Human Epidermal Neural Crest Stem Cells (hEPI-NCSC)—Characterization and Directed Differentiation into Osteocytes and Melanocytes
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- Clewes, O., Narytnyk, A., Gillinder, K.R. et al. Stem Cell Rev and Rep (2011) 7: 799. doi:10.1007/s12015-011-9255-5
Here we describe the isolation, characterisation and ex-vivo expansion of human epidermal neural crest stem cells (hEPI-NCSC) and we provide protocols for their directed differentiation into osteocytes and melanocytes. hEPI-NCSC are neural crest-derived multipotent stem cells that persist into adulthood in the bulge of hair follicles. Multipotency and self-renewal were determined by in vitro clonal analyses. hEPI-NCSC generate all major neural crest derivatives, including bone/cartilage cells, neurons, Schwann cells, myofibroblasts and melanocytes. Furthermore, hEPI-NCSC express additional neural crest stem cell markers and global stem cell genes. To variable degrees and in a donor-dependent manner, hEPI-NCSC express the six essential pluripotency genes C-MYC, KLF4, SOX2, LIN28, OCT-4/POU5F1 and NANOG. hEPI-NCSC can be expanded ex vivo into millions of stem cells that remain mulitpotent and continue to express stem cell genes. The novelty of hEPI-NCSC lies in the combination of their highly desirable traits. hEPI-NCSC are embryonic remnants in a postnatal location, the bulge of hair follicles. Therefore they are readily accessible in the hairy skin by minimal invasive procedure. hEPI-NCSC are multipotent somatic stem cells that can be isolated reproducibly and with high yield. By taking advantage of their migratory ability, hEPI-NCSC can be isolated as a highly pure population of stem cells. hEPI-NCSC can undergo robust ex vivo expansion and directed differentiation. As somatic stem cells, hEPI-NCSC are conducive to autologous transplantation, which avoids graft rejection. Together, these traits make hEPI-NCSC novel and attractive candidates for future cell-based therapies and regenerative medicine.
KeywordsNeural crest Adult stem cell Human epidermal neural crest stem cell EPI-NCSC hEPI-NCSC Pluripotency Bone Melanocyte
Neural crest-derived multipotent stem cells reside in the periphery during embryogenesis and in the adult organism [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11]. One such location is the bulge of whisker follicles, as we have shown in the Wnt1-cre::R26R mouse model in which all neural crest cells and dorsal neural tube cells specifically and indelibly express β-galactosidase [12, 13, 14]. According to their location in the bulge by the epidermal outer root sheath, the stem cells were thus termed epidermal neural crest stem cells (EPI-NCSC). A neural crest stem cell molecular signature comprising 19 genes that are abundantly and uniquely expressed in mouse embryonic neural crest stem cells and EPI-NCSC, but not in epidermal stem cells or other skin-resident stem cells/progenitors was subsequently defined . The potential of EPI-NCSC has begun to be realized in mouse models of spinal cord injury, where mouse EPI-NCSC grafts led to an improvement in sensory connectivity and touch perception [16, 17, 18]. Others have also reported hair follicle derived progenitor cells, both from within  and outside [20, 21] the bulge region, the latter having shown promise in a model of sciatic nerve injury. Furthermore, multipotent progenitor cells of various ontological origin within skin and hair follicles have been identified and have been termed skin derived precursors (SKPs) [22, 23, 24, 25, 26, 27]. Similarly, other human hair follicle-derived neural crest-like cells formed spheres in culture following enzymatic digestion of intact hair follicles, and they expressed neuronal markers when injected into the mouse brain [28, 29]. It remains to be determined whether these cells are identical to hEPI-NCSC, as they are isolated from entire hair follicles and do not show any migratory behaviour. hEPI-NCSC have the distinct advantage that due to their migratory ability they can be isolated with ease as a highly pure population of multipotent stem cells.
