CD34 defines melanocyte stem cell subpopulations with distinct regenerative properties

Melanocyte stem cells (McSCs) are the undifferentiated melanocytic cells of the mammalian hair follicle (HF) responsible for recurrent generation of a large number of differentiated melanocytes during each HF cycle. HF McSCs reside in both the CD34+ bulge/lower permanent portion (LPP) and the CD34- secondary hair germ (SHG) regions of the HF during telogen. Using Dct-H2BGFP mice, we separate bulge/LPP and SHG McSCs using FACS with GFP and anti-CD34 to show that these two subsets of McSCs are functionally distinct. Genome-wide expression profiling results support the distinct nature of these populations, with CD34- McSCs exhibiting higher expression of melanocyte differentiation genes and with CD34+ McSCs demonstrating a profile more consistent with a neural crest stem cell. In culture and in vivo, CD34- McSCs regenerate pigmentation more efficiently whereas CD34+ McSCs selectively exhibit the ability to myelinate neurons. CD34+ McSCs, and their counterparts in human skin, may be useful for myelinating neurons in vivo, leading to new therapeutic opportunities for demyelinating diseases and traumatic nerve injury.

Introduction Neural crest-derived melanocyte stem cells (McSCs) are responsible for producing differentiated melanocytes during each hair follicle (HF) cycle. During embryogenesis, neural crest cells emerging from neural tube generate melanoblasts which migrate to specific destinations including eye, epidermis and developing HF where they continue to proliferate and produce pigment-producing melanocytes in early postnatal life. In growing HFs, McSCs are distinguished from hair matrix melanocytes by their location in the outer root sheath (ORS) of the bulge/ lower permanent portion (LPP) and by distinct molecular signatures, including the expression of Dct and Pax3, but low Sox10 [1]. In resting HFs, McSCs are identified based on their quiescence properties, expression of Dct, and by their localization within HF bulge/LPP and a region previously described as the subbulge region [2].
The HF is a skin appendage composed of epithelial cells, follicular cells, mesenchymal cells and pigment-producing melanocytes. During each cyclic expansion and regression, the mammalian HF proceeds through three distinct phases, anagen (growth phase), catagen, (regression phase), and telogen, (a follicular resting phase) [3,4]. The cycle is initiated for follicular expansion when HF stem cells (HFSCs) in the hair germ of telogen HFs are activated by factors including noggin (NOG), FGF-7, FGF-10 and TGF-β2 secreted from dermal papillae [5,6]. In the newly-initiated cycle, McSCs become activated by a Wnt signal from HFSCs in the surrounding vicinity to generate proliferating, committed melanocyte progenitors [7]. During fully-developed anagen, terminally-differentiated melanocytes reside in the inner core of the hair matrix, where they produce and transfer melanin to the surrounding follicular epithelial cells. During catagen, melanocytes degenerate along with the rest of the matrix and lower ORS [8] and the HF returns to the resting phase. The quiescent state of telogen HFSCs is maintained by factors including BMP6 and FGF-18 from inner bulge cells [9], BMP4 from dermal fibroblasts and BMP2 from subcutaneous adipocytes [10]. In telogen, the lower HF component consists of two regions, the bulge/LPP [11] and secondary hair germ (SHG) [12], an epithelial extension at the base of the telogen HF. These compartments can be distinguished using region-specific markers, with the bulge compartment expressing the CD34 membrane glycoprotein [13] and the SHG selectively expressing the intracellular adhesion protein P-cadherin/ Cdh3 [14]. McSCs along with other HFSCs are present in both compartments.
