Identification of Human Oral Keratinocyte Stem/Progenitor Cells by Neurotrophin - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:57 |只看该作者 |倒序浏览 |打印

作者：Takahiro Nakamuraa,b, Ken-ichi Endoa, Shigeru Kinoshitaa作者单位：aDepartment of Ophthalmology, Kyoto Prefectural University of Medicine, Graduate School of Medicine, Kyoto, Japan;bResearch Center for Regenerative Medicine, Doshisha University, Kyoto, Japan
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      【摘要】: I( O0 w  J9 A* O# I9 P% L
   This study was undertaken to determine whether human oral keratinocyte stem cells characteristically express higher levels of the low-affinity neurotrophin receptor p75 and to elucidate the function of p75 in oral keratinocytes. Examination of their expression patterns and cell-cycling status in vivo showed that p75 was exclusively expressed in the basal cell layer of both the tips of the papillae and the deep rete ridges. These immunostaining patterns suggest a cluster organization; most p75( ) cells did not actively cycle in vivo. Cell sorting showed that cells in the p75( ) subset were smaller and possessed higher in vitro proliferative capacity and clonal growth potential than the p75(¨C) subset. Clonal analysis revealed that holoclone-type (stem cell compartment), meroclone-type (intermediate compartment), and paraclone-type (transient amplifying cell compartment) cells, previously identified in skin and the ocular surface, were present in human oral mucosal epithelium. Holoclone-type cells showed stronger p75 expression at both the mRNA and protein level than did meroclone- and paraclone-type cells. Among the several neurotrophins, nerve growth factor (NGF) and neurotrophin-3 stimulated p75( ) oral keratinocyte cell proliferation, and only NGF protected them from apoptosis. Our in vivo and in vitro findings indicate that p75 is a potential marker of oral keratinocyte stem/progenitor cells and that some neurotrophin/p75 signaling affects cell growth and survival.
      【关键词】 p Oral mucosa Keratinocyte Stem cell Clonal analysis Neurotrophin
   INTRODUCTION( A! k6 V) t  \6 B

Epithelial cells, such as epidermal, esophageal, corneal, and oral keratinocytes, are organized into multiple layers. Like other rapidly renewing tissues, such as the hemopoietic system, the human epithelium is constantly regenerating. In general, proliferation occurs in the basal layer of keratinocytes attached to the underlying basement membrane; cells that undergo terminal differentiation as they migrate through the suprabasal layers are shed from the tissue surface . Upon exhaustion of their proliferative potential, the rapidly proliferating TACs undergo terminal differentiation.
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In this study, we used clonal analysis and cell sorting techniques for the purpose of characterizing the tissue SC and TAC populations. Clonal analysis identified three types of keratinocytes with different capacities for multiplication in human epidermis, hair follicles, and ocular surface epithelium . Holoclones have the highest reproductive capacity, in paraclones, all cells undergo terminal differentiation within a few generations, and the behavior of meroclones is intermediate between holoclones and paraclones. Holoclones, meroclones, and paraclones are considered to be SCs, young TACs, and TACs, respectively. The holoclone-meroclone-paraclone transition is a unidirectional process that occurs during natural cell aging and during repeated subcultivation.$ Y" {& _+ H; B# R; ?

The cell-sorting technique uses phenotypic cell surface markers that distinguish differentiated cells from progenitor cells. Interesting results have been obtained in several tissue types. In human epidermis, SCs could be distinguished from differentiated keratinocytes by their higher expression of ¦Â1 integrins . For the further molecular characterization of various keratinocyte SCs, additional cell surface markers are needed.
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The human oral cavity contains masticatory mucosa, such as gingiva, and lining mucosa, such as buccal mucosa. Oral mucosal epithelium has drawn attention as a cell source for a variety of tissue-engineered reconstructions, such as oral cavity , and the transplantation of human oral epithelial sheets grown on various substrates can be useful for tissue reconstruction. In the clinical setting, the quality of the cultivated graft is the key to success, and the selection of a large number of highly proliferating oral epithelial cells, such as SCs and TACs, enhances the reproducibility, quality, and longevity of these grafts. However, little is known about oral keratinocyte SCs and TACs.