The neural crest is a transient embryonic structure that arises during neurulation at the boundary of the neural plate and the somatic ectoderm. Neural crest cells subsequently delaminate from the forming neural tube and migrate to various locations in the embryo to generate a wide array of progeny. Neural crest derivatives include craniofacial bone/cartilage, meninges, tooth papillae, the autonomic and enteric nervous systems, most primary sensory ganglia, endocrine cells such as the adrenal medulla, smooth musculature of the cardiac outflow tract and great vessels and pigment cells (melanoyctes) of the skin and internal organs [30, 31].
Skeletal abnormalities, bone trauma, osteoarthritis and deteriorating joints are examples of conditions, which severely compromise an individual’s quality of life and ability to perform daily tasks of living. Derivation of replacement bone and cartilage is thus of particular interest. Osteogenic differentiation by various methods has been reported for mesenchymal stem cells, adipose cells and bone marrow stem cells [32, 33, 34] with others also reporting osteogenic differentiation of neural crest stem cells derived from human embryonic stem cells (hESC) , SKPs [36, 37], mouse ESC  and cord blood . The advantage of hEPI-NCSC is that the neural crest has the physiological ability to generate bone, as a subset of cranial bones are of neural crest origin [31, 40, 41]. Likewise, melanoyctes are established neural crest derivatives. In hairy skin, they give hair its colour by injecting melanin granules into keratinocytes that form new hair.
In the current study, we present an in-depth characterization of human equivalents of mouse EPI-NCSC. By comparison analyses, we show their neural crest origin. By taking advantage of the migratory ability of neural crest cells, we have developed a culture method that yields a highly pure population of stem cells. Further studies showed that hEPI-NCSC express all six established pluripotency markers, which may make hEPI-NCSC a convenient source for reprogramming into iPS cells requiring minimal manipulation. Furthermore, proof-of-principle studies show that hEPI-NCSC can undergo efficient directed differentiation into osteocytes and melanocytes. The former are implicated in regenerative medicine in orthopaedics, whereas the latter could prove useful components of bioengineered skin.
Materials and Methods
Bulge Explants and Isolation of hEPI-NCSC
The bulge of adult human hair follicles [42, 43] were micro-dissected as we have described previously [13, 14, 15] from pubic hairy skin. De-identified biopsies were obtained with ethical approval (REC REF: 08/H0907/1) from consenting individuals undergoing repeat elective Caesarean sections. The Donor age bracket was 28–41 years. Briefly, hair follicles were dissected and mechanically cleaned of dermal and adipose tissues. The dermal papilla and matrix were removed and discarded, the bulge region excised, cut into 2–3 pieces and placed onto CellStart (Invitrogen, Paisley, UK Cat# A10142-01) coated 24-well or 35 mm plates where they adhered to the substratum within 1 h. The explants were incubated in a humidified atmosphere at 37°C, 5% CO2 and 5% O2. To protect cells against oxidative stress, we routinely culture neural crest stem cells at low oxygen tension (see e.g., ref 44). It needs to be noted that 5% oxygen does not constitute hypoxia for hEPI-NCSC, as hypoxia is defined as O2 tension below the normoxic value in a given tissue  and oxygen tension in hair follicles ranges between 2.5% and 0.1% O2. . Culture medium was NeuroCult XF (Stem Cell Technologies, Grenoble, France Cat# 05761) supplemented with 10 ng/ml rhFGF2 (R&D Systems, Abingdon, UK Cat# 233-FB), 20 ng/ml rhEGF (R&D Systems Cat# 236-EG), 1X ITS+3 (Sigma, Poole, UK Cat# I-2771), 1% (v/v) FBS (HyClone, Thermo Fisher, Cramlington, UK Cat# SH30070.02), 1X GlutaMAX (Invitrogen, Cat# 35050–038), 1X Penicillin/Streptomycin (Sigma Cat# P0781) and 2.5 μg/ml Amphotericin B (Sigma Cat# A2942). Four days post onset of emigration of hEPI-NCSC, bulges were removed with a bent tungsten needle and cells either fixed with 4% paraformaldehyde (PFA) for indirect immunocytochemistry as described below, or dissolved in TRIzol® (Invitrogen, Cat# 15596–018) for RNA isolation, or isolated by trypsinisation for sub-culturing. Briefly, for trypsinisation, cultures were rinsed with PBS-EDTA before addition of trypsin (Worthington Biochemical Corporation, Lakewood, NJ, USA. Cat# LS003703) at 500 μg/ml in PBS-EDTA. Trypsin treatment was stopped with trypsin inhibitor (Sigma Cat# T6522; 1 mg/ml) that was dissolved in culture medium and the cell suspension collected. The same culture medium was used for ex vivo expansion of hEPI-NCSC.
