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Keywords:

  • p75;
  • Oral mucosa;
  • Keratinocyte;
  • Stem cell;
  • Clonal analysis;
  • Neurotrophin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

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(−) 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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

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 [1]. Stem cells (SCs) facilitate the maintenance of self-renewing tissues; they are critical for replenishing and maintaining the cell balance (homeostasis) within tissues and for regenerating damaged tissue. Based on previous studies of several tissue types, criteria for keratinocyte SCs prescribe that they are relatively undifferentiated both ultrastructurally and biochemically; retain a high capacity for long-term, error-free self-renewal; have high proliferative potential; cycle slowly or rarely in vivo; are stimulated to proliferate in response to injury and certain growth stimuli; and are usually found in well-protected, highly vascularized and innervated areas [2, [3], [4], [5]6]. It is generally known that SCs infrequently divide, yet they can proliferate soon in response to injury and certain growth stimuli, resulting in one SC and one transient amplifying cell (TAC) with limited proliferative potential. TACs are responsible for most routine proliferative activities; they are capable of extensive expansion of the cell population [7, 8]. Upon exhaustion of their proliferative potential, the rapidly proliferating TACs undergo terminal differentiation.

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 [9, [10]11]. 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.

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 β1 integrins [12, 13] and the combination of high levels of α6 integrin and low-to-undetectable levels of the transferring receptor (CD71) [14, 15]. For the further molecular characterization of various keratinocyte SCs, additional cell surface markers are needed.

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 [16], epidermis [17], and especially ocular surface reconstruction [18, [19], [20], [21], [22]23], 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 [24]; its function is to mediate cell survival, apoptosis, and intercellular signaling in neuronal tissues [25, 26]. There is experimental evidence that p75 is also involved in controlling the fate of murine keratinocyte SCs through cell-cell interactions [27] and that it characterizes esophageal keratinocyte SCs in vitro [28]. However, its function in various human keratinocyte SCs remains to be fully elucidated.

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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Tissues

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 1–2 hours of harvest.

Cell Culture

For culture of the human oral epithelial cells, we used our previously reported system [18, [19]20]. 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°C, 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.

Clonal Analysis

For clonal analysis, we applied the method of Barrandon and Green [9]. Secondary cultures of oral epithelial cells were used. Briefly stated, single cells, isolated under an inverted microscope, were inoculated into 12-well plates that contained a feeder layer of mitomycin C (MMC)-inactivated NIH-3T3 fibroblasts. After 7 days, a single clone was identified under an inverted microscope and photographed. Each clone was then divided into three parts; 5/8 of the clone was used for real-time polymerase chain reaction (PCR) assay, and 1/8 was subjected to immunocytochemical analysis to evaluate the cell characteristics. The other 1/4 of the clone was transferred to an indicator dish, fixed 10–12 days later, and stained with 0.1% toluidine blue for classification of the clonal type, which was determined by the percentage of aborted colonies [9]. When 0%–5% 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 <100% of the colonies were terminal, the clone was classified as a meroclone.

Antibodies and Reagents

The following mouse monoclonal antibodies (mAbs) were used: anti-p75 (dilution, ×200) (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 (×1) (Progen, Heidelberg, Germany, http://www.progen.de), anti-keratin 4 (×200)/10 (×50)/13 (×200) (Novocastra Ltd., Newcastle upon Tyne, U.K., http://www.novocastra.co.uk), anti-integrin α6 (×200)/β4 (×500)/α3 (×50)/β1 (×500) (Chemicon), CD34 (×50) (BD Biosciences, San Diego, http://www.bdbiosciences.com), CD71 (×50) (YLEM, Roma, Italy, http://www.ylem.it), BCRP (×10) (Kamiya Biomedical, Seattle, http://kamiyabiomedical.com), and anti-Ki67 (×100) (Dako, Kyoto, Japan, http://www.dako.com). Rabbit polyclonal antibodies were also used: anti-ZO1 (×25) (Zymed, South San Francisco, CA, http://www.invitrogen.com) and anti-Ki67 (×50) (Abcam). Secondary antibodies were Alexa Fluor-488 goat anti-mouse or rabbit IgG (×1,500) and Alexa Fluor-594 goat anti-mouse or rabbit IgG (×1,500) (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).

