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J Dent Res 86(4):341-346, 2007
© 2007 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Isolation of Human Oral Keratinocyte Progenitor/Stem Cells

K. Izumi, T. Tobita, and S.E. Feinberg*

Department of Oral and Maxillofacial Surgery, University of Michigan Health System, B1-208 TC, Box 0018, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0018, USA.

* corresponding author, sefein{at}med.umich.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Progenitor/stem cell populations of epithelium are known to reside in the small-sized cell population. Our objective was to physically isolate and characterize an oral keratinocyte-enriched population of small-sized progenitor/stem cells. Primary human oral mucosal keratinocytes cultured in a chemically defined serum-free culture system, devoid of animal-derived feeder cells, were sorted by relative cell size and characterized by immunolabeling for ß1 integrin, nuclear transcription factor, peroxisome proliferator-activated receptor-gamma, and cell-cycle analysis. Sorted cells were distinguished as progenitor/stem cells by functional assays and their ability to regenerate an oral mucosal graft. Small-sized cells demonstrated the lowest expression of peroxisome proliferator-activated receptor-gamma, the highest colony-forming efficiency, a longer long-term proliferative potential, an enriched quiescent cell population, and the ability to regenerate an oral mucosal graft, implying that the small-sized cultured oral keratinocytes contained an enriched population of progenitor/stem cells.

KEY WORDS: oral mucosa • keratinocyte • progenitor cell • stem cell • isolation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The identification and isolation of epidermal stem cells have been goals in regenerative medicine. An analysis of growth potential of primary human neonatal foreskin keratinocytes and their serially cultured cells showed that cells smaller than 11 and 20 µm, respectively, were the most clonogenic and had the greatest colony-forming ability (Barrandon and Green, 1985). Others noted, both in vivo and in vitro, that these smaller skin cells showed progenitor/stem cell characteristics (Kim et al., 2004; Li et al., 2004; Webb et al., 2004; Youn et al., 2004). In addition, the size of cultured oral keratinocytes increased as their proliferative potential decreased—a sign of cellular differentiation and/or senescence (Kang et al., 2000), indicating that smaller cultured human oral keratinocytes may harbor the progenitor/stem cell population.

The objective of this study was to physically isolate and characterize the small-sized cultured oral keratinocyte population for the presence of a progenitor/stem-cell-enriched population that could be used to fabricate a human ex vivo-produced oral mucosa equivalent (EVPOME) suitable for intra-oral grafting, consistent with the US Food and Drug Administration’s (FDA) guidelines for cell-based therapy/combinational products (http://www.fda.gov/oc/combination/OCLove1dft.html). The isolated oral mucosal progenitor/stem-cell-enriched subpopulation was characterized by functional assays and evaluated for the presence or absence of peroxisome proliferator-activated receptor-gamma (PPAR{gamma}), which has been shown to be a nuclear transcription factor involved in epithelial differentiation (Westergaard et al., 2001).


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Procurement of Human Oral Mucosa
Keratinized oral mucosa was obtained from persons undergoing tooth extraction and/or minor dento-alveolar surgery. The protocol for harvesting human oral mucosal tissue was approved by a University of Michigan Internal Review Board. All individuals signed informed consent before the tissue samples were procured.

Primary Oral Keratinocytes and Serial Cultures
Briefly, mucosal tissue was digested overnight with 0.04% trypsin solution at room temperature, and transferred into 0.0125% trypsin-inhibitor. Dissociated oral keratinocytes were re-suspended in a chemically defined culture system and seeded into one T-25 flask. For serial cultures, cells were detached in 0.025% trypsin/EDTA (additional details in Appendix 1).

Immunohistochemistry
Non-cultured oral mucosal tissue and a day 11 EVPOME (D11E) were fixed in 10% neutral formalin. Paraffin-embedded specimens were cut into 5-µm sections. Unsorted oral keratinocytes (passage 2) were utilized to fabricate D11E as previously described (Izumi et al., 2003). Briefly, cells were re-suspended in EpiLife® supplemented with Epilife defined growth supplement (EDGS) (Cascade Biologics, Portland, OR) and 1.2 mM calcium, and 1.25 x 105 cells/cm2 were seeded onto the basement membrane side of AlloDerm® (LifeCell Corp., Branchburgh, NJ, USA), pre-soaked with human type IV collagen (5 µg/cm2) (Sigma-Aldrich, St. Louis, MO, USA). The composite of oral keratinocytes and AlloDerm® was cultured submerged for 4 days, then raised to an air-liquid interface for an additional 7 days. Immunostaining was performed as described previously for both ß1 integrin (clone K-20, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and PPAR{gamma} (clone E-8, 1:100) (Izumi et al., 2000). For ß1 integrin, the antigen retrieval was carried out in a 1 mM EDTA (Sigma) solution, at pH 1.5-2.0. Human skin and AlloDerm® were used as positive and negative controls, respectively.