Recently, by using tetracycline-regulated expression of a stable H2BGFP fusion protein from the Dct promoter in bitransgenic Dct-tTA;TRE-H2BGFP (Dct-H2BGFP) mice, we localized subbulge McSCs to the SHG, a transient structure at the base of the telogen HF [15]. Given this localization of McSCs to anatomically separate telogen HF compartments whose epithelial stem cells possess distinct characteristics, we wondered whether McSCs occupying these distinct sections were functionally different or interchangeable. We describe for the first time that McSCs can be separated into two distinct populations, corresponding to cells present in the bulge and SHG HF compartments, using CD34. We show that CD34+ McSCs from the HF bulge unexpectedly possess the ability to function as glia, forming dense myelin sheaths surrounding neurons of the myelin-deficient Shiverer mouse strain. They function less efficiently as McSCs compared to CD34-McSCs. This finding raises the question of whether all cells previously identified as McSCs uniformly possess melanocytic potential, or whether the CD34+ subset of these cells represents instead another type of neural crest-derived progenitor cell. Our findings reveal a novel developmental fate for one subset of McSCs, and suggest approaches to utilize specific, skin-derived stem cell (SDSC) populations for nerve regeneration and support.

Identification of GFP-expressing McSCs in bulge and SHG of telogen HF of Dct-H2BGFP bitransgenic mice
To identify McSCs in telogen HFs, we used Dct-H2BGFP bitransgenic mice [15]. The constitutive expression of Dct-H2BGFP mice is comparable to the expression pattern of Dct-LacZ [2,16,17] and iDct-GFP mice [18], which express the transgene in melanoblasts, melanocyte progenitors in bulge/LPP and terminally differentiated melanocytes [15]. Dct-H2BGFP cells were present in both the CD34-expressing bulge region [9] of the HF and the CD34-negative, P-cadherin-expressing SHG region [6] at the base of the telogen HF (Fig 1A and 1B, S1A-S1D Fig). [15]. Careful observation of Dct-H2BGFP cells in the bulge HF region revealed not only that these cells were present in the CD34+ region, but also express CD34 (Fig 1C). To confirm that CD34 expression was not limited to the epithelial cells of the bulge, we showed that Kit, which is restricted to HF melanocytes, and CD34 are co-localized ( Fig 1D). Given the distinction between Dct-H2BGFP cells either expressing CD34 in the bulge or lacking CD34 in the SHG, we determined formally whether each of these cell classes was neural crest-derived and from the melanocytic lineage. Dct-H2BGFP cells co-localize both with a Tyr-CreER;R26-tdTomato signal ( Fig 1E) and with a Wnt1-Cre;R26-tdTomato signal (Fig 1F), demonstrating that CD34+ and CD34-Dct-H2BGFP cells originate from neural crest and are members of the melanocyte lineage. Since our identification of CD34+ and CD34-cells as putative distinct populations of McSCs was limited to the telogen phase, we also attempted to identify CD34+ McSCs selectively throughout the HF cycle. These cells could be identified at the follicular morphogenesis stage (P8), at the first telogen (P21), and during anagen stages at both P30 and P70 (S2A- S2F  Fig). CD34-McSCs were also present at the P21 telogen stage. The SHG does not remain as a recognizable anatomic HF feature during anagen, making it difficult to establish whether individual cells beneath the bulge during these stages retain this identity during anagen. Nonetheless, analysis of expression of the differentiated melanocyte markers Tyr and Tyrp1 in HF anagen at P30 shows a clear difference of expression in differentiated bulb melanocytes compared to bulge McSCs where they are not detectable at this stage (S3A and S3B Fig). The persistence of CD34+ Dct-H2BGFP cells throughout the HF cycle fulfills an important criterion for these cells as McSCs.