Cell Fractionation

The p75(+) and p75(−) 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 (×400) at 0°C for 15 minutes. The cells were then carefully washed with MACS buffer (phosphate-buffered saline [PBS] supplemented with 2 mM EDTA and 0.5% bovine serum albumin), incubated with rat anti-mouse IgG1 microbeads (×5) at 4°C 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(−) cell fraction was again washed with MACS buffer and stained with Alexa Fluor-488 goat anti-mouse IgG (×1,500) at 4°C 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).

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 [28]. The expected product sizes were 230 base pairs (bp) for p75 and 541 bp for β-actin.

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 ×10 phase objective [29]. Cell areas were measured randomly (200 cells per fraction) using Scion Image software (Scion Corp., Frederick, MD, http://www.scioncorp.com/) and statistically analyzed.

5-Bromo-2′-deoxyuridine Cell Proliferation Assay

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 [20, 30]. Analysis was on the 6th day of passage (n = 3). Cultured cells were incubated with 10 μM BrdU-labeling solution (20 hours, 37°C), washed with 250 μl of PBS containing 10% serum per well, fixed with 70% ethanol in hydrochloric acid (30 minutes, −20°C), 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 × 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.

Colony-Forming Efficiency

The clonal growth ability of each isolated cell fraction was determined by colony-forming efficiency (CFE) assay. Cells (2 × 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.

Ex Vivo Expansion of Isolated Cells

To culture isolated cell fractions we used our previously reported system [18, [19]20]. Isolated cells (1 × 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 × 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).

Immunohistochemistry

Immunohistochemical studies followed our previously described method [18, 20]. 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).

Real-Time PCR

Quantitative real-time PCR for p75, integrin β1, integrin α6, 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 β1, integrin α6, CD71, ABCG2, and β-actin were from Applied Biosystems. For relative quantification, we used the ΔCT method (Applied Biosystems). Analyses were performed in a sequence detector (ABI Prism 7000) using the accompanying data analysis software.

Apoptosis Assay

The p75(+) cells were seeded at a density of 2 × 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) [31]. The cells were directly stained by the In Situ Apoptosis Detection Kit (terminal deoxynucleotidyl transferase dUTP nick-end labeling [TUNEL] assay; Takara).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

In Vivo Expression Patterns and Cell-Cycling Status of p75(+) Human Oral Keratinocytes

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 ± 3.41 (five different fields from six different donors [total, 30 sections] were analyzed).

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Figure Figure 1.. Immunolocalization of p75 and p75-Ki67 in human oral mucosal tissues. (A–C): 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–F): 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(−) and Ki67(+) cells among all p75(+) cells were 96.63% ± 2.69% and 3.37% ± 2.68%, respectively (F). The difference was statistically significant (∗, p < .001, t test). Asterisks and arrows indicate clusters of p75(+) and Ki67(+) cells, respectively. Arrowheads indicate p75(+) Ki67(+) cells. Scale bars = 100 μm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; PI, propidium iodide.

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Slow or infrequent cycling is one of the unique features of adult SCs [32, [33], [34], [35], [36]37]. 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 (5–10 sections each from four different donors) showed that among the p75(+) cells, the proportion of Ki67(−) cells was significantly higher (96.63% ± 2.69%) than that of Ki67(+) cells (3.37% ± 2.68%) (∗, p < .001, t test), indicating that in vivo, most p75(+) cells were not actively cycling cells (Fig. 1F).

Characteristics of p75(+) and p75(−) Cell Fractions

To examine the characteristics of the p75(+) and p75(−) 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).

As the highest clonogenicity, a feature of SCs, is reportedly found in the smallest keratinocytes [29], we measured the cell size in each isolated fraction using Scion Image software. Under an inverted microscope, p75(+) cells were clearly smaller than p75(−) cells (Fig. 2A). The average size of p75(+) cells was significantly smaller than that of p75(−) cells (102.52 ± 46.85 vs. 346.84 ± 134.67 μm2; ∗, p < .001, t test) (Fig. 2A).

