Name: y-27632 enriches the yield of human melanocytes from adult skin tissues
Uploaded: 2020-07-08T13:00:00.000+00:00
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This work was supported by The National Key Research and Development Program of China (2017YFA0104604), The General Program of National Natural Science Foundation of China (81772093), Military Logistics Research Project (AWS17J005), the Key Program of Shandong Province Natural Science Foundation (ZR2019ZD36) and The Key Research and Development Program of Shandong Province (2019GSF108107) to X.W. Guangdong Basic and Applied Basic Research Foundation (2019A1515110833) and The Fundamental Research Funds of Shandong University (2019GN043) to J.M.

The use of adult foreskin tissues in this protocol has been approved by the Human Research Ethics Committee (No.2015120401, date: May 12, 2015).
NOTE: Perform all the following procedures in a sterile environment to prevent contaminations of cells and cultures.
1. Preparations
<ol>
	<li>Gather fresh adult foreskin tissues from circumcision surgeries in 15 mL tubes containing 10 mL of phosphate buffer saline (PBS) and store in 4 &#176;C. 
	NOTE: Separate the tissues as detailed below within 24 h after their excision.</li>
	<li>Prepare reagents and culture medium.
	<ol>
		<li>Prepare 3% P/S (penicillin/streptomycin) as the washing solution. Mix 50 mL of PBS with 1.5 mL of penicillin (100 U/mL) and streptomycin (100 mg/L) (P/S).</li>
		<li>Prepare the termination solution (neutralization solution) for neutralization of enzymatic digestion. Supplement 1% P/S, 10% fetal bovine serum (FBS) into DMEM medium.</li>
		<li>Prepare TIVA medium to culture melanocytes: Ham&#8217;s F12; 10% FBS; 1x P&#8260;S&#8260;glutamine; 50 ng/mL 12-O-tetradecanoyl phorbol-13-acetate (TPA); 1 x 10-4 M 3-isobutyl-1-methyl xanthine (IBMX); 1 &#956;M Na3VO4; 1 x 10-3 M N-6,2&#8242;-O-dibutyryladenosine-3&#8242;,5&#8242;-cyclic monophosphate (dbcAMP).</li>
	</ol>
	</li>
</ol>
2. The conventional method
<ol>
	<li>Skin tissue pretreatment
	<ol>
		<li>Rinse each adult foreskin tissue once with 10 mL of 75% ethanol (alcohol) for 30 s, and then rinse twice with 10 mL of washing solution (3% P/S), for 5 min each. 
		NOTE: Wash foreskin tissues with 10 mL of PBS in the beginning if there is a lot of blood. All washes are prepared in 100 mm cell culture dishes.</li>
		<li>Scrape each foreskin tissue with a surgical blade to remove subcutaneous adipose tissues and loose connective tissues in the cover of a 100 mm cell culture dish. 
		NOTE: Use a scalpel to scrape the fat layers away until only the thin epidermis and the dense dermis remain.</li>
		<li>Transfer each adult foreskin into another 100 mm culture dish with the dermis side down. 
		NOTE: Cut each adult foreskin tissue into 3-4 mm wide strips using a scalpel blade to make the dispase work more efficiently.</li>
		<li>Add 2.5 mg/mL dispase to each 100 mm culture dish with adult foreskin tissues and incubate for 16 to 20 h at 4 &#176;C. 
		NOTE: Each gram of tissue is digested with 10 mL of dispase solution.</li>
	</ol>
	</li>
	<li>Separation of the epidermis from the adult foreskin tissue
	<ol>
		<li>After digestion for 16 to 20 h, separate the epidermis from the dermis using tweezers. Grab one side of the dermal edge of the tissue with one tweezer and the corresponding position of the epidermal part with another tweezer and gently peel off the epidermis.</li>
		<li>Wash the epidermis 3x with washing solution (3% P/S), and then transfer the epidermis into a tube.</li>
		<li>Submerge the epidermis by adding 5 mL of 0.05% trypsin into the tube and incubate in a water bath for 30 min at 37 &#176;C. 
		NOTE: Shake the tube to ensure the epidermis is completely submerged before incubation.</li>
	</ol>
	</li>
	<li>Collection and culture of primary cells
	<ol>
		<li>Add an equal volume of termination solution (10% FBS, 1% P/S in DMEM) to neutralize the trypsin and pipette the solution up and down 10-15 times to separate the epidermal cells.</li>
		<li>Pass the cell suspension into a new 50 mL tube through a 100 &#181;m mesh filter to remove debris.</li>
		<li>Centrifuge at 200 x g for 5 min.</li>
		<li>Remove the supernatant and add 10 mL of inoculation TIVA medium to resuspend the cells.</li>
		<li>Seed the resuspended cells into a 100 mm culture dish.</li>
		<li>Culture in a 37&#176; C incubator with 5% CO2.</li>
		<li>Change the medium every 2 days.</li>
		<li>Passage cells when they reach 80% confluence.</li>
	</ol>
	</li>
</ol>
3. The new method
NOTE: The procedure for tissue preparation and digestion in the new method is the same as described above for the conventional method, the only difference being that the isolated fresh cells are resuspended in 10 mL of TIVA medium containing 10 &#181;M Y-27632.
<ol>
	<li>Two days after seeding, replace the media with normal TIVA medium without Y-27632. After that, the culture conditions are the same between the new and the conventional methods.</li>
</ol>
4. Cell passaging
<ol>
	<li>Remove the TIVA medium and rinse each culture dish twice with PBS.</li>
	<li>Add 2 mL of 0.05% trypsin per 100 mm cell culture dish. 
	NOTE: Shake the dish to ensure adequate contact between digestive enzymes and the bottom of the dish.</li>
	<li>Digest in a 37 &#176;C incubator for 2 min.</li>
	<li>Use a microscope to check the cells, and make sure that most cells have dissociated from the cell culture dish. 
	NOTE: Gently tap the dish to release the cells to round up and float in the solution. Prolong the digestion time if most cells did not suspend after tapping, but no more than 5 more min.</li>
	<li>Transfer the melanocytes into a 15 mL tube after neutralizing the trypsin activity by adding 2 mL of termination solution. Centrifuge at 200 x g for 5 min.</li>
	<li>Resuspend each cell pellet with 10 mL of TIVA medium after slowly removing the supernatant, and then count the number of melanocytes.</li>
	<li>Plate about 1 x 106 melanocytes in 10 mL of TIVA medium per 100 mm cell culture dish.</li>
	<li>Renew the cell culture medium every 48 h.</li>
</ol>

