| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Department of Pathology and Laboratory Medicine,* University of Pennsylvania School of Medicine; and The Wistar Institute, Philadelphia, Pennsylvania
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Each hair follicle perpetually goes through three stages: growth (anagen), involution (catagen), and rest (telogen). Hair follicles not only serve as an appendage specialized for hair shaft production but also as a "niche" for tissue regeneration. Stem cells in the hair bulge, a clearly demarcated structure within the lower permanent portion of hair follicles,8-11 can generate the interfollicular epidermis, hair follicle structures, and sebaceous glands. The bulge epithelial stem cells can also reconstitute in an artificial in vivo system to a new hair follicle.12,13 Stem cells that give rise to the melanocyte lineage have been recently identified in the bulge and sub-bulge outer root sheath regions in adult mice.14,15 Nestin-positive cells are present in the bulge area in mouse and can give rise to neurons, smooth muscle cells, and melanocytes.16 Neural-crest-like stem cells have been identified in mouse whisker hair follicles, and bulge cells from mouse whisker hairs grow as adherent monolayer cells and appear to be multipotent.17,18 The hair bulge has also been found to serve as a local reservoir for mast cell precursors.19 These data suggest that the hair bulge is a unique differentiation-restricted area for adult stem cells.10,11,13 Other studies have demonstrated the potential of rodent and human dermis-derived precursors to generate neural, glial cells and smooth muscle cell derivatives.19,20 In addition, tagged skin cells have been found in multiple tissues after transplantation to blastocysts,21,22 indicating that there are cells in the skin that could be used to reconstitute tissues beyond those of the skin.
Most of the prior hair stem cell functional studies were performed using rodent tissues. In this study, we have developed a new method to isolate human adult stem cells by culturing cells from human hair follicles in a human embryonic stem cell (hESC) culture condition. We have shown that the isolated cells are capable of differentiating into neurons, smooth muscle cells, and melanocytes in specific induction medium. The differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. These cells appear to be located in the bulge area of human hair follicles.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Human hair follicles were isolated as described previously.23-25 Human scalp tissues (0.5 x 2 cm2 or less) from 13 patients (50 to 65 years old) and 3 autopsies were obtained through the Cooperative Human Tissue Network with approval from the Institutional Review Board of the University of Pennsylvania. Tissues were rinsed, trimmed to remove excess adipose tissues, cut into small pieces, and subjected to enzymatic dissociation in 12.5 mg/ml dispase (Invitrogen, Carlsbad, CA) in Dulbecco’s modified Eagle’s medium (DMEM) for 24 hours at 4°C. After treatment, the epidermis was peeled off from the dermis, and hair follicles were plucked from the dermis. Hair follicles were rinsed thoroughly with phosphate-buffered saline to prevent contaminating epidermal or dermal cells and examined under an inverted microscope. To confirm the integrity of plucked hair follicles, we fixed some hair follicles in 10% buffered formalin, embedded them in paraffin, and sectioned and stained them with hematoxylin and eosin (H&E). To obtain viable single cells from follicular epithelium, hair follicles were treated twice with 0.25% trypsin/ethylenediamine tetraacetic acid (Invitrogen) for 30 minutes at 37°C. The cell suspension was filtered through a 40-µm cell strainer (BD Falcon, Bedford, MA), and cell numbers were counted. Single cells were cultured in noncoated flasks in media designated for hESCs. Human ESC medium consisted of 80% knockout DMEM/F-12 medium (Invitrogen), 20% knockout serum replacer (Invitrogen), 200 mmol/L L-glutamine (Invitrogen), 0.1 mmol/L ß-mercaptoethanol (Sigma, St. Louis, MO), 1% nonessential amino acids (Invitrogen), and 4 ng/ml basic fibroblast growth factor (bFGF) (Research Diagnostics, Concord, MA).26,27 This medium was conditioned by using it for 48 hours as growth medium for mouse embryonic fibroblasts (MEFs) as described previously.28 Human ESC medium conditioned by MEFs was mixed with fresh hESC medium at a 3:1 ratio, sterilized by filtration, and supplemented with additional bFGF at 4 ng/ml before use.