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Figure Figure 2.. Characteristics of p75(+) and p75(−) cell fractions. (A): Under an inverted microscope, p75(+) cells are clearly smaller than p75(−) cells. The average cell size of the p75(+) cell fraction was 102.52 ± 46.85 μm2, significantly smaller than the 346.84 ± 134.67 μm2 of the p75(−) cell fraction (∗, p < .001, t test). Scale bars = 100 μm. (B): Phase-contrast of isolated cells on the 6th day of passage. p75(+) cells formed colonies with ovoid and round cells; p75(−) cells did not form colonies and presented as large elongated cells. 5-Bromo-2′-deoxyuridine enzyme-linked immunosorbent assay showed that the proliferation indexes of p75(+) and p75(−) cell fractions was 9.64 ± 0.1 and 1.16 ± 0.09, respectively. The difference was statistically significant (∗, p < .001, t test). (C): The clonal growth ability of isolated cell fractions was determined by CFE assay. Cells (2 × 103) were plated on six-well culture dishes containing a feeder layer of mitomycin C-inactivated NIH-3T3 fibroblasts. Colonies were fixed on day 7 and stained with 0.1% truidine blue. The CFEs of p75(+) and p75(−) cells were 10.1% ± 1.2% and 0.19% ± 0.07%, respectively, and the difference was statistically significant (∗, p < .001, t test). (D): On the amniotic membrane (AM) substrate, p75(+) oral epithelial cells formed four to five layers. Note the well-conserved columnar basal cells and the progressive flattening toward the surface. Cultivated p75(−) cells grew in monolayers and consisted of elongated differentiated cells. Of the total basal cells in p75(+) and p75(−) sheets, 81.3% ± 10.6% and 25.4% ± 9.9%, respectively, were Ki67-labeled. The difference in the Ki67-labeling index in the isolated cell fractions was statistically significant (∗, p < .001, t test). Scale bars = 100 μm. Abbreviations: CFE, colony-forming efficiency; PI, propidium iodide.

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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(−) cells, which did not form colonies, were large and elongated (Fig. 2B). The proliferation indexes of p75(+) and p75(−) cell fractions were significantly different (9.64 ± 0.1 vs. 1.16 ± 0.09; ∗, p < .001, t test) (Fig. 2B), as was the CFE (10.1% ± 1.2% vs. 0.19% ± 0.07%; ∗, p < .001, t test) (Fig. 2C), indicating that the p75(+) fraction manifested greater in vitro proliferative capacity than the p75(−) fraction.

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 4–5 layers exhibiting well-conserved columnar basal cells and progressive flattening toward the surface (p75(+) sheet). On the other hand, p75(−) 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(−) sheets, Ki67-labeled cells constituted 81.3% ± 10.6% and 25.4% ± 9.9%, respectively (Fig. 2D), rendering the difference in the Ki67-labeling index statistically significant (∗, p < .001, t test).

Using immunofluorescence and specific markers, we studied the morphological and biological characteristics of the p75(+) and p75(−) sheets. ZO-1, a tight junction-related component, was expressed in the apical surface on p75(+) but not p75(−) 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(−) 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(−) sheets, on the other hand, there was no or faint superficial staining (Fig. 3F, 3H). The basement membrane assembly proteins integrin α6/β4 showed linear positive staining on the basement membrane side on both p75(+) and p75(−) sheets (Fig. 3I–3L). Integrin α3 was mainly expressed in the basal cell membrane on both p75(+) and p75(−) sheets (Fig. 3M, 3N), and integrin β1 was expressed in the cell membrane of nearly all epithelial cells on p75(+) sheets; moderate or faint integrin β1 staining was observed on p75(−) 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 Figure 3.. Representative immunofluorescence micrographs of p75(+) and p75(−) oral epithelial cells grown on amniotic membrane. ZO-1 was expressed on the apical surface of the p75(+) sheet but not the p75(−) sheet (A, B). Desmoplakin was clearly expressed in the cell membrane of the p75(+) sheet and moderately expressed in the p75(−) 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(−) sheet, there was no or faint superficial staining (F, H). The basement membrane assembly proteins integrin α6/β4 showed linear positive staining on the basement membrane side of both p75(+) and p75(−) sheets (I–L). In both p75(+) and p75(−) sheets, integrin α3 was mainly expressed in the basal cell membrane (M, N). Integrin β1 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(−) sheet (O, P). Scale bars = 100 μm.

