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>

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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

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...

Watch this Scientific Journal Video about Direct Reprogramming of Mouse Fibroblasts into Melanocytes at JoVE.com

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

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, ...

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2.0K Views.  Jiangsu University. Directe herprogrammering om melanocyten te genereren is gemeld. Verschillende transcriptiefactoren worden echter in verschillende onderzoeken gebruikt. In dit protocol hebben we het aantal transcriptiefactoren gevalideerd en verminderd met een hogere herprogrammeringsefficiëntie.We hebben een direct herprogrammeringssysteem opgezet om melanocyten te genereren met behulp van slechts drie transcriptiefactoren, Mitf, Sox10 en PAX3, en hebben het kweeksysteem aangepast om het stabieler en...

Video: Direct Reprogramming of Mouse Fibroblasts into Melanocytes

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

Biology

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

Cancer Research

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

Genetics

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Neuroscience

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

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