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All authors declare no interest of conflict.

The protocol described here was based on a recent publication16. Some attention should be paid to the following critical steps to achieve the best results with the new method. First, successful separation of the epidermis and the dermis is crucial. Cut the adult foreskin tissues into 3-4 mm wide strips using a scalpel blade to make the dispase work more thoroughly and easily to separate the epidermis from the dermis. Second, when separating those two layers, contamination of the dermis should be avoided since dermal fibroblasts can also grow in melanocyte culture medium. Third, the trypsin digestion step should be less than 30 min; otherwise, it will significantly decrease the viability of the isolated cells.
This new method significantly shortens the time needed for melanocyte isolation. Human epidermal melanocytes produce melanin, which protects the skin from solar irradiation damage. Eisinger and Marko proposed the &#64257;rst practical method for the culture of human melanocytes from the epidermis5. Most importantly, the tissues used usually come from newborn babies and it is very difficult to isolate primary melanocytes from adult skin tissues. The Rho-associated protein kinase inhibitor Y-27632 significantly improves the efficiency of epidermal stem cell isolation11-13. Additionally, we also reported a method for isolating human primary epidermal cells from skin tissues in the presence of Y-2763210,11. In this protocol, Y-27632 was shown to efficiently increase the yield of primary melanocytes. Using the conventional method has a low cell recovery rate and needs around 3 to 4 weeks of culture for melanocytes to grow in sufficient numbers to passage. This new method, in which Y-27632 is added to the TIVA growth medium, increased melanocyte yield by about five times, and the melanocytes obtained can proliferate normally. We also tested the new method using a commercial medium and found similar results. Regarding the mechanism involved, a recent study demonstrated that the effect of Y-27632 to enhance the efficiency of melanocyte isolation occurs mainly through keratinocytes16.
Importantly, we obtained pure melanocytes with normal function using the new method. The Passage 1 (P1) melanocytes were nearly all stained for MITF expression indicating that pure melanocytes are obtained after the first passage. Primary melanocytes with high growth potential are very important for biological research and clinical applications. The melanocytes isolated using the new method can be induced to pigment after treatment with forskolin, an activator of adenylate cyclase that increases cyclic AMP levels, indicating these melanocytes can function normally, which will be useful for studying pigmentation defects and melanoma14.
In summary, a highly efficient method was successfully established to isolate primary melanocytes from adult skin tissues. This improved method could contribute to provide sufficient numbers of melanocytes in a rapid manner for biological research and for clinical applications.