Dct-H2BGFP cells in second telogen express the McSC markers Kit and Dct
Selective expression of CD34 by bulge Dct-H2BGFP cells suggested a strategy to separate these cells from SHG Dct-H2BGFP cells to evaluate their molecular and functional properties. Single cell suspensions prepared from shaven, dorsal skin of approximately 8-week-old (P56) mice, an age when all HFs are synchronously in the telogen [4], were incubated with anti-CD34 antibody and prepared (Fig 2A) for fluorescence-activated cell sorting (FACS). Dct-H2BGFP cells could be separated into distinct CD34+ and CD34-populations using FACS. Although the percentage yield of cells comprising these populations differed slightly between experiments, in general 0.1-0.3% of the dermal cell suspension was comprised of CD34+ and To further evaluate the specificity of these cell populations, RNA was isolated (S1G Fig), and relative gene expression for specific marker genes determined (Fig 2C). These results confirmed that Dct-H2BGFP cells expressed endogenous Dct at significantly higher levels than the basal keratinocyte gene Krt14, with Dct expression marginally higher in CD34-Dct-H2BGFP cells compared to CD34+ counterparts. Furthermore, Cd34 expression was significantly higher in the CD34+ Dct-H2BGFP cells, with expression of Cdh3, encoding P-cadherin, reciprocally elevated in the CD34-Dct-H2BGFP cells corresponding to the P-cadherin-expression in the SHG population. To further evaluate the specificity of CD34+ and CD34-Dct-H2BGFP isolated cells, we examined FACSseparated cells individually for Kit and CD34 expression with immunofluorescence.

Bulge and SHG Dct-H2BGFP cells are McSCs with distinct functional properties
The ability to separate subsets of HF Dct-H2BGFP cells during telogen, together with their origin in the melanocytic lineage, prompted us to evaluate the relative expression of the melanogenic genes Tyr, Tyrp1, Pmel17, and Mitf within CD34+ and CD34-subsets. Quantitative RT-PCR results ( Fig 2D) showed that relative expression of all four melanogenic genes studied was significantly higher in the CD34-McSCs present in the SHG compared to the CD34+ cells from the HF bulge, suggesting that the SHG McSCs are at a more advanced state of melanocytic differentiation than the cells in the bulge. To explore this finding functionally, cells were sorted, cultured in melanocyte differentiation medium, and observed after 4 and 7 days in culture. Only cultured cells in the CD34-Dct-H2BGFP cell culture exhibited visible pigmentation following these in vitro culture periods ( Fig 3A). Quantification of cell pigmentation and morphology ( Fig 3B) confirmed that significant numbers of pigmented cells only developed in the cultures of CD34-Dct-H2BGFP cells, with CD34+ Dct-H2BGFP cells principally maintaining a round, rather than dendritic, non-pigmented appearance even after 7 days of in vitro culture in melanocyte differentiation conditions. The percentage of pigmented cells from CD34+ cells increased from 0 to 5% from day 4 to day 7, while this percentage increased from 2% to 25% for CD34-cells introduced into the culture environment. Dendritic, non-pigmented cells also increased (CD34+, 13% to 27%; CD34-, 38% to 65%). Representative examples are shown in S5A Fig. CD34-McSCs also appeared to proliferate more in melanocyte differentiation medium than CD34+ McSCs ( Fig 3B and S5B Fig). These findings provided evidence that CD34+ bulge Dct-H2BGFP cells are functionally distinct from the CD34-Dct-H2BGFP cell population.
We determined whether both CD34+ and CD34-Dct-H2BGFP cells, previously characterized as quiescent [15], were McSCs. We introduced subsets of cells into amelanocytic skin and observed transplanted skin for durable pigmentation. Equal numbers of CD34+ and CD34-McSCs isolated from Dct-H2BGFP telogen HFs were transplanted into Mitf Mi-wh/Mi-wh neonatal mouse skin which was then engrafted onto nude mice ( Fig 3C). CD34-McSCs repigmented the amelanocytic murine HFs with high efficiency compared to CD34+ McSCs. 3/4 grafts of CD34+ cells and 4/4 grafts of CD34-cells demonstrated pigmentation at two months following graft placement. Pigmentation within the grafts was sustained, with durable pigmentation observed over 6 months. Pigmented foci were observed beginning at 2 weeks following engraftment, and in general did not expand or reduce in size over time. However, the pigmented foci were significantly larger following CD34-cell transfer compared to CD34+ cell transfer (Fig 3D and 3E).