For self-renewal, dissociated individual cells from hair spheres were serially diluted in hESC medium in uncoated 96-well plates. Each well was assessed microscopically for the presence of a single cell. The wells containing no cells or more than one cell were excluded. The human ESC line H9 was obtained from the WiCell Research Institute, Inc., (Madison, WI). Cells were cultured on mitotically inactivated MEF feeder layers as described previously.26,27 Embryoid bodies were derived from hESCs as described previously.28
Differentiation Assays
Hair spheres and attached cells were enzymatically dissociated into single cells before plating onto tissue culture-grade plastic coated with 10 ng/ml fibronectin (melanogenic differentiation) or 0.1% Matrigel (BD Biosciences, San Jose, CA; neuronal and smooth muscle differentiation) in differentiation medium. Melanogenic differentiation medium exclusively differentiated hESCs into melanocytic lineages.29 It contains dexamethasone (0.05 µmol/L; Sigma), insulin-transferrin-selenium (1x; Sigma), linoleic acid-bovine serum albumin (1 mg/ml; Sigma), low-glucose DMEM (30%; Invitrogen), MCDB 201 (20%; Sigma), L-ascorbic acid (10–4 mol/L; Sigma), conditioned media of mouse L-Wnt3a cells (American Type Culture Collection, Manassas, VA) (50%), stem cell factor (100 ng/ml; R&D Systems, Minneapolis, MN), endothelin-3 (100 nmol/L; American Peptide, Sunnyvale, CA), cholera toxin (20 pmol/L; Sigma), the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (50 nmol/L; Sigma), and bFGF (4 ng/ml; R&D Systems). For smooth muscle differentiation, dissociated cells were cultured in a medium containing 90% knockout DMEM/F-12 medium, 1% nonessential amino acids solution, 10% fetal calf serum, and 10 ng/ml transforming growth factor-ß1 (R&D Systems). Neuron differentiation medium and steps were used as previously described.20
Immunocytochemistry and Immunohistochemistry
Cells were fixed with 4% paraformaldehyde and stained with primary antibodies specific for Oct4 (mouse monoclonal, 1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA), Nanog (goat polyclonal, 1:100; R&D Systems), microphthalmia-associated transcription factor (MITF) (mouse monoclonal, 1:200; NeoMarkers), HMB45/SILV (mouse monoclonal, 1:500; DakoCytomation, Carpinteria, CA), microtubule-associated protein-2 (MAP2, mouse monoclonal, 1:1000; Sigma), chromogranin (rabbit polyclonal, 1:100; Zymed), Neurofilament-M (NFM) (mouse monoclonal, 1:100; a gift from Dr. Virginia Lee), glutamate (rabbit polyclonal, 1:1000; Sigma), pan-cytokeratin (mouse monoclonal, 1:500; Sigma), and smooth muscle actin (SMA) (mouse monoclonal, 1:100; Chemicon, Temecula, CA). Monoclonal antibodies against the surface markers of hESCs—stage-specific embryonic antigen stage-specific embryonic antigen (SSEA)-3 and SSEA-4—were obtained from The Wistar Institute (where they were originally developed). Isotype-matched mouse antibodies or normal rabbit IgG was used as control. After washings, primary antibody binding was detected via corresponding goat anti-mouse or anti-rabbit Alexa Fluor 488-conjugated, or donkey anti-goat Alexa Fluor 568 secondary antibodies (1:600 to 1:800; Invitrogen). Cells were counterstained with either Hoechst 33258 (Invitrogen) or TO-PRO-3 (Invitrogen) to show nuclei. Staining was observed by a Nikon E600 upright fluorescence microscope or Leica TCS SP2 confocal microscope. Hematoxylin and eosin, Fontana-Mason (melanin), and tyrosinase (TYR) (monoclonal, 1:75; Novocasta Lab Ltd., Newcastle On Tyne, UK) staining was performed on 5-µm sections following standard protocols. Human vascular smooth muscle cells (HUVSs), human primary neurons, and epidermal keratinocytes served as positive or negative controls. Oct4 (goat polyclonal, 1:50; Santa Cruz Biotechnology) and cytokeratin 15 (C8/144B, mouse, 1:50; Dako) immunohistochemical stains were performed on consecutive normal human scalp sections as previously described.30,31
Collagen Gel Contraction Assay
After 14 days of differentiation into smooth muscle cells, dissociated single cells were embedded at 4.5 x 104 cells/well in 3 ml of a buffered collagen solution and added to tissue reconstruct trays (Organogenesis, Canton, MA). The buffered collagen solution consisted of bovine collagen type I (Organogenesis) at a final concentration of 0.83 mg/ml in minimal essential medium with Earle’s Salts (BioWhittaker), 200 mmol/L L-glutamine, 10% fetal calf serum, and 7.5% sodium bicarbonate (BioWhittaker). Human epidermal keratinocytes and HUVSs (American Type Culture Collection) were used as negative and positive controls, respectively. The diameters of collagen gels were measured every 24 hours after seeding.