To promote differentiation of bone/cartilage cells in clonal culture, the culture medium was supplemented with BMP2 (10 ng/ml). For differentiation into neuronal cells, NGF (20 ng/ml), TGF-β2 (1 ng/ml) and forskolin (10 μM) were added. Neuregulin-1 (10 nM) and CNTF (10 ng/ml) were added to promote differentiation into Schwann cells.
Real Time Quantitative RT-PCR (qPCR)
For reverse transcription, total RNA was extracted with TRIzol® reagent and treated with DNaseI (Invitrogen Cat# 18068). cDNA was synthesised using the SuperScript III First Strand synthesis kit (Invitrogen Cat# 18080–051), with Oligo (dT) priming according to manufacturer’s instructions. For quantitative real time PCR (qPCR), reactions consisted of either RT2 Real-Time SYBR Green/ROX MasterMix (SABiosciences/Tebu-Bio, Peterborough, UK. Cat# 103PA-012) or SYBR Green JumpStart qPCR MasterMix (Sigma Cat# S4438), first Strand cDNA template and appropriate RT2 qPCR Primer Set (SABiosciences/Tebu-Bio). Thermo-cycling conditions were: 95°C, 10 min followed by 40 cycles of 95°C, 15 s, 60°C, 60 s and 70°C, 30 s, performed on an Applied Biosystems 7900HT thermocycler. Melting curve analysis showed a single amplification peak for each reaction. Ct values for targets were normalised to the average Ct values of four housekeeping genes (HKG) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Tata box binding protein (TBP), succinate dehydrogenase complex subunit A (SDHA) and Glucose-6-phosphate dehydrogenase (G6PD) and expressed as percentage thereof. Fold changes in expression were calculated using the ΔΔCt method. Information on primer pair sequences and source is provided in Supplemental Table 1. RNA from human embryonic stem cells was used from cells of the H9 line, which was maintained exactly as described .
Cultures were fixed with ice-cold 4% (w/v) PFA in phosphate buffered saline (PBS, Invitrogen, Cat# 20012) at room temperature (RT) for 30 min, followed by three 20 min PBS washes, blocked with 2% (v/v) normal goat serum (Sigma Cat# G9023) in PBS at RT for 20 min. Primary antibody was added, diluted appropriately in PBS, 0.1% (v/v) Triton X-100 and incubated overnight at 4°C. Cultures were then rinsed four times with PBS for 20 min each, followed by addition of secondary antibody diluted 1:200 in PBS and incubated in the dark for 2 h at RT. Secondary antibody was removed and four, 20 min PBS washes performed followed by mounting with Vectashield plus DAPI (Vector Laboratories, Peterborough, UK Cat# H-1500) and cover-slipped.
