First published online April 24, 2008
Stem Cells
Vol. 26 No.
7
July 2008, pp.
1743
-1748
doi:10.1634/stemcells.2007-0866; www.StemCells.com
© 2008 AlphaMed Press
TISSUE-SPECIFIC STEM CELLS |
A Novel Isolation Technique of Progenitor Cells in Human Corneal Epithelium Using Non-Tissue Culture Dishes
Seiichi Yokooa,
Satoru Yamagamia,
Takashi Shimadab,c,
Tomohiko Usuid,
Taka-aki Satob,c,
Shiro Amanod,
Makoto Araied,
Junji Hamuroe
Departments of aCorneal Tissue Regeneration and
dOphthalmology, Tokyo University Graduate School of Medicine, Tokyo, Japan;
bLife Science Laboratory, Shimadzu Corporation, Kyoto, Japan;
cDivision of Advanced Clinical Proteomics, Institute of Medical Science, University of Tokyo, Tokyo, Japan;
eDepartment of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan
Key Words. Progenitor • Corneal epithelium • Epidermal growth factor • Laminin-5
Correspondence:
Correspondence: Satoru Yamagami, M.D., Ph.D., Corneal Tissue Regeneration, Tokyo University Graduate School of Medicine, Tokyo, Japan. Telephone: 81-3-5800-8660; Fax: 81-3-5800-9839; e-mail: syamagami-tky{at}umin.ac.jp
Received on October 29, 2007;
accepted for publication on April 14, 2008.
First published online in STEM CELLS EXPRESS April 24, 2008.
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ABSTRACT
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The existence of adult stem cells or progenitor cells in the human corneal epithelium (i.e., self-renewing squamous cells) has long been suggested, but these cells have not yet been isolated. Here we describe a novel isolation technique using non-tissue culture dishes to enrich progenitor cells, which are able to reconstitute a three-dimensional human corneal epithelial equivalent from single cells in serum-, feeder-, and bovine pituitary extract-free medium. These cells showed original tissue-committed differentiation, a high proliferative capacity, and limited self-renewal. Laminin-5 was measured by mass spectrometric analysis. Pretreatment of cells with anti-laminin-5 antibody demonstrated that laminin-5 was important in allowing corneal epithelial progenitor cells to adhere to non-tissue culture dishes. Hydrophilic tubes (used for cell collection throughout this study) are essential for efficient isolation of adherent corneal epithelial progenitor cells expressing laminin-5. These findings indicate that our new technique using non-tissue culture dishes allows the isolation of progenitor cells from human corneal limbal epithelium and that laminin-5 has a critical role in the adhesion of these cells.
Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION
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Adult stem cells or progenitor cells have been isolated from various tissues in humans [1]. Reconstitution of a three-dimensional tissue from a single adult stem cell or progenitor cell, however, has not yet been accomplished for any human tissue, although the functional reconstitution of murine tissues from adult stem cells has been reported [2, 3]. We have investigated the events involved in self-renewal of human corneal progenitor cells by assessing growth at the single-cell level (clonogenicity) and in vitro differentiation potential.
The corneal epithelium is composed of self-renewing squamous cells. It covers the surface of the eye and represents the outer barrier against microbes and harmful substances. This epithelium is maintained by a population of adult stem cells residing in a part of the cornea called the limbus. Cells from the corneal limbus have been amplified ex vivo to repair injuries or genetic defects of the corneal surface with transparent epithelium, thus avoiding corneal keratinization and vascularization with a poor visual outcome [4, 5]. Corneal epithelium can be reconstituted successfully by using autologous or allogenic limbal tissue under circumstances where adult stem cells are deficient, such as in Stevens-Johnson syndrome, ocular pemphigoid, and chemical burns [6, 7]. However, little is known about the limbal adult stem cells or progenitor cells in the human corneal epithelium.