CD34+ bulge McSCs have characteristics of neural crest stem cells
The previous experiments demonstrated that both CD34+ and CD34-Dct-H2BGFP cells are McSCs, while exhibiting differences in their expression of melanogenic genes and their efficiency of HF pigmentary unit reconstitution. To characterize differences between CD34+ bulge McSCs and CD34-SHG McSCs more completely, we performed RNA-seq and analyzed the gene expression profiles of telogen-stage CD34+ and CD34-McSCs. Using an absolute log 2 -fold � 1 cutoff, differential gene expression analysis of the RNA-seq reads obtained from these cells demonstrated an expected upregulation of Cd34, a bulge marker, in CD34+GFP+ cells and Cdh3, a SHG marker, in CD34-GFP+ cells. Global hierarchical clustering and volcano plot analysis showed differential expression of 3373 genes between bulge and SHG McSCs (S6A Fig, S2 and S4 Tables). Ingenuity pathway analysis (IPA) showed that 'Axonal Guidance Signaling' and two embryonic stem cell pluripotency categories were overrepresented in genes more highly expressed in CD34+ McSCs, whereas representation in the 'Melanocyte Development and Pigmentation Signaling' cetagory was more equally weighted (S7 Fig and S3 Table), suggesting a differentiation state difference between the cell subsets. Since melanocytes are derived from the neural crest, we selected candidate neural crest stem cell-related genes from the literature to investigate their gene expression differences further by inspection and in comparison with known melanocyte development and differentiation genes. We found that CD34-McSCs expressed higher levels of melanogenic genes such as Mitf, Tyr, Tyrp1, Pmel, Pax3, Mc1r, Erbb3, Sox10, Melan-A, and Slc45a2, consistent with the higher expression of Tyr, Tyrp1, Pmel17, and Mitf determined by qRT-PCR ( Fig 4B). In contrast, CD34+ McSCs expressed higher levels of neural crest stem cell markers Nr2f2, Nr2f1 [19], Ngfr (p75) [20], Twist1, Twist2, Snai1 [21], Sox9 [22], EdnrA [23], Gli1 [24], Bmp2, Bmp4, and Bmp7 [25,26]. These subsets of cell type-characteristic genes organize into distinct hierarchical clusters ( Fig  4A). Quantitative RT-PCR analysis of expression of select genes in CD34+ and CD34-McSCs validated the RNA-Seq results (S6B and S6C Fig).
In a prior study, skin-derived cells expressing neural crest cell markers p75 and Sox10 grew as spheroids in culture under non-adherent conditions. These cells were reported to exhibit both melanocyte and glial differentiation potential [27]. Based upon this observation, we tested the ability of CD34+ and CD34-cells to grow as spheroids under non-adherent conditions in neural crest stem cell (NCSC) medium. Both populations of cells were capable of growth as spheroids, although spheroids from CD34+ McSCs were larger than those from CD34-McSCs (Fig 4C and 4D). The percentage of initially plated CD34+ and CD34-cells that formed spheroids was 0.43% and 0.33% respectively ( Fig 4C). Cells growing as spheroids were placed in adherent, neural crest cell culture conditions and studied for expression of proteins characteristically expressed by distinct, neural crest-derived cell types, such as Gfap as a marker of glial cells, Tuj1 antigen (β3-tubulin) as a marker of neurons, and α-smooth muscle actin (α-Sma) as a marker of myofibroblasts [20], as well as the primitive keratin Krt15. Only adherent cells derived from CD34+ McSCs expressed this spectrum of neural crest-derived cell markers ( Fig  4E). Adherent cells derived from CD34+ and CD34-McSC spheroids both showed expression of the melanocyte marker Tyrp1, although only cells derived from CD34-McSCs also revealed visible pigmentation (Fig 4F). Quantification of the expression of markers in individual cells ( Fig 4G) showed that of all the neural crest-derived cell type markers expressed in cells derived from CD34+ McSCs, Gfap was the most frequently expressed. CD34+ McSCs also selectively expressed nestin, a neuronal stem cell marker, at higher levels (S10A and S10B Fig). Tyrp1 was  Fig 4G). The diversity of neural crest cell lineage markers expressed selectively by CD34+ McSCs suggested that these cells might possess the capability of functioning as either a neural crest stem cell or as a non-melanocytic neural crest-derived cell.
