Name: feeder-free derivation of melanocytes from human pluripotent stem cells
Uploaded: 2016-03-03T15:00:00
Description: Watch this Scientific Journal Video about Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells at JoVE.com

This work was supported by a fellowship for melanoma researchers from the Joanna M. Nicolay Foundation and by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award F31. This work was further supported through grants from NYSTEM and the Tri-institutional stem cell initiative (Starr Foundation).

NOTE: The melanocyte protocol outlined here was first demonstrated by Mica et al.
1. Preparation of Culture Medium, Coated Dishes and Maintenance of hPSCs
<ol>
	<li>Medium Preparation 
	Note: Store all medium at 4 &#176;C in the dark for up to 2 weeks. Filter all medium for sterilization.
	<ol>
		<li>Prepare DMEM/10% FBS. Mix 885 ml DMEM, 100 ml FBS, 10 ml Pen/Strep and 5 ml L-Glutamine. Filter for sterilization.</li>
		<li>Prepare hESC-medium. Mix 800 ml DMEM/F12, 200 ml KSR, 5 ml L-Gl...

<ol>
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For the successful differentiation of melanocytes from hPSCs the following suggestions should be taken into consideration. First and foremost, it is essential to work under sterile culture conditions at all times. Additionally, it is important to start with pluripotent, fully undifferentiated hPSCs; if the starting population contains differentiated cells the yield will invariably drop as the contaminants cannot be directed towards melanocytes and may even further disrupt the properly differentiating cells.
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Human pluripotent stem cells (hPSCs) provide a platform to mimic normal differentiation in a scalable fashion for disease modeling, drug screening, and cell replacement therapies 1-6. Of particular interest, hPSCs open up avenues for studying difficult to isolate or rare/transient cell types where patient samples are scarce. Furthermore, induced pluripotent stem cells (iPSCs) enable researchers to study development and disease modeling in a patient specific manner to unravel unique mechanisms 1,2,7-11. The previously published protocol for differentiation of melanocytes from hPSCs requires up to 6 weeks of differentiations and involves culturing cells with conditioned medium from L-Wnt3a cells 12. The protocol first presented by Mica et al. and described here produces pigmented cells in three weeks and removes the ambiguity and inconsistencies associated with conditioned medium.
Melanocytes are derived from the neural crest, a migratory population of cells unique to vertebrates. The neural crest is defined during gastrulation and represents a population of cells at the edge of the neural plate, bordering between the neural and non-neural ectoderm. During neurulation, the nervous tissue evolves from a neural plate to form neural folds, which converge at the dorsal midline resulting in the neural tube 13,14.
The neural crest cells emerge from the roof plate of the neural tube, opposite the notochord, and undergo an epithelial to mesenchymal transition before migrating away to give rise to a diverse population of differentiated cells. The fates of the crest cells are defined in part by the anatomic location of the roof plate along the body axis of the embryo. Neural crest cell derivatives include lineages characteristic of both mesoderm (smooth muscle cells, osteoblasts, adipocytes, chondrocytes) and ectoderm cells (melanocytes, Schwann cells, neurons) 14. Neural crest stem cells upregulate the transcription factor SOX10 and can be isolated by fluorescence-activated cell sorting with antibodies to p75 and HNK1.
The neural crest cells fated to become melanocytes pass through a melanoblast stage and upregulate KIT and MITF (microphthalmia-associated transcription factor) 6,21 MITF is a master regulator of melanocyte development and is a transcription factor responsible for controlling much of melanocyte development 22-24. Human melanoblasts migrate to the basal layer of the epidermis where they reside either in the hair bulge or surrounded by keratinocytes in the epidermis (forming pigmentation units) to serve as precursors to the mature, pigmented melanocytes. The differentiation and maturation of melanoblasts into pigmented melanocytes occurs concomitant with colonization of the hair bulb and expression of the melanin production pathway (TYRP1, TYR, OCA2 and PMEL) 25,26.
Isolating human melanocytes and melanoblasts from patients is expensive, difficult and limiting in quantity. This protocol enables researchers to differentiate hPSCs (induced or embryonic) into melanocytes or melanocyte precursors in a well defined, rapid, reproducible, scalable, and inexpensive method without cell sorting. The protocol was used previously to identify disease-specific defects when differentiating iPSCs from patients with pigmentation disorders .