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Isolation and Clonal Analysis of Oral Keratinocyte SCs

To determine whether holoclones, meroclones, and paraclones, previously identified in human epithelium [9, [10]11, 38], are also present in human oral mucosal epithelium, we isolated single cells from eight different primary cultures obtained from eight different donors (four buccal mucosa, four gingiva). We analyzed 396 clones; of these, 56.9% ± 22.8% formed original clones (Table 1). Photographs of representative holoclones, meroclones, and paraclones are shown in Figure 4. According to Barrandon and Green [9] and Pellegrini et al. [11], 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% ± 12.5% were holoclones, 34.9% ± 10.5% were meroclones, and 41.5% ± 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.

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Figure 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 β1 was expressed in the cell membrane of all three clonal types (J–L). Scale bars = 100 μm.

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Table Table 1.. Classification of clonal types
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To investigate the expression of p75 and integrin β1 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 β1 was expressed in all three clonal types (Fig. 4J–4L). 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 < .05, Mann-Whitney U test); all three clonal type cells exhibited integrin β1 mRNA expression. These findings showed that p75 was strongly expressed in holoclone-type cells, suggesting p75 as a potential marker for oral keratinocyte SC-containing populations.

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 < .01, t test). From our immunohistochemical results, there were no differences regarding the effect of neurotrophins on cell differentiation. All p75(+) cells incubated with neurotrophins showed the expression of mucosal-specific keratin 13 but not keratinization-specific keratin 10 (supplemental online Fig. 3). TUNEL assay indicated that neurotrophins other than NGF fail to protect p75(+) oral keratinocytes from UV-induced apoptosis (Fig. 6). Our in vitro findings indicate that some neurotrophin/p75 signaling affects cell growth and survival but not cell differentiation.

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Figure 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 < .01, t test). Abbreviations: BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor; NT, neurotrophin.

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Figure 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 < .01, t test); however, the other neurotrophins do not prevent cell death (B). Abbreviations: BDNF, brain-derived neurotrophic factor; NGF, nerve growth factor; NT, neurotrophin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

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(−) 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.

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 [12, 13] reported that SC distribution is nonrandom but varies by the specific phenotype of epithelial tissue. They found that cells brightly stained for integrin β1, 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.

These cells, as was the case in our p75(+) cells, existed as clusters in tissue, rather than as sporadic single cells. Lavker and Sun [4, 7] studied the morphological and functional characteristics of epidermal SC located at different sites. Keratinocytes at the bottom of the rete ridges in foreskin and interfollicular scalp are protected, pigmented, and ultrastructurally primitive. They express less integrin β1 than basal cells in the shallowest portion (tips of the papillae) of these areas of the epidermis. However, the latter tend to be more specialized ultrastructurally and are more vulnerable to environmental insults than basal cells located in the deep rete ridges. Our in vivo and in vitro findings indicate that p75 is a potential marker of oral keratinocytes in both stem and progenitor cells, so regarding the p75 expression pattern, we do not consider that there are two stem cell niches, but rather either the deep or shallow portion of the rete ridges are stem cell-containing compartments. We posit that from the recent reports using a cell surface marker for Dsg3 [39] and K15 [40], 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.

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 β1 [12, 41], integrin α6/CD71 [14, 15], and side population (SP) cells [42, [43], [44]45]. We also investigated the expression of CD34, a mouse bulge cell marker, and found that like previous reports on human bulge cells [46, [47]48], it was not expressed in any layer of human oral mucosal epithelium (data not shown). To elucidate the relationship between p75 and integrin β1, integrin α6/CD71, and SP, we investigated their expression level in all isolated p75(+) and p75(−) 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 β1 and ABCG2 mRNA in the p75(+) cell fraction was higher than that of the p75(−) cell fraction (supplemental online Fig. 4A, 4B; ∗, p < .01). Although there was some tendency toward a decreased CD71 mRNA level in p75(+) cell fractions, the level of both integrin α6 and CD71 mRNA was not statistically significant (supplemental online Fig. 4C, 4D). Immunohistochemical analysis showed that integrin β1 was expressed in their basal and suprabasal cell layers (supplemental online Fig. 5B). The expression of p75 was observed in the restricted region of the basal cell layer (supplemental online Fig. 5A, 5C, 5F, ∗). Interestingly, immunohistochemistry showed that CD 71 was mainly expressed in the basal and suprabasal cells of human oral mucosal epithelium, and negative or low levels of CD71 cells were sporadically observed in the deep rete ridges (supplemental online Fig. 5D, ∗). Furthermore, BCRP1 was observed in the restricted region of the basal cells of human oral mucosal epithelium (supplemental online Fig. 5G, ∗). Based on these findings, the expression pattern of the proposed epidermal SC markers was partially overlapped but somewhat different from that of p75, suggesting that p75(+) oral keratinocytes may have different features from the proposed epidermal SC markers. However, these results alone do not allow us to fully explain the differences between p75 and proposed epidermal keratinocyte SC markers, and additional studies are under way in our laboratory to determine the precise character of p75(+) human oral keratinocytes.