Primary antibodies used were: rabbit anti-MSX2 (1:200) (CeMines, Evergreen, CO, USA. Cat# AB/HD19), rabbit anti-SOX10 (1:100) (CeMines Cat# AB/HMG4), mouse anti-NESTIN (1:200) (BD Biosciences, Oxford, UK. Cat# 61158), goat anti-MYO10 (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA. Cat# SC-23137), mouse anti-ETS1 (1:200) (Santa Cruz Biotechnology Cat# SC-56674), rabbit anti-ADAM12 (1:200) (Santa Cruz Biotechnology Cat# SC-25579), goat anti-CRMP1 (1:200) (Santa Cruz Biotechnology Cat# SC-46872), goat anti-αβ-crystallin (CRYAB) (1:200) (Santa Cruz Biotechnology Cat# SC-22391), goat anti-UFD2/UBE4B (1:200) (Santa Cruz Biotechnology Cat# SC-30840), goat anti-thimet oligopeptidase (THOP1) (1:200) (Santa Cruz Biotechnology Cat# SC-30579), rabbit anti-NANOG (1:1000) (Abcam, Cambridge, UK. Cat# ab21624), mouse anti-KLF4 (1:100) (Abcam Cat# ab75486), rabbit anti-LIN28 (1:1000) (Abcam Cat# ab46020), mouse anti-SOX2 (1:100) (Abcam Cat# ab75485), rabbit anti-OCT4 (1:1000) (Abcam Cat# ab19857), mouse anti-C-MYC (1:200) (Abcam Cat# ab32), mouse anti-βIII-tubulin (TUBB3) (1:200) (Millipore, Watford, UK. Cat# MAB1637), rabbit anti-tyrosine hydroxylase (TH) (1:200) (Santa Cruz Biotechnology Cat# SC-14007), rabbit anti-alpha smooth muscle actin (SMA) (1:200) (Abcam Cat# ab32575), mouse anti-alpha smooth muscle actin (SMA) (1:200) (Sigma Cat# A5228), rabbit anti-GFAP (1:1000) (Abcam Cat# ab7260), mouse anti-collagen type II (COL2A1) (1:200) (Santa Cruz Biotechnology Cat# SC-59958), rabbit anti-RUNX2 (1:100) (Abcam Cat# ab80238), mouse anti-osteocalcin (BGLAP) (1:200) (Abcam Cat# 13418), rabbit anti-osteopontin (SPP1) (1:200) (Abcam Cat# ab8448) and rabbit anti-DCT (1:1,000) (Abcam Cat#ab74073).
Secondary antibodies used were: DyLight-488 conjugated goat anti-rabbit IgG (Jackson, Newmarket, UK. Cat# 111-485-144), DyLight-594 conjugated goat anti-mouse IgG (Jackson Cat# 115-515-146), DyLight-488 conjugated donkey anti-goat IgG (Jackson Cat# 705-485-147) and DyLight-594 conjugated donkey anti-goat IgG (Jackson Cat# 705-515-147).
Cells from primary explants were detached by trypsin treatment, seeded at clonal density (60 cells per 35 mm plate) onto CellStart treated 35 mm dishes and allowed to attach overnight. The next morning, single cells were identified and circled using a diamond tipped circle scribe (circle diameter 4 mm). Single cells in circles that overlapped with another circle were excluded from analysis. Greater than 90% were single cells and circles overlapped rarely. Clones were then cultured in NeuroCult NSA (Stem Cell Technologies, Cat# 05752), 1X ITS+3, 1% (v/v) FBS, 1X GlutaMAX, 1X Penicillin/Streptomycin and 2.5 μg/ml Amphotericin B plus addition of specific growth factors for differentiation of multiple cell types for up to 42 days at 37°C, 5% CO2, 5% O2. Human growth factors were from R&D Systems; BMP2 (Cat# 355-BEC-010/CF), NGF (Cat# 256-GF-100/CF), TGF-β2 (Cat# 302-B2-002/CF), Neuregulin-1 (Cat# 377-HB-050/CF) and CNTF (Cat# 257-NT-010/CF). Forskolin was from Sigma (Cat# F6886). Cultures were fixed with 4% PFA for 30 min and subsequently processed for indirect immunocytochemistry as described for cell type specific markers. For serial cloning, clones were subcloned as follows. A sterile cloning ring was dipped into sterile vacuum grease and placed on top of the clone. The clone inside the cloning ring was rinsed twice with three drops of PBS each and finally three drops of trypsin were added. The cells were then detached by gentle trituration and re-seeded at clonal density.