Pre-fluorescence-activated Cell-sorting (FACS) Immunolabeling for ß1 Integrin
Oral keratinocytes from serial cultures were re-suspended in staining buffer [1% bovine serum albumin (BSA), Fisher Biotech, Fair Lawn, NJ, USA] and 0.1% NaN3 in HBSS (Cambrex BioScience, Walkersville, MD, USA) (106 cells/mL). Procedures could not always be performed with the same cell population because of the small sizes of the tissue samples. Cells were incubated with anti-ß1 integrin antibody for 30 min on ice, followed by incubation with 5 µL/100 µL of R-Phycoerythrin (RPE)-conjugated goat anti-mouse IgG2a (Caltag Laboratories, Burlingame, CA, USA) for 30 min. Isotype-matched normal mouse IgG2a, RPE-conjugated, was used as the negative control for ß-integrin (Santa Cruz Biotechnology). After samples were washed thoroughly, propidium iodide (PI) (50 µg/mL, Sigma) was added, and samples were stored at 4°C until subsequent cell-sorting by FACS.

Cell-sorting by FACS
Cells were sorted into 3 groups on the basis of "relative" forward scatter (FSC) by means of a FACSVantage SE (Becton Dickinson Inc., San Jose, CA, USA). From a scatter plot, 3 gates were set to sort cells in equal proportions (approximately 25% each) after PI-positive cells (19.1 ± 11.7%) and debris were eliminated. Meanwhile, RPE fluorescence was analyzed. Sorted-cell groups are referred to as "Large", "Medium", and "Small". Mean FSC values of each sorted cell group and 3 sizes of micro-beads [15.41 and 21.14 (Bangs Laboratories, Inc., Fishers, IN, USA) and 29.5 µm (Polysciences, Inc., Warrington, PA)] were obtained at every cell-sorting for calibration. We made a linear equation line (R2 > 0.99) by plotting the FSC and diameter of each bead to estimate average cell diameter in each group.

Post-FACS Analysis
Sorted, retrieved cells, not used for subsequent functional assays, were fixed and permeabilized with 70% ice-cold ethanol and stored at -4°C for PPAR{gamma} immunolabeling (N = 20) and cell-cycle analysis (N = 17). We then determined the percentages of PPAR{gamma}-positive cells based on the background control (normal mouse IgG1, FITC-conjugated, Santa Cruz Biotechnology), and the DNA content of each sorted cell group (additional details in Appendix 2).

Functional Assays
    Colony-forming Efficiency (CFE)
We plated 5.0 x 103 sorted, retrieved cells, from passages 3 to 8, into a six-well plate (Costar, Corning, NY, USA), cultured them another 7 days in EpiLife® with EDGS that was changed 4 days post-plating. Cells were fixed for 10 min with methanol and stained with 2% crystal violet (Baker Chemical, Phillipsburg, NJ, USA). Under the microscope, colonies consisting of 16-49 cells and greater than 50 cells were counted separately. This assay was performed 6 times.

    Long-term Growth Potential
We plated 2.0 x 104 sorted, retrieved cells, from passages 4 to 6, onto a 60-mm culture dish with 2-mm grids, pre-coated with type IV collagen (100 µg/mL). They were allowed to attach for 20 min, and non-adherent cells were removed. Adherent cells were subcultured when the diameter of one of the cell colonies reached 10 mm. Harvested cells were seeded onto another culture vessel at a density of 5.0 x 103 cells/cm2. When cell density reached 70-80% confluence, they were serially passaged (up to 1.5 x 106 cells seeded) until cells lost their proliferative capacity. The assumptive total cell output, days of culture after plating, and cumulative population doublings were determined. Since up to 1.5 x 106 cells were re-plated at each passage, the cell outputs were calculated based on the assumption that all the cells from the previous passage had been re-plated (additional details in Appendix 3).

Fabrication of EVPOME
We used retrieved, sorted oral keratinocytes from passage 8 to determine their ability to generate a D11E.