Under steady-state conditions, SCs divide infrequently in vivo [49, 50]. 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.

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 [51, 52]. These keratinocytes also exhibited the highest clonogenicity; cultured keratinocytes isolated from human epidermis formed clones when they were 11 μm or less in diameter, and they terminally differentiated when they were 12 μm or more in diameter [29]. The average diameter of our p75(+) cells was 11.4 ± 3.8 μm, which is consistent with the report of Barrandon and Green [29]. Based on these findings and on the reported size of corneal epithelial SCs [53], we suggest that cell size is a potential indicator of keratinocyte SCs, not only of the epidermis, but also of other epithelial cell types.

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 [28]. However, SC-rich fractions from adult rat epidermis [54] and neonatal human foreskin [55] manifested substantially lower CFE than the source populations. Moreover, in standard tissue cultures, exhaustion of the proliferative potential of keratinocytes can be slowed by amending the culture conditions [56]. These discrepancies may be related to species differences (human and rodent), the cell isolation techniques used, and the biological state of the keratinocyte SCs in culture. Although the cell-sorting procedure using specific cell-surface markers is useful [12, 55], the isolated cells are released from the control mechanisms regulating the in vivo SC niche that renders them slow-cycling.

Although cultured human keratinocytes may not retain all their in vivo characteristics after removal from their microenvironment, they are clonogeneic [12, 13, 49] and able to generate three distinct clonal forms due to the varied self-renewal capacities of individual keratinocytes [9]. 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 β1, an epidermal SC marker, was expressed in all three clonal-type cells. According to De Luca et al. [57] and Nguyen et al. [58], keratinocyte SCs completely disappeared in most mitogenic tissue culture environments and exhibited marked changes in their expression of integrin and other markers. Cell-surface receptor molecules are particularly useful for isolating SC compartments; however, reliance on cell adhesion molecules alone may be inadequate for SC isolation because almost all basal keratinocytes express these molecules, which are essential for biological processes, to various degrees [14, 59, [60]61]. Therefore, markers specific for in vivo keratinocyte SCs may differ from those of keratinocyte colonies grown in vitro.

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 [62, [63]64]. 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 [65, 66]. The elucidation of the role of neurotrophin/p75 signaling in oral keratinocyte stem cells is important for better understanding the mechanism of stem cell behavior. In our series, one of the representative neurotrophin molecules, “NGF,” can support keratinocyte cell proliferation and protect the cells from apoptosis. These results were consistent with the previous reports using other cell types [31, 66, [67], [68], [69], [70], [71]72]. We also pointed out that effects of neurotrophins are mediated via the activation of two different classes of plasma membrane receptor, high-affinity Trk tyrosine kinase receptors (TrkA, TrkB, and TrkC) and low-affinity p75 neurotrophin receptors that form complexes. For example, p75 modulates cell death in different models [73, [74]75]. Proapoptotic signaling occurs when p75 is expressed alone without the coexpression of Trk receptors [76, 77]. 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 [37, 78] and human [46] 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.

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

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.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supplemental_Figure_1.pdf153KSupplemental Figure 1
Supplemental_Figure_2.pdf20KSupplemental Figure 2
Supplemental_Figure_3.pdf95KSupplemental Figure 3
Supplemental_Figure_4.pdf20KSupplemental Figure 4
Supplemental_Figure_5.pdf256KSupplemental Figure 5
Suppplemental_Legends.pdf76KSupplemental Legends

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