FACS Isolation of Melanocyte Stem Cells from Mouse Skin CURRENT STATUS: POSTED

Melanocyte stem cells (MeSCs) are crucial for generating mature melanocytes that colour the skin and hair. Dysfunction of MeSCs can result in conditions such as hair greying, hypo- or hyper-pigmentation disorders, and melanoma. Here we describe a fluorescence-activated cell sorting (FACS) strategy for isolating MeSCs from mouse skin. The isolated MeSCs can be used in a multitude of experiments including gene expression analysis, transplantation, and others.


Introduction
Pigmentation of the skin and hair are one of the most diverse traits in the animal kingdom and are crucial for camouflage, signal, and photoprotection from UV light. In mammals, melanocyte stem cells (MeSCs) maintain pigment-producing melanocytes that give the skin and hair distinct colours.
Mutations in melanocyte lineages can lead to the formation of melanoma, one of the most dangerous and aggressive forms of skin cancers.
The melanocyte lineage also offers an accessible and easily trackable system to study stem cell biology and tissue regeneration in mammals. MeSCs are located in the hair follicle at a region known as the bulge. As the hair follicle enters the regenerative phase (anagen), MeSCs transiently proliferate to generate pigment-producing melanocytes, which migrate downwards to the hair bulb to colour the growing hair from the root 1 . At the destruction phase (catagen), differentiated melanocytes are destroyed, sparing only the MeSCs once the hair follicle returns to the resting phase (telogen).
Much of our understanding of MeSC biology and its regulation relies on our ability to robustly isolate a pure population of MeSCs. In mouse skin, MeSCs have traditionally been isolated using reporter lines, such as Dct-EGFP or Tyr-CreER together with Rosa-reporter lines [2][3][4] . However, it can be laborious and time-consuming to breed reporter lines, especially to bring in additional alleles to study molecular changes of MeSCs when harbouring different mutations.
Another issue which hinders efficient isolation of MeSCs is the low number of MeSCs in the skin. Each hair follicle only contains about ten MeSCs. In total, MeSCs make up ≤1% of total cells in the skin.
Thus, the MeSC population can be easily contaminated by other cell populations during fluorescent activated cell sorting (FACS). Our protocol addresses this issue by depletion of other abundant cell 3 types (immune cells, dermal fibroblasts, and epidermis) first, before enriching MeSCs from the remaining cells.
Our strategy design was initially guided by and fine-tuned using the Tyr-CreER; Rosa-lsl-H2BGFP mouse line. We first compared the GFP signal with known surface markers which recognize cell types in the epithelium and dermal compartment using FACS. In addition, we conducted immunofluorescent staining on tissue sections to confirm the specificity of selected markers. From these analyses, we identified MeSCs to be positive for CD117 (cKit), with modest expression of integrin alpha-6 (α6 mid ), but negative for CD45, CD34, CD140a, and Sca1 (Figures 1, 2). CD117 is also expressed by mast cells, which are excluded by their expression of CD45. In the anagen skin, CD117 is also expressed by the matrix cells of the hair follicle and mature melanocytes, so an additional step to remove the hair bulb is necessary to enrich for MeSCs in anagen. The purity of these MeSCs has been validated by qRT-PCR as well as RNAseq 5 .
The following protocol describes a step-by-step process for isolating MeSC populations from mouse skin. Flip the skin so that it is now dermal side up, and gently scrape off dermal cells using a scalpel.

2.3)
Collect the solution containing dermal cells in a 50 mL tube using a serological pipette. To ensure the collection of all dermal cells, add sterile DPBS to the culture dish and collect the DPBS containing cells into the same 50mL tube (final volume: 40mL). Preparation of dermal cells will be continued in steps 2.5-2.9.

2.4)
Add 10 mL of trypsin-EDTA 0.25% to the same culture dish with the skin dermal side down.
Shake the dish at 37 ºC for 30 minutes. Preparation of epidermal cells will be continued in steps 2.10-2.13.

2.5)
While the epidermal layer is under trypsin digestion, spin down the dermal cells collected from step 2.4 at 400g for 10 minutes at 4ºC.

2.6)
After centrifugation, gently pour out the supernatant and add 10mL of trypsin-EDTA 0.25% to the 50 mL tube containing the pellet. Resuspend the pellet gently, screw on the lid tightly, and then seal the tubes shut using parafilm to ensure that there is no leakage. Shake the sealed tubes at 37 ºC for 15 minutes.

2.7)
Add 15mL of FACS buffer to the dermal cells in Trypsin-EDTA 0.25%. Mix thoroughly using a serological pipette to quench the reaction.

2.13)
Spin down dermal and epidermal cells in solution from steps 2.9 and 2.12 at 300g for 10 minutes.

2.14)
Pour out the supernatant. Resuspend epidermal cells in 0.5mL of FACS buffer and resuspend dermal cells in 0.5mL of FACS buffer.

2.15)
Combine dermal and epidermal cells, so that total cells from each harvested skin are in 1mL of FACS buffer.

3) Preparation for Negative Selection and Staining:
3.1) Prepare 5mL FACS tubes by precoating the tubes with FACS buffer. Using a serological pipette, fill the FACS tube with 2 mL of FACS buffer and then remove the liquid. Be sure not to create any bubbles and to remove all the liquid. · Prepare 5 tubes for single channel controls (one tube for each channel in step 4.1, plus an unstained control and a DAPI-stained control).

3.2)
Aliquot 50 μL of cells from step 2.15 into 5 precoated FACS tubes for single channel controls. Add an additional 150 μL of FACS buffer to each control tube (final volume per tube: 200 μL).

3.3)
Transfer the remaining cells from step 2.15 into a new precoated 5mL FACS tube.

4) Depletion using Negative Selection:
· Only perform depletion on the cells reserved for sample(s), not the single channel controls.

Anticipated Results
The frequency of MeSCs isolated using this protocol should be between 0.2%-1%, or about 5000-  Melanocyte stem cells express CD117 in both telogen and anagen. Tyr-CreER; Rosa-lsl-tdTomato mice were induced 3X with tamoxifen during 1st telogen. Representative images of hair follicles in telogen following induction and in the immediately following anagen show colocalization of tdTomato (red) and CD117 (green) in melanocyte stem cells. Scale bar: 50μm.