitiligo.</a> Lancet. 386 (9988), 74-84 (2015).</li>
<li>Picardo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vitiligo%5BTitle%5D)+AND+%22Nature+Reviews.+Disease+Primers%22%5BJournal%5D)">Vitiligo.</a> Nature Reviews. Disease Primers. 1, 15011(2015).</li>
<li>Speeckaert, R., van Geel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vitiligo%3A+An+update+on+pathophysiology+and+treatment+options%5BTitle%5D)+AND+%22American+Journal+of+Clinical+Dermatology%22%5BJournal%5D)">Vitiligo: An update on pathophysiology and treatment options.</a> American Journal of Clinical Dermatology. 18 (6), 733-744 (2017).</li>
<li>Cortelazzi, C., Pellacani, G., Raposio, E., Di Nuzzo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vitiligo+management%3A+combination+of+surgical+treatment+and+phototherapy+under+reflectance+confocal+microscopy+monitoring%5BTitle%5D)+AND+%22European+Review+for+Medical+and+Pharmacological+Sciences%22%5BJournal%5D)">Vitiligo management: combination of surgical treatment and phototherapy under reflectance confocal microscopy monitoring.</a> European Review for Medical and Pharmacological Sciences. 24 (13), 7366-7371 (2020).</li>
<li>Mohammad, T. F., Hamzavi, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Surgical+therapies+for+vitiligo%5BTitle%5D)+AND+%22Dermatologic+Clinics%22%5BJournal%5D)">Surgical therapies for vitiligo.</a> Dermatologic Clinics. 35 (2), 193-203 (2017).</li>
<li>Bishnoi, A., Parsad, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+and+molecular+aspects+of+vitiligo+treatments%5BTitle%5D)+AND+%22International+journal+of+molecular+sciences%22%5BJournal%5D)">Clinical and molecular aspects of vitiligo treatments.</a> International journal of molecular sciences. 19 (5), 1509(2018).</li>
<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+decade+of+transcription+factor-mediated+reprogramming+to+pluripotency%5BTitle%5D)+AND+%22Nature+Reviews.+Molecular+Cell+Biology%22%5BJournal%5D)">A decade of transcription factor-mediated reprogramming to pluripotency.</a> Nature Reviews. Molecular Cell Biology. 17 (3), 183-193 (2016).</li>
<li>Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pluripotent+stem+cell-based+cell+therapy-promise+and+challenges%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Pluripotent stem cell-based cell therapy-promise and challenges.</a> Cell Stem Cell. 27 (4), 523-531 (2020).</li>
<li>Xu, J., Du, Y., Deng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+lineage+reprogramming%3A+strategies%2C+mechanisms%2C+and+applications%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Direct lineage reprogramming: strategies, mechanisms, and applications.</a> Cell Stem Cell. 16 (2), 119-134 (2015).</li>
<li>Ieda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+reprogramming+of+fibroblasts+into+functional+cardiomyocytes+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142 (3), 375-386 (2010).</li>
<li>Gascón, S., Masserdotti, G., Russo, G. L., Götz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+neuronal+reprogramming%3A+achievements%2C+hurdles%2C+and+new+roads+to+success%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Direct neuronal reprogramming: achievements, hurdles, and new roads to success.</a> Cell Stem Cell. 21 (1), 18-34 (2017).</li>
<li>Atkinson, P. J., Kim, G. S., Cheng, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+cellular+reprogramming+and+inner+ear+regeneration%5BTitle%5D)+AND+%22Expert+Opinion+on+Biological+Therapy%22%5BJournal%5D)">Direct cellular reprogramming and inner ear regeneration.</a> Expert Opinion on Biological Therapy. 19 (2), 129-139 (2019).</li>
<li>Kurita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+reprogramming+of+wound-resident+cells+generates+skin+epithelial+tissue%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">In vivo reprogramming of wound-resident cells generates skin epithelial tissue.</a> Nature. 561 (7722), 243-247 (2018).</li>
<li>Yang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+conversion+of+mouse+and+human+fibroblasts+to+functional+melanocytes+by+defined+factors%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors.</a> Nature Communications. 5, 5807(2014).</li>
<li>Fehrenbach, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+tumorigenic+potential+upon+transdifferentiation+from+keratinocytic+into+melanocytic+lineage%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Loss of tumorigenic potential upon transdifferentiation from keratinocytic into melanocytic lineage.</a> Scientific Reports. 6, 28891(2016).</li>
<li>Majumdar, G., Vera, S., Elam, M. B., Raghow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+streamlined+protocol+for+extracting+RNA+and+genomic+DNA+from+archived+human+blood+and+muscle%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.</a> Analytical Biochemistry. 474, 25-27 (2015).</li>
<li>Bachman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reverse-transcription+PCR+%28RT-PCR%29%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Reverse-transcription PCR (RT-PCR).</a> Methods in Enzymology. 530, 67-74 (2013).</li>
<li>Donaldson, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunofluorescence+staining%5BTitle%5D)+AND+%22Current+Protocols+in+Cell+Biology%22%5BJournal%5D)">Immunofluorescence staining.</a> Current Protocols in Cell Biology. 69 (1), 1-7 (2015).</li>
<li>Yin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-viral+vectors+for+gene-based+therapy%5BTitle%5D)+AND+%22Nature+Reviews.+Genetics%22%5BJournal%5D)">Non-viral vectors for gene-based therapy.</a> Nature Reviews. Genetics. 15 (8), 541-555 (2014).</li>
</ol>

The authors have no conflicts of interest to disclose.

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 ...

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.

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

direct reprogramming of mouse fibroblasts into melanocytes

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

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.

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</st...

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, ...

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 leap. 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 times g 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 times g 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 minus 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 times g 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 times g 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.

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Research

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Developmental Biology

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Discover direct reprogramming of mouse fibroblasts into melanocytes using Mitf, Sox10, and PAX3 transcription factors. This optimized method enhances efficiency and stability for regenerative medicine applications.

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

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