The goal of this study was to develop a simple and effective protocol to isolate melanocytes from the epidermis of adult skin for biological research and clinical applications. Melanocytes, which are located in the basal epidermis of the skin and in hair follicles, play an important role in pigmentation of the skin and hair by producing melanin1. The resulting skin pigmentation from epidermal melanocytes acts as an ultraviolet radiation &#64257;lter that reduces/prevents DNA damage to underlying cells in the skin2. The abnormal proliferation of melanocytes in the skin is quite common, such as in the formation of benign nevi (moles) in which melanocytes potentially transform to oncogenic growth followed by cellular senescence3.
Since 1957, the isolation and subsequent culture of human primary melanocytes has been possible4, but only since 1982 has there been an efficient method that can reproducibly establish cultures of human melanocytes from the epidermis5. The conventional method to isolate primary melanocytes from the epidermis involves a two-step enzymatic digestion. Briefly, the skin is initially digested with dispase to separate the epidermis from the dermis, after which the epidermis is digested with trypsin to produce suspensions containing melanocytes and keratinocytes, which can then be selectively grown in different media. Currently, the initial culture usually takes about 3 to 4 weeks for melanocytes to reach confluency using the conventional method, which is likely due to the low efficiency of their isolation. Therefore, increasing the initial production of melanocytes from adult skin tissues would be quite helpful for both laboratory research and clinical applications.
Many growth factors, most of which have been shown to be secreted by keratinocytes (e.g., &#945;-MSH, ACTH, bFGF, NGF, ET-1, GM-CSF, HGF, LIF and SCF)5-8, regulate the differentiation and proliferation of mammalian melanocytes. In the absence of feeder layers, the lack of those growth factors leads to the decreased proliferation of melanocytes and their increased apoptosis9. The co-culture of keratinocytes as feeder cells and melanocytes could lead to accelerated melanocyte proliferation and reduced apoptosis. However, the co-culture method not only demands more skin tissues to prepare keratinocytes, which is not practical, but also could not work efficiently because the culture conditions required by melanocytes does not favor keratinocyte growth, and vice-versa.
Previous studies have reported that the addition of Y-27632, a ROCK inhibitor, into the growth medium can enhance the yield of human primary epidermal cells from skin tissues10-14. Therefore, it would be interesting to test whether the isolation of human primary melanocytes would benefit from the presence of Y-27632. Indeed, the addition of Y-27632 into the inoculation TIVA medium15, which contains TPA, IBMX, Na3VO4 and dbcAMP, significantly increased the yield of primary melanocytes isolated from foreskin tissues in the initial cultures16. Compared to the conventional protocol, this new protocol is significantly more efficient in increasing the yield of melanocytes16. Therefore, this protocol describes the new method in detail using adult skin tissues based on a previous study16 in order to promote its application in basic and applied biological research.