CD34+ McSCs also resemble skin-derived precursor cells (SKPs), or skin-derived stem cells, that have been previously isolated from mammalian dermis insofar as they are skinderived, grow as spheroids in non-adherent culture conditions, and differentiate along multiple neural crest lineages [28][29][30]. Establishing whether SKP differentiation closely resembles the differentiation profile of CD34+ McSCs that we have previously demonstrated could provide insight into the relationship between SKPs and CD34+ McSCs. Given the ability of SKPs to undergo glial differentiation [28,29], we wondered whether the differentiation profile of CD34+ McSCs, with their expression of multiple NCSC-associated genes, would most closely resemble SKPs as a stem cell isolated from adult skin or a more primitive embryonic NCSC. We compared the frequencies of distinct lineage markers expressed by CD34+ McSCs and murine SKPs, grown first as non-adherent spheroids, then differentiated in neural crest differentiation medium [31], to determine whether these cell populations are identical or merely overlap. We included a comparison with embryonic neural crest stem cells (eNCSCs) with this analysis. CD34+ McSCs expressed nestin, fibronectin, neuron-specific β-tubulin (Tuj-1), and α-Sma at similar frequencies to murine SKPs (Table 1, S8 Fig). However, CD34+ McSCs expressed p75/Ngfr at a much higher frequency than murine SKPs (86% vs. 3.5%). eNCSCs expressed p75/Ngfr at a similar high frequency (65%). Both the Schwann cell/oligodendroglial markers Gfap and CNPase were also more frequently expressed in CD34+ McSCs compared to SKPs (44% vs 3% and 31% vs. 3%, respectively). eNCSCs expressed these markers at an intermediate frequency (9% and 11%, respectively). When SKPs and CD34+ McSCs were instead differentiated in SKP medium [32], CD34+ McSCs again expressed higher levels of p75/Ngfr, Gfap, and CNPase than SKPs. SKP medium was unable to support the growth of eNCSCs (S1 Table). Although CD34+ McSCs located in the telogen bulge did not express Gfap   Fig). These data support a model whereby CD34+ McSCs represent a subset of murine SKPs enriched for glial differentiation, and also mirror several defined properties of eNCSCs consistent with their profile as a neural crest-like stem cell.

De novo myelination of myelin-deficient, Shiverer neurons by CD34 + McSCs
To determine whether CD34+ McSCs could recapitulate an activity of a neural crest-derived cell type other than a melanocyte, we adapted a culture system, previously used to study the ability of oligodendroglial cells (ODCs) to myelinate neurons in vitro [33], to assess their ability to function as glia. We isolated dorsal root ganglia (DRG) from either wild-type or C3Fe. SWV-Mbp shi/shi /J (Shiverer, shi/shi) mice. Mice of the shi/shi genotype lack myelin basic protein (Mbp) and develop a "shivering" phenotype, or tremor, eventually dying between 3-4 months of age. CD34+ and CD34-McSCs, and rat ODCs as positive controls, were co-cultured with neurites extending from wild-type or shi/shi DRGs and studied for their ability to express Mbp, a marker of oligodendrocytes [34] and Schwann cells [35] in a neuronal distribution ( Fig 5A).