Here we describe a novel technique for the isolation of progenitor cells from the human corneal limbal epithelium by using non-tissue culture dishes for floating culture, which obtains epithelial cells without contamination by fibroblasts or other types of cells. We also identified a critical component of the extracellular matrix that explains the success of this technique, by performing mass spectrometric analysis.
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MATERIALS AND METHODS
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Isolation of Adherent Cells from Human Tissues
This study was conducted in accordance with the Declaration of Helsinki. Donor corneas were obtained from the Rocky Mountain Lions' Eye Bank at 3–5 days after harvesting. The ages of the donors were 32–68 years. The central corneal epithelium, the limbal tissues, including the epithelium and fibroblasts at the sclerocorneal junction, and Tenon's capsule (containing subconjunctival fibroblasts) from the donor corneas were cut into small pieces approximately 2–3 mm in diameter. These tissue pieces were incubated overnight at 37°C in basal culture medium (Dulbecco's modified Eagle's medium-Ham's F-12 medium, 1:1; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) with 20 ng/ml epidermal growth factor (EGF; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 0.02% type IA collagenase (Sigma-Aldrich), B27 (Invitrogen) [8], 100 units/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B. Hydrophilic tubes (Sumilon Stem Full; Sumitomo Bakelite Co., Ltd., Tokyo, http://www.sumibe.co.jp/english), but not normal tubes (BD Falcon 15-ml conical tubes; BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), were used during cell collection to prevent adhesion to the inside of the tube in all experimental procedures, unless otherwise noted. Cells were treated with 0.02% trypsin/ethylenediaminetetraacetic acid (EDTA) for 10 minutes at 37°C and then were dissociated into single cells by pipetting. The viability of the isolated cells was >90% by trypan blue staining (Wako Chemical, Osaka, Japan, http://www.wako-chem.co.jp/english). Cell numbers were determined by using a Coulter counter. After washing in basal medium, completely disaggregated single cells were cultured overnight at a density of 500 cells per cm2 in serum- and feeder cell-free medium containing B27 and 20 ng/ml EGF in uncoated dishes for floating culture (untreated plate: 24 wells or 60 x 15-mm dish; BD Biosciences). To remove the nonadherent cells, uncoated dishes were washed twice with phosphate-buffered saline (PBS) and then were washed with basal culture medium containing B27 [8]. Basal culture medium containing B27 for the third washing was used to protect the surface of non-tissue culture dishes from desiccation, which would have led to cell death during cell processing. Subsequent culture was done in basal medium containing B27 and EGF, which was selected because it has been reported that basal limbal epithelial cells show significant epidermal growth factor receptor (EGFR) expression [9, 10]. The EGFR is a member of a family of receptor tyrosine kinases that are localized to the plasma membrane of cells in the basal layer of stratified squamous epithelium [11]. All non-tissue culture dishes were checked using a phase-contrast microscope after washing, but aggregated cells were not detected in the non-tissue culture dishes. For passage of the cells, adherent colonies derived from single cells were treated with collagenase and detached by incubation with trypsin/EDTA for 1 hour, and dissociated single cells were cultured overnight in basal medium containing B27 and EGF in the non-tissue culture dishes. After washing dishes and checking cell-aggregation on the dishes as described above, culture was done in basal medium containing B27 and EGF. For reconstitution of a three-dimensional corneal epithelial equivalent tissue from a single cell, adherent colonies were detached with EDTA alone and placed in six-well Transwells (Corning Enterprises, Corning, NY, http://www.corning.com).