The p75 molecule, a low-affinity neurotrophin receptor, is a member of the tumor necrosis factor receptor superfamily . However, its function in various human keratinocyte SCs remains to be fully elucidated.
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We investigated the fate of p75( ) cells to determine whether this subset is critical for stem- or progenitor-cell lineages in human oral keratinocytes. Using clonal analysis and cell sorting, we evaluated the p75 expression patterns of human oral mucosal tissue and attempted to identify and isolate human oral keratinocyte stem/progenitor cells. We then investigated the effect of neurotrophin/p75 signaling in isolated oral keratinocytes. We found that human oral keratinocyte stem/progenitor cell phenotypes are characterized by their expression of p75 and that some neurtrophin/p75 signaling affected cell proliferation and survival.4 M+ d; [) z7 m$ O( x* o
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MATERIALS AND METHODS$ i8 T" E5 ^# n& @" W% U
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Tissues$ k6 [  o' h$ _; Z2 P
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Oral tissues were obtained from healthy volunteers and as superfluous tissue from patients undergoing oral surgery. We followed the tenets of the Declaration of Helsinki; all donors provided proper informed consent for biopsy. All samples were processed within 1¨C2 hours of harvest.6 u( {3 B' B  N( L) E4 b

Cell Culture' C' l/ f6 S6 L& K

For culture of the human oral epithelial cells, we used our previously reported system . Briefly stated, submucosal connective tissues were removed with scissors to the extent possible; the resulting samples were cut into small explants. These were incubated (37¡ãC, 1 hour) with 1.2 IU of Dispase (Roche, Indianapolis, http://www.roche.com) and treated with 0.05% trypsin-EDTA solution (10 minutes, room temperature) to separate the cells. The cell suspension was filtered through a cell dissociation sieve (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) to remove unsatisfactory segments; this yielded a suspension of purified oral epithelial cells. Isolated cell suspensions were then subjected to cell-sorting assay and clonal analysis.
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Clonal Analysis
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For clonal analysis, we applied the method of Barrandon and Green . When 0%¨C5% of the colonies were terminal, the clone was classified as a holoclone. When all colonies were terminal or when no colonies formed, the clone was classified as a paraclone, and when >5% but 1 y# N) n; m3 v9 t- j

Antibodies and Reagents' |4 j' z/ c0 k* a# r/ }
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The following mouse monoclonal antibodies (mAbs) were used: anti-p75 (dilution, x200) (Chemicon, Temecula, CA, http://www.chemicon.com; Abcam, Cambridge, U.K., http://www.abcam.com; and Upstate Biotech, Lake Placid, NY, http://www.upstate.com), anti-desmoplakin (x1) (Progen, Heidelberg, Germany, http://www.progen.de), anti-keratin 4 (x200)/10 (x50)/13 (x200) (Novocastra Ltd., Newcastle upon Tyne, U.K., http://www.novocastra.co.uk), anti-integrin 6 (x200)/¦Â4 (x500)/3 (x50)/¦Â1 (x500) (Chemicon), CD34 (x50) (BD Biosciences, San Diego, http://www.bdbiosciences.com), CD71 (x50) (YLEM, Roma, Italy, http://www.ylem.it), BCRP (x10) (Kamiya Biomedical, Seattle, http://kamiyabiomedical.com), and anti-Ki67 (x100) (Dako, Kyoto, Japan, http://www.dako.com). Rabbit polyclonal antibodies were also used: anti-ZO1 (x25) (Zymed, South San Francisco, CA, http://www.invitrogen.com) and anti-Ki67 (x50) (Abcam). Secondary antibodies were Alexa Fluor-488 goat anti-mouse or rabbit IgG (x1,500) and Alexa Fluor-594 goat anti-mouse or rabbit IgG (x1,500) (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). The following neurotrophins were used: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) (Sigma-Aldrich).
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Cell Fractionation
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The p75( ) and p75(¨C) cells were separated using magnetic cell sorting with the indirect microbead system (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). The oral epithelial cell suspensions were labeled with anti-p75 mAb (x400) at 0¡ãC for 15 minutes. The cells were then carefully washed with MACS buffer (phosphate-buffered saline  supplemented with 2 mM EDTA and 0.5% bovine serum albumin), incubated with rat anti-mouse IgG1 microbeads (x5) at 4¡ãC for 15 minutes, and exposed to the magnetic field of a permanent magnet on columns containing a ferromagnetic matrix. To increase the purity of the positive fraction, our protocol combined depletion columns and positive selection columns.