In co-cultures with rat DRGs, CD34+ McSCs selectively exhibited Mbp expression in their vicinity in a neuronal pattern (Fig 5B) To identify the injection site histologically, we found the disrupted retinal pigmented epithelium (RPE) layer corresponding to the injection, (S11C Fig, right panels), and focused on sections from this region to identify transferred, CTG-labelled CD34+ and CD34-McSCs. In 2/2 shi/shi eyes receiving CD34+ McSCs, CTG-labelled cells co-expressing Mbp could be observed (S11C Fig, top row). However, in 2/2 shi/shi mice receiving CD34-McSCs we observed that CTG-labelled, CD34-McSCs do not co-express Mbp (S11C Fig, middle row). In a potentially more relevant neurodegenerative model, CD34+ and CD34-McSCs were cultured as non-adherent spheroids and introduced intracranially into the brain tissue of approximately 6-8 week-old shi/shi mice (n = 5, Fig 6A). Significant foci of Mbp expression were principally detected in brains injected with CD34+ McSCs, at the site of CTG vital dyelabeled CD34+ McSCs (Fig 6B and 6C). Quantification of co-localization of the CTG vital dyelabeled cells with Mbp showed that co-localization was observed in 82% of CTG+ cells following CD34+ McSC injection, versus 19% of CTG+ cells following CD34-McSC injection  Fig 6E). Shi/shi brain that was not injected with cells as a negative control showed no Mbp signal (S12A Fig). In addition, groups of CTG-labeled cells could be found in regions of the central (Fig 6C) and caudal brain, following injection into a rostral site, perhaps indicating some ability of injected cells to move or migrate following their introduction. Mbp expression colocalizes with neurofilament H (NeuH), a neuronal marker [36], in these foci (Fig 6D). EM analysis revealed evidence of dense myelin sheath surrounding neurons of CD34+ McSCtransplanted shi/shi brain (Fig 6F and S12C Fig)  In co-culture with DRG neurites and in murine brain, they expressed Mbp in a neuronal pattern similar to primary oligodendroglial cells and form a dense myelin sheath surrounding the neuronal axon. These data indicate that CD34+ McSCs are not exclusively committed to terminal melanocytic differentiation, readily producing myelin in a neural environment, a property normally associated with myelinating Schwann cells or oligodendroglial cells of the glial lineage. This could indicate that CD34+ McSCs contain glial cell precursors in addition to melanocyte precursors, or bipotent or multipotent progenitor cells derived from neural crest. Single-cell analysis will be required to understand further the heterogeneity and developmental potential of CD34+ McSCs.
Our data are consistent with the description of activities of other skin-derived stem cells but identify a specific cell with neural crest stem cell properties. These include studies which found evidence of p75/Sox10+ neural crest-like cells in adult murine skin HFs [27] and nestin-GFP expressing cells isolated from the HF bulge, which also exhibit the ability to express markers of multiple neural crest lineages [37]. The pluripotent, nestin-expressing GFP cells isolated from CD34+ HF bulge, which transdifferentiate largely into Schwann cells, were transplanted into the gap region of severed sciatic nerve, greatly enhancing the rate of nerve regeneration and restoring the nerve function [38]. Melanoblasts isolated from skin possess multipotency and self-renewal capabilities. The Kit-positive and CD45-negative cells isolated from embryonic melanoblast and neonatal skin HFs, when cultured on stromal cells, formed colonies containing neurons, glial cells and smooth muscle cells, together with melanocytes [39]. There is also evidence for endogenous bipotent embryonic precursors to the glial and melanocyte lineage in avians [40,41] and in mice [41]. Our results demonstrate that a melanocyte lineage-specific cell defined by Dct, Kit, and CD34 expression in the adult HF bulge represents a neural crestlike stem cell in the skin and provide comprehensive, genome-scale characterization of its expression profile.
The skin-derived stem cells (SDSCs) isolated from mouse and human skin [28][29][30] exhibit glial cell differentiation and express Mbp in vitro similar to CD34+ McSCs isolated from HFs of Dct-H2BGFP mice. Mouse and human SDSCs from mammalian dermis grow as spheroids in non-adherent culture, express nestin, and possess multipotency to generate neural and mesodermal progeny [29]. When transplanted into shi/shi mice, SDSCs from human-and mouse-derived Schwann cells repaired peripheral nerve damage and myelinated neuronal axons of the dysmyelinated brain [42]. However, the cell of origin of these skin-derived cells with unique regenerative properties has not yet been identified specifically. CD34+ McSCs may represent a highly-specific subset of SDSCs. Although desert hedgehog (Dhh) is selectively expressed by myelinating Schwann cells [43] it was not differentially expressed between the two McSC subsets. This may reflect the fact that its expression is either only activated in the peripheral environment, or that other factors may be responsible for hedgehog pathway activity in CD34+ McSCs as suggested by their higher expression of Gli1 (Fig 4B). Mbp expression was higher in CD34+ McSCs, although the higher level of expression was not statistically significant in the RNA-Seq profiles (S4 Table). Mbp could be detected both in vitro (Fig 5C) and in vivo (Fig 6B) at low levels in cells derived from CD34-McSCs, although expression in these environments was more robust and more clearly in a neuronal distribution when derived from CD34+ McSCs. Nonetheless, robust myelination was correlated strongly with CD34+ McSCs in vitro ( Fig 5D) and in vivo (Fig 6E and S12C Fig). Environmental cues are likely to be extremely important for determining the terminal differentiation characteristics of CD34+ and CD34-McSCs in a manner that cannot be readily predicted from their extant gene expression profile differences. Our ability to define with specific cellular markers the identity of these cells, not relying simply upon their growth characteristics and functional properties, will facilitate their comparison with McSCs in other species and enhance efforts to define highly-specific human skin-derived stem cell subsets with unique regenerative properties that can be leveraged for these therapeutic purposes.