<table><tbody><tr><td>Alexa fluor-594 donkey anti-mouse IgG</td><td>Thermo Fisher Scientific</td><td>A21203</td><td>For Immunofluorescence</td></tr><tr><td>Alexa fluor-594 donkey anti-rabbit IgG</td><td>Thermo Fisher Scientific</td><td>R37119</td><td>For Immunofluorescence</td></tr><tr><td>Cell Culture Dish</td><td>Eppendorf</td><td>30702115</td><td>For cell culture</td></tr><tr><td>Cell Strainer</td><td>Corning incorporated</td><td>431792</td><td>Cell filtration</td></tr><tr><td>15 mL Centrifuge Tube</td><td>KIRGEN</td><td>171003</td><td>For cell centrifuge</td></tr><tr><td>50 mL Centrifuge Tube</td><td>KIRGEN</td><td>171003</td><td>For cell centrifuge</td></tr><tr><td>CO2 Incubator</td><td>Thermo Scientific</td><td>51026333</td><td>For cell incubating</td></tr><tr><td>Constant Temperature Shaker</td><td>Shanghai Boxun</td><td>150036</td><td>For water bath</td></tr><tr><td>DAPI</td><td>Abcam</td><td>ab104139</td><td>For Immunofluorescence</td></tr><tr><td>Dispase</td><td>Gibco</td><td>17105-041</td><td>For melanocyte isolation</td></tr><tr><td>DMEM</td><td>Thermo Scientific</td><td>C11995500</td><td>Component of neutralization medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Biological Industries</td><td>04-001-1AC5</td><td>Component of neutralization medium</td></tr><tr><td>Fluorescence microscope</td><td>Olympus</td><td>5E44316</td><td>For Immunofluorescence</td></tr><tr><td>Forskolin</td><td>MCE</td><td>HY-15371</td><td>Induce pigmentation</td></tr><tr><td>Glutamine</td><td>Thermo Fisher Scientific</td><td>25030081</td><td>melanocyte culture medium</td></tr><tr><td>Ham&#039;s F12</td><td>Thermo Scientific</td><td>11330032</td><td>melanocyte culture medium</td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>5C42258</td><td>For cell microscopic observation</td></tr><tr><td>3-isobutyl-1-methyl xanthine (IBMX)</td><td>Sigma</td><td>17018</td><td>melanocyte culture medium</td></tr><tr><td>mouse anti-human MITF</td><td>Abcam</td><td>ab12039</td><td>For Immunofluorescence</td></tr><tr><td>Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S6508</td><td>melanocytes culture medium</td></tr><tr><td>N6,2&#039;-O-dibutyryladeno-sine 3&#039;,5&#039;-cyclic monophosphate (dbcAMP)</td><td>Sigma</td><td>D0627</td><td>melanocyte culture medium</td></tr><tr><td>12-O-tetradecanoyl phorbol-13-acetate (TPA)</td><td>Sigma</td><td>79346</td><td>melanocyte culture medium</td></tr><tr><td>Penicillin Streptomycin</td><td>Thermo Scientific</td><td>15140-122</td><td>Antibiotics</td></tr><tr><td>rabbit anti-human Ki-67</td><td>Abcam</td><td>ab15580</td><td>For Immunofluorescence</td></tr><tr><td>Phosphate buffered solution</td><td>Solarbio Life Science</td><td>P1020-500</td><td>Washing solution</td></tr><tr><td>Sorvall ST 16R Centrifuge</td><td>Thermo Scientific</td><td>75004380</td><td>Cell centrifugation</td></tr><tr><td>TC20TM automated cell counter</td><td>Bio-Rad</td><td>1450102</td><td>Automatic cell counting</td></tr><tr><td>0.05% Trypsin</td><td>Life Technologies</td><td>25300-062</td><td>For melanocyte dissociation</td></tr><tr><td>Y-27632</td><td>Sigma</td><td>Y0503</td><td>For melanocyte isolation</td></tr></tbody></table>

y-27632 enriches the yield of human melanocytes from adult skin tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