Human Skin Reconstructs
Human skin reconstructs were generated as described previously.32 Briefly, dermal reconstructs consisted of bovine collagen type I with embedded dermal fibroblasts. Cells treated in melanocyte differentiation medium for 14 days were seeded into tissue reconstruct trays together with keratinocytes onto dermal reconstructs at a 1:5 ratio of melanocytic cells to keratinocytes. Reconstructs containing undifferentiated cells were used as a control. Two weeks later, reconstructs were harvested and fixed in 10% neutral buffered formalin for 2 to 3 hours, processed by routine histological methods, and embedded in paraffin.
RNA Isolation and Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated using a Cells-to-cDNAII kit (Ambion, Austin, TX). Per kit protocol, 20 ng of total RNA was used for the first-strand cDNA synthesis. Real-time PCR was performed on an iCycler (Bio-Rad, Hercules, CA) using 1 µl of cDNA and iQ SYBR Green reagents (Bio-Rad). Standard real-time PCR protocol was used with melting curve analysis to ensure amplification specificity. Human epidermal keratinocytes and embryonic bodies at day 4, derived from human embryonic stem cells (H9), were used as negative and positive controls, respectively, to amplify stem cell markers. Human epidermal keratinocytes and melanocytes were used as negative and positive controls respectively, to amplify melanocytic markers MITF and TYRP1. Epidermal keratinocytes and human neurons were used as negative and positive controls, respectively, for neuronal markers. Keratinocytes and HUVSs were used as negative and positive controls, respectively, for smooth muscle cell markers. Relative expression levels were calculated using 18S rRNA expression levels for comparison. The primer sequences are listed in Table 1
.
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
We cultured human hair follicle-derived cells in hESC medium, which is sufficient to maintain hESCs in an undifferentiated state without the need for feeder cells.28
Sixteen fresh human scalp tissues were used in this study. Differential interference contrast and histological examinations showed that plucked human hair follicles were intact with little dermal contamination (Figure 1, A–D)
. The follicular epithelium was trypsinized and filtered to obtain a single-cell suspension containing cells of varying size and shape. Dissociated single cells were pelleted and cultured in conditioned hESC medium. Most of the single cells did not proliferate and gradually died. A few cells attached to the plate and continued to grow slowly as monolayer cells. Some small round cells proliferated and became 2- to 3-cell aggregates in suspension after 3 to 5 days. These aggregates slowly increased in size, gradually attached to the plate, and after 40 to 50 days, generated three-dimensional sphere-like structures, which we termed hair spheres (Figure 1, E–H)
. A hair sphere normally contains a sphere at its core and sprouting adherent fibroblastic cells at the periphery. We were able to isolate sphere-forming cells from 8 of 13 surgical specimens and 0 of 3 autopsy specimens. The number of spheres formed was directly proportional to the amount of scalp tissue used. Approximately in 104 to 105 seeded follicular cells were capable of forming a hair sphere. We termed these sphere-forming cells human hair follicle stem cells (hHFSCs). Therefore, hESC culture medium can be used to isolate and expand a subpopulation of sphere-forming cells from human hair follicles.