Differentiation into Osteocytes
Cells were isolated by trypsinisation and seeded onto CellStart-treated 35 mm plates at 2.5 × 103 cells per plate. Cultures were grown in AdvanceSTEM osteogenic differentiation medium (HyClone; Thermo Fisher Cat# SH30877.KT), 1X GlutaMAX, 1X Penicillin/Streptomycin and 2.5 μg/ml Amphotericin B, with 50/50 medium exchanges on alternate days. Cultures were incubated in a humidified atmosphere at 37°C, 5% CO2 and either 5% O2 or ambient air for up to 35 days. Alizarin Red S staining of fixed cultures was performed to detect deposition of calcium. Briefly, cultures were washed twice with PBS at RT, stained for 2 h at RT with 2% (w/v) Alizarin Red S, pH 4.2 (Sigma Cat# A5533), followed by three PBS washes and then visualised with an inverted microscope.
Differentiation into Melanocytes
For in vitro differentiation of hEPI-NCSC into melanocytes, cells were treated with 100 nM Endothelin-3 (Sigma Cat# E9137), 20 nM Cholera Toxin (Sigma Cat# C8052), 16.2 mM 12-O-tetra-decanoylphorbol-13-acetate (TPA) (Sigma Cat# 79346), for up to 17 days at 37°C, 5% CO2,10% O2. The DOPA reaction was performed to enhance the dark melanin hue. Briefly, cultures were fixed with 4% PFA for 20 min at RT, followed by three rinses with PBS and incubated at 37°C with 5 mM DOPA (Sigma Cat# D9628) for 3 h. Cells were post-fixed with 4% PFA for 20 min at RT, rinsed with PBS and visualised with bright field light microscopy.
Neural Crest Origin
Multipotency and Self-renewal
Self-renewal is an important aspect of stemness. Self-renewal capability of hEPI-NCSC was determined by serial cloning. Primary clones were detached with trypsin and re-seeded at clonal density, which lead to secondary clones. The procedure was repeated to establish tertiary clones. Clone-forming ability was maintained at high levels, as 70.7 ± 7.9% of secondary clones and 54.0 ± 11.7% of tertiary clones consisted of fast-growing motile cells (Supplemental Table 2). Double stains with cell-type specific antibodies showed that secondary clones contained multiple cell types as well (Supplemental Figure 1). The presence of multiple cell types in secondary clones shows that hEPI-NCSC can undergo self-renewal. Taken together, we have thus shown that hEPI-NCSC are multipotent stem cells.
Expression of Pluripotency Genes
Ex vivo Expansion
hEPI-NCSC can be expanded into millions of stem cells without an overall significant loss of stem cell markers (Fig. 2 A, B). Ex vivo expanded cells continued to express the neural crest stem cell molecular signature, pluripotency genes, SOX10, SNAI2, TWIST, MS1 (Musashi), P75NTR, TERT, nestin, and some early lineages genes at the RNA level (Fig. 2A). Expression of the neural crest stem cell molecular signature, SOX10 and nestin, was also tested by immunocytochemistry. All genes were expressed at the protein level (Supplemental Figure 2). Notably, in vitro clonal analysis showed that the majority of in vitro expanded cells remain multipotent and thrive in clonal culture; 53.2 ± 3.6% of clone-forming expanded cells generated clones that contained multiple cells types; 12.3 ± 2.6% died and 34.5 ± 3.0% stopped dividing (Supplemental Figure 3). While early lineage markers were expressed in cells from primary explants and in ex vivo expanded cells, they were not expressed at the protein level (see e.g., dopachrome tautomerase; Supplemental Figure 4). The changes in gene expression levels observed in expansion culture are likely due to donor-specific differences (see also Fig. 6), as due to technical issues the data for primary explants and ex vivo expanded cells were derived from tissue of different donors. On average, three million cells per bulge explant were obtained within 28 days. As illustrated in Fig. 2 B, expansion did not level off at 28 days and therefore, if desirable, could be continued for longer periods of time. Overall, we show that hEPI-NCSC can be expanded ex vivo efficiently and reproducibly and that they retain stemness. An attractive feature of the expansion protocol is its short duration. Changes in the karyotype are thus less of a concern in hEPI-NCSC than in cell lines that are passaged multiple times. The high numbers of stem cells that can be obtained through ex vivo expansion make testing in animal models of human disease and future applications feasible.