Statistical Analysis
Data were assessed by either repeated-measures analysis of variance (ANOVA) adjusted with Tukey’s adjustment or by a regression analysis followed by a linear mixed model. Poisson’s regression analysis, in place of repeated-measures ANOVA, was used for data not showing normal distribution.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Study Population of Keratinized Oral Mucosal Samples
Samples were taken from nine males and eight females ranging in age from 11 to 74 yrs, with a mean age of 38.6 yrs. Of the 17 samples, 9 were analyzed at 2 different passages, for a total sample size of 26. Passages of each sample ranged from 1 to 8, with an average of 4.1 ± 1.8. Days in culture ranged from 14 to 42, with an average of 26.6 ± 7.3 days. Numbers of passage and total days in culture varied among samples.

ß1 Integrin, PPAR{gamma} Expression in Native Oral Mucosal Tissue and D11E
ß1 integrin expression was confined to the basal layer (Fig. 1AGo). PPAR{gamma} expression was seen in the suprabasal layers but was absent in the basal layer (Fig. 1BGo). The PPAR{gamma} activity was mainly located in the cytoplasm, with minimal nuclear staining. The D11E showed an expression pattern similar to that seen in native oral mucosa (Figs. 1C, 1DGo).


Figure 1
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  		Figure 1. Immunohistochemical staining of non-cultured oral mucosa. (A) ß1 integrin, (B) PPAR{gamma}. ß1 integrin staining was more intense in the basal layer and a few layers above the basal layer at the tops of the ridges than seen at the bases of the ridges. The differential PPAR{gamma} expression was more distinct in the basal layer at the tops of the ridges. D11 EVPOME: (C) ß1 integrin, (D) PPAR{gamma}. D11 EVPOME showed an expression pattern similar to that seen in native oral mucosa.

 
Profiles in Size-sorted, Cultured Oral Keratinocytes
Cultured oral keratinocytes demonstrated a direct increase in cell size and granularity with increases in cell passage, based on the increasing forward and side scatters (Fig. 2AGo). The mean cell diameters of 18 sorted samples were 61.0 ± 2.7 µm, 46.3 ± 1.7 µm, and 33.9 ± 0.9 µm for Large, Medium, and Small cells, respectively. All cell-size groups showed a direct increase in percentage of PPAR{gamma}-positive cells with an increase in passage number (Fig. 2BGo).


Figure 2
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  		Figure 2. Characteristics of different cell sizes as determined by FACS analysis. (A) Representative FACS dot plot of cultured oral keratinocytes at passages 2 and 4. Shown are the 3 gates (Small, Medium, Large) for sorting cells by relative cell size in diameters in equal proportions (approximately 25%) (left panels). The subpopulation indicated by a black arrow in the dot diagrams was debris eliminated by the gate in the side-scatter vs. forward-scatter, as well as PI-fluorescence before cell-sorting. (right panels) ß1 integrin expression at passage 2 in each size of sorted cells. ß1 integrin was expressed 99.4 ± 0.6% in Large cells, 99.5 ± 0.4% in Medium cells, and 99.4 ± 0.5% in Small cells. (B) Representative PPAR{gamma} expression at passages 4 (72.8% in Large cells, 57.3% in Medium cells, 44.0% in Small cells) and 6 (84.2% in Large cells, 83.9% in Medium cells, 49.5% in Small cells) in each size of sorted cells. Among the cell size groups, the different proportion of PPAR{gamma}-positive cells was statistically significant (79.9 ± 25.5% in Large cells, 57.6 ± 34.7% in Medium cells, 37.4 ± 32.6% in Small cells) (overall p < 0.0001). Dotted line drawn at the top 3% of the total population of background control indicates the borderline showing positive FITC fluorescence when compared with background control. Note shift of the histograms to the right as the passage number increases, indicating that a larger percentage of cells is PPAR{gamma}-positive. (C) Linear regression analysis with a linear mixed model, showing relationship between proportion cells in S + G2M phases and days in culture in each size group. Passage numbers ranged from 1 to 8. The longer Small cells were in culture, the more cycling cells increased. In contrast, the longer Medium and Large cells were in culture, the more non-cycling cells increased. (D) Linear regression analysis with a linear mixed model, showing relationship between proportions of cells in S + G2M phases and cell diameters in each size group. Passage numbers ranged from 1 to 8. Within Small cells, the smaller they were, the more quiescent. In contrast, within Medium and Large cells, the smaller they were, the more cycling occurred.

 
The results obtained from cell-cycle analyses of the proportions of cells in S and G2M phases for each cell-size group demonstrated that a significant relationship existed between "days in culture" and "cell diameter" (Figs. 2C, 2DGo). Only the Small cells showed a positive correlation of percentage of cells in S and G2M phases with the number of days in culture, with an increase in cell diameter indicating that the longer they were in culture, the more proliferative they became.

