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.