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	<tbody>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>apo human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid (L-AA)</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>B27 (B27 Supplement)</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-bp</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>4423</td>
			<td>6 mM</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>50 mg/ml</td>
		</tr>
		<tr>
			<td>cAMP (cyclicAMP)</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D2915-100MG</td>
			<td>50 &#956;M</td>
		</tr>
		<tr>
			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco-Life Technologies</td>
			<td>11985-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 - Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12</td>
			<td>Gibco-Life Technologies</td>
			<td>1133--032</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 powder</td>
			<td>Invitrogen</td>
			<td>12500-096</td>
			<td></td>
		</tr>
		<tr>
			<td>EDN3 (Endothelin-3, human)</td>
			<td>American Peptide Company</td>
			<td>88-5-10B</td>
			<td>100 &#956;M</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td>200 &#956;g/ml</td>
		</tr>
		<tr>
			<td>gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td>0.1% in PBS</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Human insulin</td>
			<td>Sigma</td>
			<td>I2643</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS+ Universal Culture Supplement Premix&#160;</td>
			<td>BD Biosciences</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>KSR (Knockout Serum Replacement)</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>Invitrogen</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Dissolve 1:20 in DMEM/F12</td>
		</tr>
		<tr>
			<td>MCDB201 Medium</td>
			<td>Sigma</td>
			<td>M6770</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM minimum essential amino acids solution</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-8105M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Poly-L Omithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td>15 mg/ml&#160;</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>0.032g in 100ml 100% ethanol</td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2 (Recombinant human FGF basic)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-001MG/CF</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1814</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF (Stem Cell Factor, recombinant Human)&#160;</td>
			<td>Peprotech Inc.</td>
			<td>300-07</td>
			<td>50 &#956;g/ml</td>
		</tr>
		<tr>
			<td>TYRP1 (G-17) Antibody</td>
			<td>Santa Cruz</td>
			<td>10443</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>TYRP2 Antibody</td>
			<td>Abcam</td>
			<td>74073</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Gibco-Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1254</td>
			<td>10 mM</td>
		</tr>
	</tbody>
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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 protocol provides a method for deriving fully pigmented, mature melanocytes from hPSCs in an in vitro feeder-free, cost efficient, and reproducible manner. In contrast to the previously established Fang et al. protocol for hPSC-derived melanocytes, the outlined protocol does not require conditioned medium and decreases the time requirement. The Fang et al. protocol utilized conditioned medium from a WNT3A-producing murine cell line and took up to 6 weeks to...

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Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

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 ...