Functional Assays
The results showed a highly significant capacity of the Small cells to give rise to larger colonies (Fig. 3AGo), and to have a longer-term proliferative potential than either the Medium or Large cells (Figs. 3B, 3CGo). Small cells showed a more enhanced replicative lifespan than did larger cells, although all samples used in the long-term growth potential assay had a limited replicative lifespan (Fig. 3DGo).


Figure 3
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  		Figure 3. Progenitor/stem cell functional assays. (A) Colony-forming efficiency assay. Left bars show colonies consisting of 16-49 cells. Right bars show colonies consisting of greater than 50 cells. N = 10; Passage 3 (N = 1), Passage 4 (N = 5), Passage 6 (N = 3), Passage 8 (N = 1). Higher colony efficiency of both size colonies is seen with the Small cells. (B) Long-term growth potential. (1) Assumptive total output of cells after cell-sorting (N = 7). Type IV collagen-attached cells out of the 2.0 x 104 sorted Small cell subpopulation yielded cells up to 5.0 x 108 folds, and were significantly more productive. Out of 7, 3 failed further propagation in Medium and Large cells. (C) Long-term growth potential. (2) Days in culture after cell-sorting (N = 7). Large, 26.4 ± 26.6; Medium, 40.1 ± 49.7; Small, 121.1 ± 43.9 days (Mean ± SD). Small cell subpopulations significantly survived longer than Medium and Large cell subpopulations in vitro. (D) Long-term growth potential. (3) Cumulative population doublings after cell-sorting (N = 7). Small cells had an enhanced replicate lifespan. In contrast, the lifespan of Medium and Large cells diminished earlier than that of Small cells. In B, C, and D, each number indicated a different person’s sample.

 
Fabrication of D11E
The Large cells could not form an epithelial monolayer, and the Medium cells formed only a poorly organized epithelial layer, while only the Small cells were capable of regenerating a highly stratified and well-organized epithelial layer on the AlloDerm® (Figs. 4A, 4B, 4CGo).


Figure 4
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  		Figure 4. EVPOME fabricated by size-sorted cells from unsorted passage 8 (hematoxylin-eosin staining). (A) Large cells show no evidence of an intact epithelial cell layer. (B) Medium cells show a thin monolayer of cells. (C) Small cells are the only ones that show an intact stratified epithelial layer of cells similar to D, an EVPOME produced by unsorted passage 2 cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The colony-forming ability, in vivo and in vitro, of foreskin keratinocytes decreased as they enlarged, and ceased when they reached a specific cell diameter (Barrandon and Green, 1985). Sorting non-cultured keratinocytes by cell size to enrich for a stem cell subpopulation has been used for rodent skin (Pavlovitch et al., 1991; Dunnwald et al., 2003; Zhou et al., 2004) and human corneal keratinocytes (Romano et al., 2003; De Paiva et al., 2006). It appears that physical separation based on cell size may be a practical way of enriching for a progenitor/stem cell subpopulation from cultured oral keratinocytes. Nakashima et al.(2004) suggested that the best way of manipulating cells for clinical use would be either by physical or pharmacologic means, since these modalities would be consistent with FDA regulatory guidelines (Guidance for industry and FDA current good manufacturing practice for combination products, http://www.fda.gov/oc/combination/OCLove1dft.html, Sept, 2004).

The pattern of ß1 integrin expression of non-cultured oral mucosa was slightly different between the top and base of the ridges. This is consistent with previous reports for interfollicular epidermis and esophageal epithelium (Watt and Hertle, 1994; Seery and Watt, 2000), implying that ß1 integrin expression may be associated with oral keratinocyte proliferative capacity (Morasso and Tomic-Canic, 2005). This correlated with the expression pattern seen in both EVPOME and cultured oral keratinocytes.

This study is the first to show PPAR{gamma} expression in normal oral mucosa, cultured oral keratinocytes, and an EVPOME. The number of cells, in vitro and in vivo, expressing cytoplasmic PPAR{gamma} increased as the sizes of the keratinocytes increased, correlating with a more differentiated or senescent cell. In normal skin, PPAR{gamma}-immunoreaction was present in the cytoplasm of the basal cells, but was seen in the nuclei of the stratum granulosum (Westergaard et al., 2003). The PPAR{gamma} expression is most likely correlated to keratinocyte differentiation, and the polyunsaturated fatty acids induced PPAR{gamma} translocation from the cytoplasm to the nucleus (Jiang et al., 2000). Essential fatty acid deficiency in our culture system appeared to play a similar role in cytoplasmic PPAR{gamma} expression, because of its correlation with cell differentiation. The ability to modify PPAR{gamma} expression through pharmacological manipulation might be a potential approach to block differentiation of oral keratinocytes in vitro.