Validation of the Cell-Sorting Procedure

Flow Cytometry and Immunofluorescence. After the MACS procedure, each p75( ) and p75(¨C) cell fraction was again washed with MACS buffer and stained with Alexa Fluor-488 goat anti-mouse IgG (x1,500) at 4¡ãC for 20 minutes for subsequent flow cytometric analysis (FACSCalibur; BD Biosciences) and immunofluorescence study. The cells were coverslipped using antifading mounting medium and examined under an immunofluorescence microscope (Olympus, Tokyo, http://www.olympus-global.com).# @$ [; `/ ^3 S; K; ^; H6 ~

Reverse Transcription PCR. Total RNA was isolated from each isolated cell fraction using TRIzol reagent in accordance with the manufacturer's protocol. Complementary DNA was generated by mixing the extracted RNA (1 µg/µl per sample) with a random hexamer primer and AMV Reverse Transcriptase XL (Takara, Tokyo, http://www.takara.co.jp). The primers and PCR conditions were followed the previously reported method . The expected product sizes were 230 base pairs (bp) for p75 and 541 bp for ¦Â-actin.
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Measurement of Cell Area

Each isolated cell fraction was centrifuged and resuspended in culture medium. Cells (approximately 103 cells) in 10 ml of medium were placed in 100-mm culture dishes and photographed under an inverted microscope using a x10 phase objective . Cell areas were measured randomly (200 cells per fraction) using Scion Image software (Scion Corp., Frederick, MD, http://www.scioncorp.com/) and statistically analyzed.( E& h) {+ c5 k) k9 y
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5-Bromo-2'-deoxyuridine Cell Proliferation Assay
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The proliferative capacity of each isolated cell fraction was determined by 5-bromo-2'-deoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA) cell proliferation assay (Amersham Biosciences, Freiburg, Germany, http://www.amersham.com) using a previously reported protocol . Analysis was on the 6th day of passage (n = 3). Cultured cells were incubated with 10 µM BrdU-labeling solution (20 hours, 37¡ãC), washed with 250 µl of PBS containing 10% serum per well, fixed with 70% ethanol in hydrochloric acid (30 minutes, ¨C20¡ãC), and incubated with 100 µl of monoclonal antibody against BrdU (90 minutes), and then 100 µl of peroxidase substrate was added to each well. BrdU absorbance in each well was measured directly with a spectrophotometric microplate reader at a test wavelength of 450 nm and a reference wavelength of 490 nm. This measure of the degree of cell proliferation we termed the proliferation index. Each sample was cultured in triplicate.

The proliferative capacity of isolated p75( ) cells incubated with several neurotrophins (NGF, NT-3, NT-4, and BDNF) was also analyzed using the same procedure. The p75( ) cells were seeded at a density of 2 x 102 cells per cm2 for 48 hours. Next, the cells were preincubated with the aforementioned neurotrophins (1, 10, and 100 ng/ml, respectively) for an additional 48 hours and then analyzed.6 M4 F$ {. [4 Y/ k0 t. Q
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Colony-Forming Efficiency% j  A2 \9 T( j; O( H1 _% p
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The clonal growth ability of each isolated cell fraction was determined by colony-forming efficiency (CFE) assay. Cells (2 x 103) were plated on six-well culture dishes that contained a feeder layer of MMC-inactivated NIH-3T3 fibroblasts (n = 3). The colonies were fixed on day 7, stained with 0.1% truidine blue, and counted independently by three investigators; the data were then averaged. Each sample was cultured in triplicate. CFE was defined as the ratio of the number of colonies to the number of viable cells seeded." _7 `' W' ?+ X; Y0 f8 O
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Ex Vivo Expansion of Isolated Cells
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To culture isolated cell fractions we used our previously reported system . Isolated cells (1 x 105 cells per well) were then seeded onto denuded amniotic membrane (AM) spread on the bottom of culture inserts, and cocultured for 10 days with mitomycin C-inactivated 3T3 fibroblasts (2 x 104 cells per cm2). The culture medium consisted of a defined keratinocyte growth medium (ArBlast, Kobe, Japan,http://www.arblast.jp) supplemented with 5% fetal bovine serum. The percentage of Ki67( ) cells in the basal layer of cultivated epithelium was determined on tissue sections. We analyzed five different fields from samples obtained from three different donors (15 areas per donor).
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Immunohistochemistry9 P4 F# L, B) b% [$ B: y1 ~1 h
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Immunohistochemical studies followed our previously described method . Briefly stated, 3-µm-thick cryostat sections were placed on gelatin-coated slides, air-dried, and rehydrated in PBS at room temperature for 15 minutes. To block nonspecific binding, the tissues were incubated with 2% bovine serum albumin (room temperature, 30 minutes). The sections were then incubated (room temperature, 1 hour) with the appropriate primary antibody (simple antibody or a mixture of antibodies for double staining) and washed (three times) in PBS containing 0.15% Triton X-100 for 15 minutes. Control incubations were with the appropriate normal mouse and rabbit IgG (Dako) at the same concentration as the primary antibody; the primary antibody for the respective specimen was omitted. After staining with the primary antibody, the sections were incubated with the appropriate secondary antibodies (room temperature, 1 hour), washed several times with PBS, coverslipped using antifading mounting medium containing propidium iodide or 4,6-diamidino-2-phenylindole (Vectashield; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), and examined under a confocal microscope (Olympus Fluoview).
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Real-Time PCR