Directed Differentiation of hEPI-NCSC into Osteocytes
Directed Differentiation of hEPI-NCSC into Melanocytes
For future applications, it needs to be shown that ex vivo expanded hEPI-NCSC can be frozen, stored frozen, thawed and subsequently grown in culture again. We show here that this is indeed the case. Cells were frozen in 90% FBS, 10% DMSO in a Nalgene freezing container (Sigma Cat# C1562) to −80°C overnight and then transferred to liquid Nitrogen. Subsequently, cells were thawed and re-cultured. Trypan blue stain showed 87% cell viability after thawing and the cells continued to proliferate (Supplemental Figure 5). We thus show that hEPI-NCSC can be frozen by inexpensive means and thawed again with high yield.
In-depth analyses performed in this study showed that the bulge of human hair follicles contains multipotent neural crest-derived stem cells, which can be expanded ex vivo and differentiated into osteocytes and melanocytes with high efficiency. Analogous to their equivalent counterparts in mice, the stem cells are termed human epidermal neural crest stem cells (hEPI-NCSC).
hEPI-NCSC have many desirable features, the sum of which makes them a highly attractive type of somatic stem cell. As remnants of an embryonic tissue, the neural crest, hEPI-NCSC have the well-recognized physiological ability to generate a wide array of cell types and tissues. This innate high level of multipotency, combined with the expression of pluripotency genes and efficient ex vivo expansion render this stem cell type conducive not only to autologous transplantation but potentially also to efficient reprogramming into iPS cells.
hEPI-NCSC are readily accessible in the hairy skin by minimal invasive procedure. The patient’s own hEPI-NCSC could therefore be harvested, expanded ex vivo and then used for autologous transplantation. hEPI-NCSC are particularly relevant for treatment in spinal cord injury, as neural crest stem cells are ontologically closely related to spinal cord stem cells. Our studies in mouse models of spinal cord injury showed that EPI-NCSC grafts caused a significant improvement in sensory connectivity and touch perception, that they can provide neurotrophic support and angiogenic activity, and that they possibly modulate scar formation by synthesis and release of metalloproteases [16, 18]. It is expected that hEPI-NCSC have similar properties.
Bulges can be dissected with ease. Unlike other procedures [21, 29], the isolation technique does not require enzymatic digestion, as in the anagen phase the bulge region reaches into the dermis. Therefore one does not have to deal with the rather inaccessible, collagen-rich dermis, as is necessary with isolating stem cells from levels above the bulge or from intact hair follicles. By taking advantage of their migratory ability, hEPI-NCSC can be harvested as a highly pure population of stem cells without an overt need for further purification, as non-stem cells are not proliferative and within a short time are outgrown by the stem cells and left behind upon subculture. Furthermore, optimization of culture conditions provided bulge explants that produce emigrating hEPI-NCSC with high yield. Ease of isolation, high yield, and rapid ex vivo expansion, along with high purity of the cell population are highly desirable features of hEPI-NCSC and important considerations for cost-effective stem cell production.
We show here that hEPI-NCSC express six established pluripotency genes. Notably, the exact expression levels vary in a donor-specific manner. Age is unlikely to account for these differences, as the age bracket of the three donors tested was 3 years only. The expression of all six pluripotency genes, C-MYC, KLF4, SOX2, LIN28, POU5F1/OCT4, NANOG and SOX10 was not expected and seemingly is unprecedented in somatic stem cells. Comparison experiments indicate, however, that the genes are expressed at substantially lower levels in hEPI-NCSC than in H9 embryonic stem cells. Moreover, it remains to be determined whether the pluripotency genes are present in their activated state in hEPI-NCSC. Of particular interest is the observation that virtually all cells expressed at least four of the pluripotency genes, C-MYC, LIN28, POU5F1/OCT4 and NANOG. Conversely, the fact that fewer cells expressed KLF4 and SOX2 indicates that there is heterogeneity within the cell population with regard to expression of pluripotency genes at the protein level.