The unique properties of CD34+ McSCs in the bulge, with their ability to myelinate neurons and express markers of non-melanocytic lineages, may be relevant to their role in this HF region where Shh-expressing neurons approach the HF [44] and where the arrector pili muscle attaches [45]. CD34+ McSCs may provide regenerative support to maintain the function of these extrafollicular tissues in the HF environment. Although McSCs have previously been described both in the bulge and the subbulge/SHG regions [2,15,46,47], some recent studies have emphasized selectively the contribution of the subbulge/SHG subset either in the follicular onset of proliferation and differentiation [7] or in UV-induced emergence from quiescence and melanomagenesis [48]. These and other reports on McSCs have not distinguished between the bulge and subbulge/SHG subsets and their distinct functional properties. Given the heterogeneity that has been described inherently in melanoma cells and their treatment-resistant subpopulations [49], recognition of the variation in McSC phenotypes may be especially important to further define the contribution of normal McSCs to cancer development.

Ethics statement
All mouse procedures were approved by the Institutional Animal Care and Use Committee protocol of the University of Maryland School of Medicine (IACUC protocols 0917008, 1014005).

Isolation of HF McSCs from subepidermal skin and fluorescence activated cell sorting (FACS)
Dorsal skin samples were obtained from transgenic mice at the indicated ages immediately following euthanasia and fat was removed from the dermis using fine forceps. Defatted skin was incubated in 0.5% of trypsin (USB) dissolved in PBS at 37˚C for 30 min. Epidermis was peeled away from the dermis following incubation, and the remaining dermis including HFs was cut into small pieces. The dermal fragments were incubated in digestion medium containing 0.2 mg/mL Liberase Thermolysin low (Roche) in a 37˚C water bath for 45 to 60 min. The digested dermal mixture was added into PBS containing 0.05% DNase (Sigma) and 5% FBS. Single cells were extracted from the dermis by repeated plunging with a 60 mL syringe followed by filtration through 40 μm nylon mesh (BD Falcon). The dermal cell suspension is prepared in 5% FBS/PBS prior to incubation with respective antibodies and FACS. This procedure was done under aseptic conditions [15].
To separate the hair follicle bulge/LPP and SHG cells of Dct-H2BGFP bitransgenic mice using FACS, the cell suspension was incubated with Alexa 647-conjugated anti-CD34 antibody in for 30 min at 4˚C. 7-AAD was added to the CD34-labeled dermal cell suspension in 5% FBS/PBS to discriminate between live and dead cells, and cell sorting was performed using BD FACSAria1 (Becton-Dickenson) on a 100 μm nozzle at 20 psi low sheath pressure. Sorted cells were counted and used either for primary cultures or for quantitative realtime PCR (qRT-PCR) by extracting RNA from respective cell populations.