The overall goal of this protocol is to differentiate human pluripotent stem cells into fully pigmented, mature melanocytes. This method can help identify key factors during melanocyte development. Additionally, this protocol will enable us to identify factors that have gone awry during disease development, as well as screen for important drugs and during drug discovery.The main advantage of this protocol is that it enables us to produce large numbers of fully pigmented melanocytes. Whereas isolating melanocytes from patients usually results in relatively few cells. After aliquoting and freezing gelatinous protein, thaw and resuspend a one milliliter aliquot in 19 milliliters of DMEM f12.Then introduce five milliliters of this mixture onto a single 10 centimeter tissue culture dish. After preparing several such plates proceed to incubate them for one hour at room temperature. Next, begin to prepare plates coated with PO, laminin, and fibronectin by first adding PBS containing 15 micrograms per milliliter PO to a culture dish.Then incubate the plates overnight at 37 degrees Celsius in a humidified incubator. Afterwards, aspirate the solution and wash the dishes three times with 1X PBS. Continue coating by adding 10 milliliters of PBS containing one microgram per milliliter mouse laminin 1 and two micrograms per milliliter fibronectin.Then, incubate the dishes as previously described. To begin, add 0.1%gelatin in PBS to a 10 centimeter dish. And leave it at room temperature for five minutes.As this plate is being coated, place frozen mouse embryonic fibroblasts, or MEFs in a 37 degree Celsius water bath to quickly thaw them. Next, aspirate the gelatin from the dish and plate MEFs at a density of approximately 50, 000 cells per square centimeter in DMEM supplemented with 10 percent fetal bovine serum. After incubating the dish overnight at 37 degrees Celsius aspirate the medium and wash the prepared MEF plate with 1X PBS.Proceed to add 10 milliliters of previously prepared hESC medium containing human pluripotent stem cells abbreviated as hPSCs, to a 10 centimeter dish. After seeding cells at a density of 5 million cells per dish incubate the plates at 37 degrees Celsius in five percent carbon dioxide. Proceed to feed the cells daily with 10 milliliters of fresh hESC medium.Prior to commencing differentiation inspect the plate for any colonies that appear to contain differentiated cells irregular borders, or transparent centers. Under a dissecting microscope in a laminar flow hood mechanically dislodge any such irregular colonies with a pipette. To perform coating with gelatinous protein such as Matrigel, first aliquot and freeze the protein in one milliliter parts in order to avoid repetitive freeze thaw cycles.Then, thaw and resuspend a one mililliter frozen aliquot with 19 milliliters of DMEM f12. Proceed to plate five milliliters of this mixture onto a 10 centimeter dish and incubate the dishes for one hour at RT.Afterwards, aspirate the gelatinous protein immediately before plating the cells and wash with DMEM F12. As this dish is being coated select a plate where hPSCs are at approximately 80%confluency.Proceed to aspirate the hESC medium. And then wash the cells with PBS. After removing the PBS, add three milliliters of 0.05%trypsin-EDTA to the plate.Proceed to vigorously shake the dish horizontially for two minutes until the MEFs are observed under a microscope to begin to lift off as single cells. During this time, expect the hPSC colonies to remain attached to the plate. Proceed to aspirate the trypsin once the MEFs have lifted but before the hPSC colonies have detached.Then, to the plate, add 10 milliliters of hESC medium containing 10 micromolar Y-27632 dihydrochloride a ROCK inhibitor. Afterwards detach the cells by pipetting up and down over the colonies. Next return to the dish with a gelatinous protein and aspirate the solution.Wash this dish with DMEM f12 to remove any clumps. And into it plate hPSCs at a 1 to 2 ratio. Then, to the plate, add hESC medium containing 10 micromolar Y-27632 dihydrochloride up to 10 milliliters.Incubate the cells at 37 degrees Celsius overnight. Feed the cells daily with ROCK inhibitor supplemented medium. When the hPSCs reach 80%confluency proceed to initiate neural differentiation and designate this time point as Day 0.To commence differentiation feed the cells on Day 0 and Day 1 with 10 milliliters of KSR differentiation containing 100 nanomolar LDN-193189 of the MP inhibitor and 10 micromolar SB 431542, an inhibitor of activin and Nodal. Continue on Day 2 by replacing the medium with that containing the same concentrations of the BNP and activin and Nodal inhibitors plus three micromolar CHIR99021, a Wnt Agonist. Then, on Day 3, feed the cells with KSR differentiation medium, supplemented with only 10 micromolar SB 431542 and three micromolar CHIR99021.After preparting a mixture of 75%KSR differentiaion medium and 25%N2 differentiation medium which also contains three micromolar of the Wnt Agonist. Feed the cells with 15 milliliters of this liquid on Day 4 and 5. Next, on Days 6 and 7 feed the cells with 15 milliliters of medium consisting of 50%KSR differentiation medium and 50%N2 differentiation medium which has also been supplemented with three micromolar CHIR99021 25 nanograms per milliliters BMP4 and 100 nanomolar EDN3.Then, on Days 8 and 9 feed the cells with 20 milliliters of 25%KSR differentiation medium and 75%N2 differentiation medium supplemented with the same concentrations of factors used on Days 6 and 7. Proceed to continue with neural differentiation on Day 10 by feeding the cells with 20 milliliters of N2 differentiation medium containing three micromolar CHIR99021 25 nanograms per milliliter BMP4 and 100 nanomolar EDN3. On Day 11, aspirate the solution from dishes previously coated with PO lamanin 1, and fibronectin.Then leave the plates uncovered in a tissue culture hood for approximately 10 to 15 minutes, allowing them to dry completely. Proceed to remove the medium from the Day 11 cells and wash them with PBS. Then, add four milliliters of cell detachment solution to the dish and incubate it at 37 degrees Celsius for 25 minutes.Following incubation add five milliliters of full melanocyte medium to the dish. And resuspend the cells by manually pipetting up and down until they have lifted off the plate. Transfer the suspension to a 15 milliliter tube and spin the cells down for five minutes at 200 Gs.After counting cells using Triypan Blue Exclusion and a hemocytometer, resuspend them in full melanocyte medium at a density of 2X10 to the 6th cells per milliliter.Plate 10 microliter droplets of the cell suspension onto the dried, coated dishes. Ensure that the droplets are close to one another but not touching, have well defined edges and do not run. To allow the cells to adhere, let the droplets stand at room temperature for 10 to 20 minutes.Afterwards, slowly add 10 milliliters of full melanocyte medium to the dish being careful not to disturb the attached cells. Then, move the dish to the incubator. Feed the cells every two to three days with full melanocyte medium.And expect to observe pigmentation in clusters of cells by the end of the first week. Once a week, passage cells by first associating them with cell detachment solution as previously described. After collecting and centrifuging the cells wash them twice with plain neurobasal medium.Then, replate the cells at a ratio of one to six in full melanocyte medium on dishes coated with PO, lamanin 1, and fibronectin. Around 30 days of differentiation the cells will appear deeply pigmented. Which is easily visualized when plated at high density or pelleted when passaging.Shown here is a bright field image of cells from a transgenic psox10 GFP human embryonic stem cell line cultured until Day 11 of the described differentiation protocol before cells are replated. Evident in these cultures are dark defined ridges of cells. When these cultures were assessed for GFP signal the dark ridges were enriched for sox10 positive cells.As sox10 is a transcription factor upregulated by neural crest stem cells and maintained in melanoblasts these results suggest that by Day 11 this differentiation method enriched for cells that will eventually give rise to melanocytes. As evidence of this, by Day 20 of the protocol cultures contained unpigmented, maturing, and fully mature pigmented melanocytes. At Day 25, the same types of cells are observed in cultures.The presence of maturing and pigmented melanocytes in these preparations are confirmed through staining with two melanin production proteins. TYRP2, which serves as a marker of both melanoblasts and melanocytes and TYRP1 which is a late stage melanocyte marker only expressed here in pigmented melanocytes. Once mastered, this protocol can be employed to differentiate human pluripotent stem cells into fully pigmented mature melanocytes in 25 days.The protocol proceeds through normal melanocyte development by first proceeding through the neuro crest state and as well as the melanoblast stage.

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