Figure 1 shows a schematic diagram comparing the conventional and the new methods. The procedure for tissue preparation and digestion with the new method is the same as the procedure for the conventional method, the only difference being that the isolated cells are resuspended in 10 mL of TIVA medium in the presence of 10 &#181;M Y-27632. Two days after seeding, replace the media with normal TIVA medium without Y-27632. The conventional method of isolating primary melanocytes from skin tissues usually requires about 3 to 4 weeks for melanocytes to grow in sufficient numbers to passage, but more importantly, the tissues usually come from newborn babies and it is very difficult to isolate primary melanocytes from adult skin tissues. However, using the new method, melanocytes from adult tissues are observed in significantly greater numbers than melanocytes isolated using the conventional method. Microscopic views were taken at days 3, 6 and 8 when isolating the melanocytes from adult skin tissue using the conventional method and the new method (Figure 2A). In these microscopic views, the new method significantly increased the yield of melanocytes at days 6 and 8. Melanocyte numbers were then quantified by counting at least 10 different microscopic fields at days 3, 6 and 8. The results showed that the number of melanocytes isolated using the new method is much higher than the number of melanocytes isolated using the conventional method at days 6 and 8 (Figure 2B). After 8 days of culture, the melanocytes were harvested using trypsin and then counted with an automated cell counter in each single 100 mm dish, which revealed that the new method increased the melanocyte yield by about five times (Figure 2C). Microscopic views taken at day 9, one day after passaging, showed that the passaged melanocytes proliferated normally (Figure 2D), which was confirmed by Ki67 staining (Figure 2E).
The enhanced yield of melanocytes by Y-27632 during the initial culture after isolation has been shown through the regulation of keratinocytes in a previous publication16. As shown in Figure 3, Y-27632 did not increase the growth and proliferation of passaged melanocytes (Figure 3A-B) and did not inhibit the apoptosis of melanocytes (Figure 3C-D). However, Y-27632 significantly increased keratinocyte attachment (Figure 3E-H) and also promoted its surviving (Figure 3I) in the melanocyte-culture TIVA media. Either co-culture with keratinocytes in the presence of Y-27632 or the conditioned medium derived from Y-27632 treated keratinocytes could significantly enhance the growth of melanocytes (Figure 3J).
To further characterize the melanocytes isolated using the new method, MITF, a specific melanocyte marker, was analyzed using immunofluorescence (IF) staining to check the purity of melanocytes after initial passaging. Figure 4A shows that the percentage of cells expressing MITF at passage 1 was nearly 100%, indicating that pure melanocytes were obtained after the first passage by the new method, which is similar to the conventional method. To test whether melanocytes isolated using the new method can function normally, they were treated with forskolin (FSK) for 2 days in vitro, which induced the levels of pigmentation of melanocytes (Figure 4B). Taken together, these data suggest that the melanocytes isolated using the new method can function normally and can produce pigment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61226/61226fig01.jpg" /> 
Figure 1: Comparison of the new method and the conventional method.&#160;This scheme shows a comparison of the conventional method with the new isolation method. The only difference is that the isolated fresh cells are resuspended in 10 mL of TIVA medium in the presence of 10 &#181;M Y-27632. Two days after seeding, the medium is replaced with normal TIVA medium without Y-27632. The new method usually achieves a density of melanocytes suitable for passaging at around day 8, while the conventional method usually takes about 20 days to grow sufficient numbers of melanocytes for passaging. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61226/61226fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61226/61226fig02.jpg" /> 
Figure 2: The new isolation method increases the production of primary melanocytes.&#160;(A) Representative images of melanocytes prepared by the conventional method (top row) and by the new method (bottom row) at days 3, 6 and 8 after the initial inoculation. (B) Quantification of melanocyte numbers prepared using the conventional method and the new method by counting melanocyte numbers in at least 10 different microscopic fields (cell number/vision) at days 3, 6 and 8. All experiments were performed in triplicate and results are expressed as the average number of cells per microscopic field. (C) After culture for 8 days, melanocytes were harvested using trypsin and the number of melanocytes prepared using the conventional method and the new method were counted using a cell counting plate. The number of cells was calculated from triplicate experiments, and the results show the average number of cells per 100 mm dish. (D) After counting the number of melanocytes using an automated cell counter, melanocytes prepared using the conventional method or the new method were seeded in new dishes and microscopic photos were taken at day 9. (E) Melanocytes isolated using the new method at passage 0 or at passage 1 were fixed and stained with Ki67 antibody (red) and DAPI (nuclei, blue). (B-C), Student&#8217;s t-test was used to analyze differences between the new and the conventional method, ** P &#60; 0.01, ***P &#60; 0.005. All scale bars represent 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61226/61226fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61226/61226fig03v5.jpg" /> 
Figure 3: Y-27632 enhances melanocyte growth through regulation of keratinocytes.&#160;(A) Pure melanocytes (passage 3) were treated with 10 &#956;M Y-27632 for 12, 24, 48, and 60 h and the number of melanocytes was analyzed by CCK-8 assay. (B) Pure melanocytes (passage 3) were treated with 10 &#956;M Y-27632 for 48h and then were fixed for Ki67 staining. Quantification of percentage of Ki67 positive cells among total live cells indicated with DAPI nuclear staining. (C-D) Pure melanocytes (passage 3) were treated with 10 &#956;M Y-27632 for 48 h and then were either stained for apoptotic cells with Annexin V-FITC and propidium iodide followed by FACS analysis (C) or fixed for TUNEL assay. (E) Isolated epidermis after trypsin digestion was plated with TIVA medium with or without Y-27632 (10 &#956;M) and 2 days later immunofluorescence analysis of keratinocytes was performed with a pan-cytokeratin antibody (pan-ck). (F) Quantification of keratinocytes in the Y-27632-treated and control dishes from (E) as percentage of keratinocytes in a total of 500 cells counted. (G) Pure passage 3 keratinocytes were seeded in TIVA medium with or without Y-27632 (10 &#956;M). Images of epidermal cells are shown at one day after seeding. (H) Quantification of attached keratinocytes in the Y-27632-treated and control dishes from (G) as the percentage of attached cells in a total of 5000 cells seeded. (I) Pure passage 3 keratinocytes were plated with TIVA medium in the presence or absence of Y-27632. The microscopic images were obtained at 24 h. (J) Left panel: Equal numbers of pure passage 3 melanocytes were plated with TIVA medium in 4 groups with addition(s) as indicated: 1) Y-27632 (10 &#181;M) alone (Y in graph), 2) passage 3 keratinocytes alone , 3) keratinocytes and Y-27632 (10 &#181;M) (K+Y), and 4) no addition (negative control, con). Relative fold changes in melanocyte proliferation compared to the negative control were determined by counting the number of melanocytes at 24 h and 48 h after plating. Right panel: Conditioned TIVA media were obtained from the 4 groups of passage 3 melanocyte cultures in (left panel) at 48 h after plating. Then, equal numbers of passage 3 melanocytes were plated in 4 groups, each with one of the conditioned media. Relative fold changes in melanocyte proliferation compared to the negative control were determined by counting the number of melanocytes at 24 h and 48 h after plating. (A-J) All experiments have been repeated for 3 times, *p&#60;0.05, **p&#60;0.01, ***p&#60;0.005. All Bars in the images represent 100 &#181;m. The figure has been modified from Mi et al.16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61226/61226fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61226/61226fig04.jpg" /> 
Figure 4: Characterization of melanocytes isolated using the new method.&#160;(A) Representative images of immunofluorescence staining of MITF (red) in melanocytes prepared using the new or the conventional method. DAPI stains nuclei (blue). (B) Cell pellets were collected from melanocytes prepared using the new method and were treated for 2 days with forskolin (FSK) or DMSO vehicle as a control (con). All scale bars represent 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61226/61226fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues at JoVE.com