|
We then performed limiting dilution assays to test whether single cells derived from hair spheres were able to re-form spheres. Hair spheres were enzymatically or mechanically dissociated into single cells and seeded into 96-well plates at 0.5 to 1 cell/well. Only wells containing a single cell were observed daily for proliferation. Some wells contained large flattened cells that proliferated into monolayer cultures with fibroblastic morphology, but they were unable to reform spheres. Small round cells in some wells were able to regenerate adherent spheres. They started to proliferate 2 to 4 days after seeding, and a small aggregate developed after 20 to 30 days. A typical hair sphere appeared after approximately 8 weeks, indicating their capacity for self-renewal (Figure 1, I–L)
. Approximately 1% of the seeded disassociated hair sphere cells were capable of forming a new hair sphere. Human hair follicle-derived stem cells maintained sphere-forming capacity within a period of 8 months in the hESC medium, and then they gradually became flattened and stopped proliferating, suggesting that these cells have a finite life span in the hESC medium in vitro.
Hair Spheres Express Neural Stem Cell and Neural Crest Stem Cell Markers
To characterize cells in the hair spheres, we studied their gene and protein expression. Neural crest stem cells and nestin-positive neural stem cells have been identified in the adult gut, dermis, and mouse hair follicles.16,17,20,33
We examined hair spheres for the expression of nestin (NES) as well as genes transcribed in embryonic neural crest stem cells.34
Real-time RT-PCR analyses showed that hair spheres expressed NES as well as the neural crest stem cell markers SLUG, SNAIL, TWIST, SOX9, and BMP4 (Figure 2C)
. We performed nestin immunocytochemical staining on hair spheres and disassociated hair sphere cells and demonstrated that nestin-positive cells were present in the hair sphere (Figure 2, A and B)
with approximately 10% of the cells being nestin positive. The results demonstrate that cells in the hair spheres express both neural and neural crest stem cell markers.
|
Unlike epithelial and melanocytic stem cells, hHFSCs cultured in hESC medium did not express any lineage-specific markers such as MITF and tyrosinase-related protein 1 (TYRP1) for melanocytes, MAP2 and ß3-tubulin for neurons, calponin and desmin for smooth muscle cells, cytokeratin for keratinocytes, or CK20 for Merkel cells (data not shown).
Because hHFSCs can survive and expand in hESC medium, we tested whether these cells express Nanog and Oct4. Nanog and Oct4 are transcription factors involved in stem cell self-renewal and are thought to be the intrinsic factors that mediate pluripotency in embryonic stem cells.35-39
However, their expressions might not be as restricted as previously thought. Oct4 expression has been demonstrated in adult stem cells, and rare positive cells can be identified in skin.40
We found that a few cells within the hair spheres were positive for these transcriptional factors by immunocytochemistry and confocal microscopy (Figure 2A)
. To confirm the observation, we dissociated hair spheres into single cells and plated them onto coated glass chamber slides. The resulting cultures contained heterogeneous populations of small round cells (3 to 5%), intermediate cells (15 to 20%), and large flattened cells (75 to 82%). Confocal microscopy revealed that most of the small round cells were positive for Nanog or Oct4 (3 to 5%). Oct4 staining showed a characteristic punctate nuclear staining pattern as previously described,40
and Nanog, a homogenous nuclear staining pattern.35,37
Rare small round cells were positive for both Nanog and Oct4 (data not shown). A few intermediate and large flattened cells also expressed Oct4 (Figure 2B)
. Unlike hESCs, hHFSCs do not express SSEA-3 and -4 (data not shown). Thus, hHFSCs are distinctively different from previously identified lineage-specific epithelial or melanocytic stem cells from hair follicles.