Quantitative real-time PCR for p75, integrin ¦Â1, integrin 6, CD71, and ABCG2 was performed using an ABI Prism 7000 instrument (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Total RNA was isolated with the RNeasy Micro Kit (Qiagen, Tokyo, http://www1.qiagen.com) (n = 5). We then performed quantitative real-time reverse transcription (RT)-PCR using QuantiTect Probe RT-PCR kits (Qiagen). Primers and probes for p75, integrin ¦Â1, integrin 6, CD71, ABCG2, and ¦Â-actin were from Applied Biosystems. For relative quantification, we used the CT method (Applied Biosystems). Analyses were performed in a sequence detector (ABI Prism 7000) using the accompanying data analysis software.
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Apoptosis Assay

The p75( ) cells were seeded at a density of 2 x 102 cells per cm2 on a chamber slide for 48 hours. Next, the cells were preincubated with several neurotrophins (100 ng/ml) for 48 hours, with or without UV irradiation (25 mJ per cm2)  assay; Takara).9 m  Y' W8 k2 G

RESULTS
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In Vivo Expression Patterns and Cell-Cycling Status of p75( ) Human Oral Keratinocytes; K1 w5 k0 L/ Q( }5 y

The in vivo expression patterns of p75 in human oral mucosal epithelium were investigated by indirect immunofluorescence. We used p75 antibodies from three companies (Chemicon, Abcam, and Upstate Biotech) and found no differences in the immunohistochemical results. We obtained tissues from the buccal mucosa (representing lining mucosa), where rete ridges are comparatively shallow and gentle, and gingiva (representing masticatory mucosa), where they are steep and deep. In the buccal mucosa, we noted intensive p75 expression primarily in the basal cell layer of the tips of the papillae (Fig. 1A); in some parts, p75 was expressed in deep rete ridges (Fig. 1B). In the gingiva, it was exclusively expressed in the basal cell layer of both the tips of the papillae and the deep rete ridges (Fig. 1C). In both tissues, p75 was expressed in the cell membrane. The immunostaining patterns suggest cluster (patch) organization, and clusters of brightly fluorescent basal cells were interspersed with stretches of basal cells with little or no fluorescence. The percentage of p75( ) cells per total oral keratinocytes was 7.35 ¡À 3.41 (five different fields from six different donors  were analyzed).
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Figure 1. Immunolocalization of p75 and p75-Ki67 in human oral mucosal tissues. (A¨CC): In buccal mucosa, rete ridges are relatively shallow and gentle; p75 expression was intense primarily in the basal cell membrane of the tips of the papillae (A) (*). There was p75 expression in some deep rete ridges (B) (*). In the gingiva, rete ridges are deep and steep; p75 was expressed exclusively in the basal cell layer of the tips of the papillae and the deep rete ridges (C) (*). Patches of brightly fluorescent basal cells were interspersed with stretches of basal cells showing no or little fluorescence. (D¨CF): Ki67 expression was mainly observed in the suprabasal cell layer and occasionally in the basal cell layer of oral epithelium (D, E). The percentages of Ki67(¨C) and Ki67( ) cells among all p75( ) cells were 96.63% ¡À 2.69% and 3.37% ¡À 2.68%, respectively (F). The difference was statistically significant (*, p 6 |9 o3 c3 A/ E4 H
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Slow or infrequent cycling is one of the unique features of adult SCs . To investigate the in vivo cell cycle status in human oral mucosal epithelium, we used double immunostaining with p75 and Ki67, a marker for actively cycling cells. Ki67 was primarily expressed in the suprabasal and occasionally in the basal cell layer of oral epithelium (Fig. 1D, 1E). Statistical analysis of our double-staining results on 32 sections (5¨C10 sections each from four different donors) showed that among the p75( ) cells, the proportion of Ki67(¨C) cells was significantly higher (96.63% ¡À 2.69%) than that of Ki67( ) cells (3.37% ¡À 2.68%) (*, p
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Characteristics of p75( ) and p75(¨C) Cell Fractions
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To examine the characteristics of the p75( ) and p75(¨C) cell fractions, we isolated the subsets by magnetic cell sorting and validated our results by flow cytometry, RT-PCR, and immunofluorescence (supplemental online Fig. 1). Flow cytometric analysis showed that our use of a combination of depletion and positive selection columns yielded a purity in excess of 92% for p75( ) cells in all experiments (supplemental online Fig. 1A). RT-PCR and immunofluorescence for p75 confirmed its expression at both the mRNA and protein level in the purified fraction (supplemental online Fig. 1B, 1C).  ]: X* q9 h+ O: j* v3 I
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As the highest clonogenicity, a feature of SCs, is reportedly found in the smallest keratinocytes , we measured the cell size in each isolated fraction using Scion Image software. Under an inverted microscope, p75( ) cells were clearly smaller than p75(¨C) cells (Fig. 2A). The average size of p75( ) cells was significantly smaller than that of p75(¨C) cells (102.52 ¡À 46.85 vs. 346.84 ¡À 134.67 µm2; *, p