Interestingly, but not surprisingly, hEPI-NCSC already express early lineage markers even though they are multipotent to a high degree. Expression of early lineage markers in stem cells has been described previously [19, 44, 49]. Expression of early lineage genes for multiple cell lineages, that is the neuronal, glial and melanocyte lineages (Fig. 2) may be an indication that the cells are poised to differentiate into either lineage when exposed to appropriate cues. This notion is in line with our previous data in mouse EPI-NCSC, which showed that all cells express both neuron-specific and glia-specific early lineage genes .
Neural crest cells are predestined to give rise to bone and melanocytes, as they generate craniofacial bone [40, 41] and pigment cells of the skin and internal organs . We here show that hEPI-NCSC can be differentiated efficiently into both osteocytes and into melanocytes. While other adult stem cells, such as mesenchymal stem cells [32, 34] and adipocyte stem cells [50, 51] are conducive to bone differentiation, hEPI-NCSC have the advantage that they can be accessed fast and safely in the hairy skin as a highly pure population of stem cells without the need for enrichment and purification. We conclude from our data that ex vivo expansion is best carried out at 5% oxygen, whereas according to most markers tested osteogenic differentiation is preferably performed at ambient oxygen tension, as it has been described in the literature [32, 52].
Hair cycles constantly . We have determined that hair in early and late anagen phase yield hEPI-NCSC. Epidermal stem cells are known to migrate towards the matrix [54, 55], where they give rise to new hair. Likewise, we have observed in mouse hair follicles streams of neural crest-derived cells that appear to head from the bulge towards the matrix . hEPI-NCSC are therefore highly likely to migrate within the hair follicle during anagen phase, in particular as neural crest-derived melanocytes differentiate in the matrix of the hair follicle and thus give hair its colour. During anagen phase, hEPI-NCSC may thus be in an activated stage, or they may progress into a transit amplifying-like progenitor cell compartment. The fact that the dermal sheath needs to cover the bulge in order to trigger efficient cell emigration from bulge explants suggests that there are paracrine interactions between the bulge and the dermal sheath, an intriguing observation that warrants further investigation. hEPI-NCSC from different locations in the body may differ by various criteria. This is an important notion that needs to be explored in future studies.
In summary, we have established that hEPI-NCSC are derived from the neural crest and that they represent a type of somatic multipotent human stem cell with considerable potential for future applications in cell-based therapies and regenerative medicine. The novelty and advantageous characteristics of hEPI-NCSC include their easy accessibility in the hairy skin, their high level of purity, their high degree of multipotency, rapid ex vivo expansion and efficient directed differentiation into various clinically relevant cell types. This notion is supported by application trend analysis, which shows that somatic stem cells are coming to the forefront with more than 70 human applications in 2009. Moreover, the final priority value is 60% for adult stem cells versus 27% and 13% for embryonic stem cells and iPS cells, respectively, as determined by analytical hierarchy process .
This work was supported by Medical Research Council Grant 22358, UK (MSB); USPHS grant R01 NS038500-04S1 from the National Institute of Neurological Disease and Stroke, NIH, USA (MSB); the North East England Stem Cell Institute, Newcastle University, Newcastle upon Tyne, UK (MSB) and the Plunkett Family Foundation, Milwaukee, Wisconsin, USA (MSB). We thank Majlinda Lako for providing RNA from H9 human ESC and Elena Taki for her assistance with Alizarin Red S staining and immunocytochemistry.
Conflict of interest statement
The authors declare no conflict of interest.
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