Melanocytic Differentiation of Human Hair Sphere-Forming Cells
We proceeded to induce differentiation of hHFSCs into melanocytes. Dissociated hair spheres were cultured in a melanogenic differentiation medium29
for 14 days, when 20 to 40% of cells in medium adopted a dendritic morphology typical of melanocytes (Figure 3A)
. Real-time RT-PCR revealed that the differentiated cells gained the expression of melanocyte markers MITF and TYRP1, whereas they lost expression of the stemness gene NANOG (Figure 3B)
. Melanin pigment could be detected in differentiated cells by Fontana-Mason stain (Figure 3C)
. Expression of melanocyte markers MITF and silver (HMB45) proteins was detected by immunocytochemistry (Figure 3D)
. To study whether hHFSC-derived melanocytes have acquired functions similar to normal melanocytes, we introduced the differentiated melanocytes into human skin reconstructs that mimic human skin architecture. We have previously used these organotypic cultures to study interactions among melanocytes, epidermal keratinocytes, and dermal fibroblasts32
and have shown that normal melanocytes migrated to the dermal epidermal junction. In this study, similar to normal melanocytes, hHFSC-derived melanocytes, identified by immunoreactivity for tyrosinase and melanin pigment, homed to the dermal-epidermal junction. Undifferentiated cells did not show any melanocytic phenotypes in the reconstructs (Figure 3E)
. These results suggest that, in the human skin environment, hHFSC-derived melanocytes not only produce melanin pigment but also respond to skin patterning cues in ways similar to those of normal epidermal melanocytes.
|
Next, we examined whether hHFSCs could differentiate into neurons, a type of cells not present in hair or skin. Fourteen days after culture in neuronal differentiation medium, approximately 10% of cells showed long dendritic processes and expressed MAP2, NFM, and chromogranin A (CGA) proteins (as assayed by immunocytochemistry) (Figure 4A)
. MAP2 antibody predominantly stained the dendritic processes but not the cell bodies of differentiated cells, a staining pattern characteristic of neurons. Examination of neurotransmitters showed the presence of glutamate-positive cells; however, dopamine-positive cells were not identified. We detected the expression of MAP2 and ß3-tubulin (TUBB3) genes by real-time RT-PCR. The gain in neural markers was accompanied by a loss of NANOG gene expression after neuronal differentiation (Figure 4B)
.
|
To test whether hHFSCs could differentiate into mesoderm-derived lineages, we induced smooth muscle differentiation. In smooth muscle differentiation medium, approximately 80% of hHFSCs were induced into smooth muscle cells that acquired abundant cytoplasm and displayed immunoreactivity for SMA (Figure 5A)
. After differentiation, additional smooth muscle markers CNN3 and DES were also detected by real-time RT-PCR, accompanied by the loss of NANOG gene expression (Figure 5B)
. To examine whether hHFSC-derived smooth muscle cells have acquired appropriate functions, we mixed differentiated cells into a collagen gel. Cultured HUVSs were used as a positive control. Human HFSC-derived smooth muscle cells contracted collagen gels; the contraction rate was identical to that of HUVSs (Figure 5, C and D)
, suggesting that they had acquired contracting function similar to those of HUVSs.
|
We extended our studies to identify a potential stem cell niche for hHFSCs. We performed Oct4 and cytokeratin 15 (K15) immunohistochemistry on consecutive normal human scalp tissues. K15 staining has been shown to highlight the bulge area.31,41
Rare Oct4-positive cells can be seen in the epidermis as previously described40
; however, most of the Oct4-positive cells in skin were located in the hair follicles within the area highlighted by K15 staining in both anagen and catagen/telogen hair follicles (Figure 6A)
. We then performed immunostaining on plucked human hair follicles after they were cultured in hESC medium for 6 days. Nanog- and Oct4-positive cells were readily identified in the bulge area of catagen/telogen follicles (Figure 6B)
. Expression of Nanog and Oct4 in bulge cells was further confirmed by confocal microscopy (Figure 6C)
. The results suggest that hHFSCs reside in the bulge.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Skin appears to be a stem cell-rich organ. Shih et al43 isolated dermal stem cells from human adult scalp, Toma et al44 isolated dermal stem cells from foreskin, and Belicchi et al45 isolated dermal stem cells from fetal skin.45 Neural-crest-like stem cells have been isolated from mouse whisker hair using a medium containing chick embryo extract and fetal calf serum17,18 ; these mouse hair stem cells grew as adherent monolayer cells. However, human hair follicle-derived stem cells were not able to proliferate using their medium condition (data not shown), suggesting different biological behavior of mouse and human stem cells. Using Nestin-driven green fluorescent protein (GFP) transgenic mice (Nestin is a marker for neural progenitor cells), Hoffman and colleagues16 showed that GFP-positive cells are present in the hair bulge. These cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. More recently, they implanted the GFP-expressing stem cells into the gap region of a severed sciatic nerve; these cells greatly enhanced the rate of nerve regeneration and the restoration of nerve function. These cells transdifferentiated largely into Schwann cells, which are known to support neuron regrowth.46 Our study showed for the first time that human hair follicles also contain a stem cell population that can be differentiated into neuron, smooth muscle cell, and melanocyte lineages in induction medium.