Figure 2. Characteristics of p75( ) and p75(¨C) cell fractions. (A): Under an inverted microscope, p75( ) cells are clearly smaller than p75(¨C) cells. The average cell size of the p75( ) cell fraction was 102.52 ¡À 46.85 µm2, significantly smaller than the 346.84 ¡À 134.67 µm2 of the p75(¨C) cell fraction (*, p 9 u1 Q, ~8 l9 [* D0 j: y% s

The proliferative capacity and clonal growth ability of each isolated cell fraction was determined by BrdU ELISA cell proliferation assay and CFE. Phase-contrast inspection of the isolated cells on the 6th day of passage showed that p75( ) cells formed colonies consisting of ovoid and round cells (Fig. 2B); p75(¨C) cells, which did not form colonies, were large and elongated (Fig. 2B). The proliferation indexes of p75( ) and p75(¨C) cell fractions were significantly different (9.64 ¡À 0.1 vs. 1.16 ¡À 0.09; *, p
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To evaluate their in vitro tissue-forming ability, we cultivated isolated cell fractions on an AM substrate. After 10 days of cultivation, p75( ) oral epithelial cells formed 4¨C5 layers exhibiting well-conserved columnar basal cells and progressive flattening toward the surface (p75( ) sheet). On the other hand, p75(¨C) cells grew in monolayers composed of elongated, differentiated cells (Fig. 2D). To assess the cell-cycling status of these cultivated oral epithelial cells we examined their expression of Ki67. In p75( ) and p75(¨C) sheets, Ki67-labeled cells constituted 81.3% ¡À 10.6% and 25.4% ¡À 9.9%, respectively (Fig. 2D), rendering the difference in the Ki67-labeling index statistically significant (*, p   t) x6 \' w- Z
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Using immunofluorescence and specific markers, we studied the morphological and biological characteristics of the p75( ) and p75(¨C) sheets. ZO-1, a tight junction-related component, was expressed in the apical surface on p75( ) but not p75(¨C) sheets (Fig. 3A, 3B). Desmoplakin, a cell-cell junction component, was clearly expressed in the cell membrane on p75( ) sheets; there was moderate desmoplakin expression on p75(¨C) sheets (Fig. 3C, 3D). On p75( ) sheets, nonkeratinized, mucosa-specific keratin 4 was expressed in the superficial layer and the upper half of the intermediate layer; keratin 13 was expressed in all but the basal cell layers (Fig. 3E, 3G). On p75(¨C) sheets, on the other hand, there was no or faint superficial staining (Fig. 3F, 3H). The basement membrane assembly proteins integrin 6/¦Â4 showed linear positive staining on the basement membrane side on both p75( ) and p75(¨C) sheets (Fig. 3I¨C3L). Integrin 3 was mainly expressed in the basal cell membrane on both p75( ) and p75(¨C) sheets (Fig. 3M, 3N), and integrin ¦Â1 was expressed in the cell membrane of nearly all epithelial cells on p75( ) sheets; moderate or faint integrin ¦Â1 staining was observed on p75(¨C) sheets (Fig. 3O, 3P). Thus, only the p75( ) sheets manifested normal cell differentiation and normal junctional specialization, suggesting that only the p75( ) cells possessed high in vitro proliferative capacities and normal three-dimensional (3D) tissue formation potential.
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Figure 3. Representative immunofluorescence micrographs of p75( ) and p75(¨C) oral epithelial cells grown on amniotic membrane. ZO-1 was expressed on the apical surface of the p75( ) sheet but not the p75(¨C) sheet (A, B). Desmoplakin was clearly expressed in the cell membrane of the p75( ) sheet and moderately expressed in the p75(¨C) sheet (C, D). In the p75( ) sheet, mucosal-specific keratin 4 was expressed in the superficial portion and the upper half of the intermediate layers; keratin 13 was expressed in all epithelial layers except the basal cell layers (E,G). In the 75(¨C) sheet, there was no or faint superficial staining (F, H). The basement membrane assembly proteins integrin 6/¦Â4 showed linear positive staining on the basement membrane side of both p75( ) and p75(¨C) sheets (I¨CL). In both p75( ) and p75(¨C) sheets, integrin 3 was mainly expressed in the basal cell membrane (M, N). Integrin ¦Â1 was expressed in the cell membrane of nearly all epithelial cells in the p75( ) sheet; only faint or moderate staining was observed in the p75(¨C) sheet (O, P). Scale bars = 100 µm.
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Isolation and Clonal Analysis of Oral Keratinocyte SCs
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To determine whether holoclones, meroclones, and paraclones, previously identified in human epithelium , the representative original clone of the holoclone is large, has a smooth perimeter, and contains mainly small cells (Fig. 4A); most original clones of the paraclone are small and contain large differentiated squamous cells (Fig. 4C), and the meroclone is intermediate between the holoclone and the paraclone (Fig. 4B). On indicator dishes, holoclones formed large, rapidly growing colonies, fewer than 5% of which aborted or terminally differentiated (Fig. 4D). The paraclone grew no colonies or only uniformly small, terminal colonies (Fig. 4F), and the meroclone formed growing and aborted colonies (Fig. 4E). Of the clones studied, 23.6% ¡À 12.5% were holoclones, 34.9% ¡À 10.5% were meroclones, and 41.5% ¡À 11.3% were paraclones (Table 1). Our findings indicate that holoclone-, meroclone-, and paraclone-type cells, previously identified in skin and ocular surface, also comprise the proliferative compartment of the human oral mucosal epithelium.4 Y: g8 [7 p" i' u3 W
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Figure 4. Isolation and clonal analysis of oral keratinocytes. The representative original clone of the holoclone type is large, has a relatively smooth perimeter, and contains mainly small cells (A). Most original clones of the paraclone type are small and contain large differentiated squamous cells (C). The meroclone type is intermediate between the holoclone and the paraclone (B). Toluidine blue staining (indicator dishes) clearly showed that the holoclone formed large, rapidly growing colonies, fewer than 5% of which aborted or differentiated terminally (D). The paraclone grew no colonies or only uniformly small terminal colonies (F). The meroclone formed growing and aborted colonies (E). Immunofluorescence showed that p75 was strongly expressed in the cell membrane of holoclone-type cells (G). In the meroclone-type cells, p75 was moderately expressed in some of the small cells (H). Paraclone-type cells did not express p75 (I). In contrast, integrin ¦Â1 was expressed in the cell membrane of all three clonal types (J¨CL). Scale bars = 100 µm.& k9 K7 _/ I8 M  @. w8 A/ O4 T1 H, J