For the comparison of CD34+ McSCs with murine SKPs and eNCSCs, these cell types were grown as spheroids in NCSC medium or SKP medium for 7 days in ultra-low attachment plates. The SKP medium consists of DMEM/F12 (Gibco Life technologies), B27 supplement, 40 ng/mL FGF, 20 ng/ml EGF and penicillin/streptomycin as described previously [32]. For CD34+ McSCs, murine SKPs, and eNCSC adherent cultures to evaluate neural crest lineage marker expression, cells were cultured in neural crest differentiation medium as described above or SKP differentiation conditions for a week. SKP differentiation medium consists of DMEM/F12, B27 supplement, 3% FBS and penicillin/streptomycin as described previously [32]. For SKP studies, cells were isolated by preparing a dermal single cell suspension from skin of C57BL6 mice at P56 as described above. For the isolation of eNCSCs, neural tubes were isolated from Dct-H2BGFP embryos at E9.5 and then cultured in NCSC medium. After 24 hours of incubation at 37˚C, eNCSCs migrated away from the cultured neural tube and were analyzed experimentally [51].

Dorsal root ganglion (DRG) co-cultures
Isolation and culture of DRGs from C3Fe.SWV-Mbp shi/shi /J (shiverer, shi/shi) pups was performed as described previously [52]. Shi/shi pups (P5 to P8) were euthanized according to institutional guidelines and the spine extracted. Excess muscle and bone from the spine was trimmed away and the spine placed in a Petri dish ventral side up. Using dissection scissors the spinal column was cut along the midline starting caudally in a longitudinal fashion. The spinal column was gently opened by two pairs of forceps and the spinal cord exposed. Using fine-tipped forceps the DRGs, found beneath and lateral to the spinal cord, were removed and transferred to ice cold Hank's buffered salt solution (HBSS) in a new Petri dish. DRGs were transferred to a 1.5 mL centrifuge tube containing 500 μl of ice-cold HBSS and were pelleted by spinning at 1200 rpm for 5 min at 4˚C. The supernatant was discarded and a 500 μL solution of pre-warmed DRG papain solution was added followed by incubation at 37˚C for 10 min. Following another light spin, the supernatant was discarded and 500 μL of pre-warmed Collagenase A solution was added followed by incubation at 37˚C for 10 min. Following another light spin and removal of supernatant, DRGs were washed twice with 1mL of DRGN media (DMEM containing 10% FBS). Finally, DRGs were dissociated by triturating them with a BSA-coated glass Pasteur pipette. After dissociation, the suspension was passed through a 40 μm filter into a sterile Petri dish containing 7 mL of DRGN media. This suspension was incubated at 8.5% CO 2 for 1h 15 min. At this stage many contaminating cells including fibroblast and glial cells strongly adhered to the Petri dish, enriching cell suspension for DRGs. The cell suspension containing DRGs was collected by pelleting and cultured in 10 μg/mL Laminin (and 30 μg/mL Poly-D-Lysine coated 24-well plates in DRGN media at 8.5% CO 2 , 37 2 C overnight. The next day, DRGN media was replaced with OL media (DMEM with 2% B27 supplement, 1% N2 Supplement, 1x glutamine, 0.5% FBS and 1% penicillin/streptomycin) with 10 μM 5'-Fluoro-deoxyuridine (FuDR; Sigma) to prevent the proliferation of contaminating fibroblasts and glial cells. On Day 5, full media was changed with OL media (without FuDR) and by Day 7, the DRGs had formed an extensive neurite bed, ready for co-culture.
For DRG co-cultures, CD34+ or CD34-McSCs isolated from Dct-H2BGFP mouse skin or rat oligodendroglial cells (ODC; Gibco Life technologies) were seeded onto the dense neuronal bed generated either by shi/shi or rat embryonic DRGs. For co-cultures of CD34+ or CD34-McSCs and shi/shi or rat embryonic DRGs, cells were cultured in neural crest differentiation medium containing 10μM ascorbic acid to induce myelination. For co-cultures of rat ODCs and shi/shi DRGs, cells were cultured in glial cell differentiation medium. After one week of co-culture, cells were fixed and examined for Mbp expression by immunofluorescence and myelin sheath formation by electron microscopy. Additional information is available in the S1 Text: Supplemental materials and methods.