Nanog and Oct4 are transcription factors that mediate hESC self-renewal and pluripotency.36-39 Oct4 belongs to the family of POU-domain transcription factors that are normally expressed in pluripotent cells of the developing embryo and mediate pluripotency.36,47,48 Consistent with its role in maintaining pluripotency, the reactivation of Oct4 has been correlated with efficient reprogramming of somatic cells after nuclear transfer into oocytes49,50 or cell fusion of somatic cells with ES cells.51 Moreover, ectopic expression of Oct4 in certain somatic cells has been associated with active dedifferentiation.52 The reactivation of pluripotency-associated genes has been observed in various somatic cancer cells, such as breast and colon cancers,53,54 although the relevance of this finding in adult cells is unclear. Our data demonstrate that Oct4-positive cells are present in human skin, and most of them are located in the hair follicles in vivo. It has been suggested that a subpopulation of pluripotent stem cells present in the inner cell mass of the pre-implantation embryo may never differentiate; instead, they might persist and seed adult tissues. Such rare pluripotent cells may be responsible for recent observations of unexpected adult somatic tissue plasticity.55 It is possible that these Oct4-positive cells in the hair follicles are related to these pluripotent stem cells that can perceivably give rise to follicular melanoblasts, Merkel cells, and other cells. These stem cells might generate diverse cell types during tissue renewal or repair in response to environmental cues. More research is warranted to further characterize these stem cells in the hair follicles.
The hair bulge is a stem cell niche, which can be highlighted by K15 staining. We have demonstrated that most of the Oct4-positive cells in human skin are located in the areas highlighted by K15 staining in vivo, suggesting that these stem cells are located in the bulge area, an area that provides an unique differentiation-restricted environment for adult stem cells.10,11,13 Oct4- or Nanog-positive cells are present in the bulge area when individual hair follicles are cultured in hESC medium, further suggesting that these newly identified human follicular stem cells are located in the bulge.
In conclusion, our data indicate that human hair follicles contain multipotent stem cells other than epithelial and melanocytic stem cells, and these cells are located in the bulge area. These cells show promising plasticity in ex vivo and in vitro conditions, making them potential candidates for cell engineering and cell replacement therapies. Human scalp tissues are easily accessible, and the fact that hair spheres can be generated from autologous adult tissue makes it an attractive source for individualized cell-based therapies.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by funding from Department of Pathology and Laboratory Medicine, University of Pennsylvania to X.X., grants from the National Institutes of Health (CA25874, CA80999, CA10815, and CA76674 to M.H.), and the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health.
H.Y. and D.F. contributed equally to this study.
Current address of D.F.: Celera Genomics, Rockville, MD.
Accepted for publication February 22, 2006.
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  D. Scholz and S. Ergun One more reason to save your hair Cardiovasc Res, July 1, 2008; 79(1): 1 - 2. [Full Text] [PDF]
|
|
|
|
|
|
J. Y. Liu, H. F. Peng, and S. T. Andreadis Contractile smooth muscle cells derived from hair-follicle stem cells Cardiovasc Res, July 1, 2008; 79(1): 24 - 33. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Zabierowski and M. Herlyn Melanoma Stem Cells: The Dark Seed of Melanoma J. Clin. Oncol., June 10, 2008; 26(17): 2890 - 2894. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