Table 1. Classification of clonal types  V9 O1 A6 I; Q- d/ Q

To investigate the expression of p75 and integrin ¦Â1 in these three clonal types, we performed immunohistochemical analysis and real-time PCR assay. Immunofluorescence showed that p75 was strongly expressed in the membrane of holoclone cells (Fig. 4G), moderately expressed in some of the small cells of meroclones (Fig. 4H), and not expressed in paraclone cells (Fig. 4I). In contrast, integrin ¦Â1 was expressed in all three clonal types (Fig. 4J¨C4L). We used real-time PCR to assess p75 mRNA expression (supplemental online Fig. 2) and found that compared with meroclone and paraclone cells, in holoclone cells, mean p75 mRNA expression was significantly upregulated (*, p ( Q- i( D8 q3 [4 B/ @

The Role of Neurotrophin Signaling in p75( ) Oral Keratinocytes

To evaluate the role of neurotrophin/p75 signaling in cell growth, differentiation, and survival, we performed the BrdU ELISA cell proliferation assay, immunohistochemistry for differentiation markers (keratin 10 and 13), and UV-induced apoptosis assay (TUNEL) in the presence or absence of neurotrophins in an in vitro culture system. BrdU assay indicated that NGF and NT-3 stimulated p75( ) oral keratinocyte cell proliferation (Fig. 5; *, p
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Figure 5. Neurotrophin effects on oral keratinocyte proliferation. 5-Bromo-2'-deoxyuridine (BrdU) enzyme-linked immunosorbent assay cell proliferation assay of p75 ( ) oral epithelial cells cultivated in the presence (1, 10, and 100 ng/ml) and absence of neurotrophin (NGF, NT-3, NT-4, and BDNF)-supplemented media for 48 hours (n = 5). Scale bars indicate the mean values of BrdU absorbance in each culture condition. BrdU assay indicated that NGF and NT-3 stimulated p75( ) oral keratinocyte cell proliferation (*, p
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Figure 6. Neurotrophin effects on oral keratinocyte cell survival. The p75( ) oral epithelial cells were treated for 48 hours in the presence (100 ng/ml) and absence of neurotrophin (NGF, NT-3, NT-4, and BDNF)-supplemented media before, with, or without UV irradiation, and apoptosis was measured by TUNEL staining. The percentage of TUNEL( ) cells in each culture condition was calculated by three individual experiments. Without UV conditions, neurotrophins do not seem to influence cell death in these cells (A). Under UV( ) conditions, NGF exerts a protective effect against UV-induced apoptosis (*, p 4 a# E2 }! v: k& d) b6 |  |
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The role of SCs in homeostasis, wound healing, and tumorigenesis and their identification and therapeutic use in tissue engineering, gene therapy, and the treatment of various diseases have gained attention. In view of ethical concerns surrounding the use of embryonic SCs, the identification of specific markers to isolate keratinocyte SC populations is of great value, both for a better understanding of SCs and for the development of SC-mediated regenerative therapies. We demonstrated the following points: (a) that p75( ) cell subsets are present as clusters in a specific region of the human oral mucosal epithelium and (b) that most of these cells do not cycle actively in vivo. Cell sorting showed that compared with p75(¨C) cells, p75( ) cells (c) were smaller and (d) had higher in vitro proliferative capacity and clonal growth potential. Our single-cell clonal analysis revealed (e) that three clonal types, previously identified in skin and ocular surface, also comprise the proliferative compartment of human oral mucosal epithelium and (f) that p75 is strongly expressed in holoclones at both the mRNA and protein level. Our in vivo and in vitro findings indicate that p75 may represent a novel marker for oral keratinocyte SC-containing populations." F5 ]: N! ?, X; t# K- z  B