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

y-27632-enriches-yield-human-melanocytes-from-adult-skin

Nanyang Technological University

Research

JoVE Journal

Biology

5.4K Views.  Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration. Het algemene doel van het op cellen gebaseerde protocol is om menselijke melanocytencellen te isoleren en te kweken, waardoor de tekortkomingen van conventionele methoden worden verbeterd. Van de nieuwe methode kunnen we grotere hoeveelheden primaire melanocyten cellen in een relatief korte tijd, op basis van materialen en apparatuur. Menselijke weefsels die in dit protocol worden gebruikt, zijn vers voor huidweefsels die zijn weggegooid uit besnijdenischirurgie in het ziekenhuis en worden be...

Video: Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Bioengineering

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Developmental 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

Quantification of Hypopigmentation Activity In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis.
In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Biochemistry

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 ...

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

Medicine

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as UV protective agents, skincare, cosmetics etc. A marine Pseudomonas stutzeri produces melanin without the addition of tyrosine. The feedback inhibition was observed by melanin in the culture of a melanin-producing marine bacterium, Pseudomonas stutzeri. Melanin also demonstrated microbial growth inhibition. The Han-Levenspiel model-based analysis identified uncompetitive type product inhibition of melanin on the cell growth. Tyrosinase enzyme, which produces melanin, was inhibited by melanin. The double reciprocal plot of the enzymatic reaction in the presence of different melanin concentrations revealed uncompetitive product inhibition. An adsorbent-based adsorptive bioprocess was developed to reduce the feedback inhibition by melanin. Different adsorbents were screened to select the best adsorbent for melanin adsorption. Dosage amount and time were optimized to develop the adsorptive bioprocess, which resulted in an 8.8-fold enhancement in melanin production by the marine bacteria Pseudomonas stutzeri (153 mg/L to 1349 mg/L) without supplementation of tyrosine and yeast extract.

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Here we present a protocol on adsorptive bioprocess to produce a biological product, especially a pigment that inhibits its own biosynthesis and inhibits the growth of the microorganism that produces it.

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as ...

Engineering

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.

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, ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

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