The clonogenicity of the Small cells was confirmed by the in vitro functional assays. Small cells were able to develop significantly larger, active colonies than were both the Medium and Large cells, suggesting a growth potential arising from a putative progenitor/stem cell, even though cells capable of generating larger colonies do not necessarily have a long-term proliferative potential (Kaur et al., 2004).

In Small cells, the proportion of cells in S and G2M phases remained lower, since the majority was relatively slow-cycling during their earlier days in culture, then increased as culture time increased. In contrast, during the earlier days in culture, the rapidly proliferating cell population (cells in S and G2M phases) consisted of Medium and Large cells, while the proportion continued to decline over days in culture, implying that Medium and Large cells had reached their post-mitotic stage. A significant relationship was noted between cell diameter and proportion of cells in S and G2M phases, for each cell-size group, indicating that 40 µm may be the size at which cultured oral keratinocytes lose their proliferative potential and enter into irreversible terminal differentiation. This is consistent with the results seen with asymmetrical division (Barrandon and Green, 1985), in which larger and less clonogenic Small cells (up to 40 µm) gave rise to smaller and more clonogenic progeny cells.

The most significant evidence that Small cells contained a progenitor/stem cell-enriched subpopulation was their ability to regenerate a highly stratified and well-organized EVPOME, whereas the Medium and Large cells failed to do so. The regenerative capability, as well as the long-term growth potential, in Small cells was consistent with the recent study in which Kaur et al.(2004) proposed that the assessment of stem cell activity requires the development of long-term assays that measure sustained epithelial tissue regeneration.

Cultured oral keratinocytes showing clone-forming ability in our study were smaller than 40 µm, whereas cultured keratinocytes less than 20 µm in size, from neonatal foreskin, showed clone-forming ability (Barrandon and Green, 1985). Cultured oral keratinocytes in our system showed a higher incidence of high side-scatter and FSC characteristics, compared with the profile seen with adult cultured palm keratinocytes (Wan et al., 2003). This dichotomy in size may, in part, be due to several factors: culture conditions (the presence of a feeder layer and serum), age of donor (newborn vs. adult), anatomical source of cells, and different means of cell-size measurement.

Any cell that comes into contact with foreign or undefined proteins prior to grafting to humans is unacceptable, according to the guidelines of the USFDA or the European Agency for the Evaluation of Medicinal Products (Louët, 2004), although there have been no reports on adverse events caused by foreign materials (Louët, 2004). Thus, growing either human keratinocytes or hematopoietic or embryonic stem cells in a system containing foreign materials would negate their ability to be used in humans for tissue engineering (Amit et al., 2004; Martin et al., 2005). In this study, we successfully isolated a progenitor/stem-cell-enriched population from cultured primary oral mucosal keratinocytes, by physical means, using a protocol that would be acceptable to the FDA for fabrication of an EVPOME that could be use in human clinical trials (Editorial, 2005). A clinical trial using an unsorted, cultured oral keratinocyte population is ongoing in our facility (Feinberg et al., 2005). Isolation of a small cell population would reduce the minimum number of cells necessary to seed, allowing us to fabricate a larger EVPOME with a more prolific cell population.

In conclusion, the ability to physically separate a putative progenitor/stem cell population that possesses the appropriate phenotypic markers, cell-cycle profile, and functional stem cell assays and, most importantly, has the ability to "regenerate itself"—i.e., a tissue-engineered oral mucosa (EVPOME)—will be of considerable value in the fabrication of an engineered human oral mucosa for use in intra-oral reconstructive procedures.


   ACKNOWLEDGMENTS
 
We thank Dr. Cynthia Marcelo for a critical reading of this manuscript, Dr. Kathleen Welch for statistical analysis, and Judi Schmitt for technical assistance. The authors are grateful to David J. Adams, Ann Marie Deslauriers, Karen A. Peterson, and Martin J. White for FACS operation. This study was supported by the National Institute of Dental and Craniofacial Research at the US National Institutes of Health, Grant number DE13417 (S.E.F).

Received February 20, 2006; Last revision November 16, 2006; Accepted November 24, 2006


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 DISCUSSION
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