In this study, we demonstrate for the first time that human oral keratinocytes exhibit regional diversity with respect to p75 expression. In the buccal mucosa, p75 was mainly expressed in the tips of the papillae, whereas in gingiva it was observed in both the tips of the papillae and the deep rete ridges. In the current situation, we can not exactly determine whether this means there are two stem cell niches in gingiva. The precise location of stem/progenitor cells within the epithelium is a question that remains to be settled, and its proposed answer depends on the expression pattern, cell surface markers, specific tissues, and species. Others  reported that SC distribution is nonrandom but varies by the specific phenotype of epithelial tissue. They found that cells brightly stained for integrin ¦Â1, a putative epidermal SC marker, were located in the tips of dermal papillae in human foreskin and interfollicular scalp and in the deep rete ridges of the palm.( G  f5 I3 h* w" T7 B- f0 N# H/ r

These cells, as was the case in our p75( ) cells, existed as clusters in tissue, rather than as sporadic single cells. Lavker and Sun , the location of stem and transient amplifying cells tends to be in the tips of the deep rete ridges. Additional cell biological studies are needed to clarify this point.1 x, M. V2 G8 C2 W+ q* ?) U
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It is important to clarify how high p75 expression can better define human oral keratinocyte stem/progenitor cells compared with other previously reported epidermal keratinocyte SC phenotypes, such as integrin ¦Â1 , it was not expressed in any layer of human oral mucosal epithelium (data not shown). To elucidate the relationship between p75 and integrin ¦Â1, integrin 6/CD71, and SP, we investigated their expression level in all isolated p75( ) and p75(¨C) cell fractions using real-time PCR (supplemental online Fig. 4). Moreover, we have investigated the expression of these proposed epidermal SC markers and compared them with the expression of p75 in the serial cross sections using the appropriate mouse monoclonal antibodies (supplemental online Fig. 5). Real-time PCR showed that the level of integrin ¦Â1 and ABCG2 mRNA in the p75( ) cell fraction was higher than that of the p75(¨C) cell fraction (supplemental online Fig. 4A, 4B; *, p 6 M' j4 K4 z2 ~- h! u! f
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Under steady-state conditions, SCs divide infrequently in vivo . However, they can proliferate vigorously in vitro and after appropriate stimulation in vivo. TACs, on the other hand, cycle actively. Cell kinetic studies, such as the label-retaining method, are clearly considered the gold standard by which to identify slow-cycling SCs. However, it is difficult to perform these kinds of cell kinetic studies in humans due to ethical considerations. Therefore, it is necessary to establish a useful assay to assign SC status isolated on the basis of biological markers in humans. In our study, most p75( ) basal cells did not express the proliferation-associated marker Ki67, suggesting that they did not cycle actively in vivo. Our finding that the human oral mucosal epithelium contained a small subset of p75( )-Ki67( ) cells suggests that p75, although valuable for sorting SC populations, may also identify early TAC populations on the basis of their cell-cycling status.( x3 a4 t* D. |

Others reported that cell size may distinguish SCs from TACs or differentiated cells. In the epidermis, the response to phorbol esters of the smallest keratinocytes is different from that of other cells , we suggest that cell size is a potential indicator of keratinocyte SCs, not only of the epidermis, but also of other epithelial cell types.: U- V; K& T* x- F0 x' I5 `* l

We found that isolated keratinocytes fractionated on the basis of the intensity of their p75 expression could produce different cell populations with different properties. Only cells in the p75( ) population exhibited exceptionally high proliferative potential, CFE, and 3D tissue forming-ability, features associated with SC populations and consistent with esophageal keratinocyte SCs studied in vitro , the isolated cells are released from the control mechanisms regulating the in vivo SC niche that renders them slow-cycling./ N4 U2 s: j9 u' E2 s
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Although cultured human keratinocytes may not retain all their in vivo characteristics after removal from their microenvironment, they are clonogeneic . Analysis of clonal-type frequency after a period of in vitro growth yields information on the intrinsic nature and proliferative potential of the original keratinocyte populations. The three clonal types we identified (holoclone, meroclone, and paraclone) comprise the proliferative compartment of the human oral mucosal epithelium. As p75 was intensively expressed only by holoclone cells on both the mRNA and protein level, it may specifically characterize human oral keratinocyte SC-containing population.

In contrast, integrin ¦Â1, an epidermal SC marker, was expressed in all three clonal-type cells. According to De Luca et al. . Therefore, markers specific for in vivo keratinocyte SCs may differ from those of keratinocyte colonies grown in vitro.: R$ S" y- n7 d- M6 k/ C

We also posit that it is very important to carefully interpret the results of stem cell research. Our conclusion that p75 may represent a marker for a human oral keratinocyte stem cell-containing population was mainly based on the results obtained by short-term in vitro clonogenic assay and tissue-regenerative ability, not by in vivo long-term reconstitution assay . We truly recognize that sustained epithelial tissue regeneration in an appropriate in vivo transplant model is likely to be the best functional definition of keratinocyte stem cells. Even though in vitro assay with development of culture techniques for keratinocytes is believed to reflect the extensive capacity expected of keratinocyte stem cells in vivo, at present our in vitro assay alone does not allow us to fully characterize the human oral keratinocyte stem cells. Even though it is quite difficult to transplant human oral keratinocytes onto the oral cavity site, both technically and methodologically, an additional in vivo long-term assay is needed to clarify these points.

In several cell types, the protein family of mammalian neurotrophins (NGF, BDNF, and NT-3/4/5) supports cell survival, differentiation, and plasticity . Thus, the cell death-inducing or cell-survival actions of neurotrophins are strongly dependent on the relative expression of p75 and Trk receptors on the target-cell populations. Studies are under way to elucidate the relationship between p75 and Trk receptors in keratinocytes from human oral mucosal tissue.

We believe that one way to characterize distinct cell populations is to generate global gene expression profiles. Recently, several microarray profiles of mouse  hair follicle bulge cells were reported. In these results, upregulation of p75 has not been identified in the bulge cells, yet we consider that oral mucosal epithelium is fundamentally different from bulge cells and epidermis, which may explain why upregulation of p75 was not detected. Moreover, the data represent the average characteristics of a cell population, rather than the properties of individual cells.
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Our study showed that oral keratinocytes expressing the p75( ) phenotype exhibited properties suggesting that they are equivalent to the SC-containing population. The degree of SC sorting made possible by p75 will allow the further fractionation and analysis of SC populations, thereby facilitating the identification of a set of additional SC markers unique to the epithelium. Systematic evaluation using cell-sorting techniques and clonal analysis will facilitate the identification and purification of these cells and greatly contribute to our understanding of SC biology. In addition, greater knowledge regarding SCs will provide a foundation for the development of treatments for epithelium-related diseases.0 J5 j3 J: p2 q& ^% o( W
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DISCLOSURES
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The authors indicate no potential conflicts of interest.' ^: v: n) p% ~* ~; I

ACKNOWLEDGMENTS
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We thank Yann Barrandon for advising the clonal analysis system, Narisato Kanamura and Takashi Amemiya for performing the oral biopsies, Hideo Honjyo for providing AM, and Hisayo Sogabe and Tomoko Horikiri for assisting with the culture procedures. This study was supported in part by Grants-in-Aid for Scientific Research from the Japanese Ministry of Health, Labor and Welfare (H16-Saisei-007) and by the Japanese Ministry of Education, Culture, Sports, Science and Technology (Kobe Translational Research Cluster), a research grant from the Kyoto Foundation for the Promotion of Medical Science, and the Intramural Research Fund of Kyoto Prefectural University of Medicine.& K" y7 i( _+ [
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