Immunocytochemistry of Adherent Colonies
Cells were fixed with methanol (Wako Chemical) in PBS for 10 minutes. After washing in PBS, the cells were incubated for 30 minutes with 3% bovine serum albumin (BSA) in PBS containing 0.3% Triton X-100 (BSA/PBST) to block nonspecific staining. Then, the cells were incubated for 2 hours at room temperature with specific primary antibodies (Abs) diluted in BSA/PBST. After further washing in PBS, the cells were incubated for 1 hour at room temperature with the appropriate secondary Abs diluted in BSA/PBST. Nuclei were counterstained with Hoechst 33342 (1:2,000; Invitrogen). After further washing in PBS, examination was done with a laser scanning confocal microscope (Fluoview; Olympus, Tokyo, http://www.olympus-global.com). The following Abs were used: anti-P63 monoclonal antibody (mAb) [12], mouse anti-nestin mAb (1:200; BD Biosciences), anti-microtubule-associated protein 2 (MAP-2) mAb (MAB378; Chemicon, Temecula, CA, http://www.chemicon.com), rabbit anti-β-III tubulin polyclonal antibody (pAb; 1:2,000; Covance, Princeton, NJ, http://www.covance.com), rabbit anti-glial fibrillary acidic protein (GFAP) pAb (1:400; Dako, Glostrup, Denmark, http://www.dako.com), mouse anti-
-smooth muscle actin (SMA) mAb (1:200; Sigma-Aldrich), mouse anti-human cytokeratin (CK)-3 mAb (AE-5, 1:10,000; Progen Biotechnik GmbH, Heidelberg, Germany), mouse anti-human CK-13 mAb (1:20; American Research Products, Inc., Palos Verdes, CA, http://www.arp1.com), mouse anti-human CK-12 mAb (N-16, 1:500; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), anti-laminin-5 mAb (P3E4; Chemicon), and anti-laminin-5 mAb (1:200, P3H9-2; Chemicon). As a control, mouse immunoglobulin G (IgG, 1:1,000; Sigma-Aldrich) or normal rabbit serum (1:1,000; Dako) was used instead of the primary Ab. The secondary Abs used were fluorescein-labeled goat anti-mouse IgG (Alexa Fluor 488, 1:200; Invitrogen) and fluorescein-labeled goat anti-rabbit IgG (Alexa Fluor 594, 1:400; Invitrogen). Some colonies were incubated overnight with 10 µM/ml bromodeoxyuridine (BrdU; Sigma-Aldrich) on day 10. After treatment with 2 M HCl for 1 hour, the cells were stained with fluorescein-labeled anti-BrdU antibody.
Preparation of RNA and Reverse Transcription-Polymerase Chain Reaction
Corneas from donors 45 and 62 years old were used for reverse transcription (RT)-polymerase chain reaction (PCR). Total RNA was isolated with a kit (Isogen; Nippon Gene, Tokyo, http://www.nippongene.com) according to the manufacturer's instructions. First-strand cDNA was synthesized using a Reverse Transcription System (Promega, Madison, WI, http://www.promega.com) and 1 µg of total RNA in a 20-µl reaction mixture. Then PCR was performed with cDNA polymerase (AmpliTaq Gold; Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) in a 50-µl reaction mixture. After incubation at 95°C for 9 minutes, amplification was done at 94°C for 30 seconds and then at 60°C for 30 seconds using a thermal cycler (iCycler; Bio-Rad, Hercules, CA, http://www.bio-rad.com). Products were separated on 2% agarose gel and stained with ethidium bromide. The primer pairs are described in Table 1.
SDS-Polyacrylamide Gel Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 4%–12% gradient gel. After separation on the gel, proteins were visualized by using a SilverQuest silver staining kit (Invitrogen) according to the manufacturer's protocol. Protein bands were excised and were destained with 15 mM potassium ferricyanide (III) and 50 mM sodium thiosulfate for 20 minutes. After washing each piece of gel twice with water, reductive alkylation was performed for 60 minutes at 56°C with 10 mM dithiothreitol in 100 mM ammonium bicarbonate and for 45 minutes at room temperature with 55 mM iodoacetamide in 100 mM ammonium bicarbonate. After washing off any remaining reagents, the gel pieces were dehydrated with acetonitrile and dried in a centrifugal evaporator. Each dried gel piece was rehydrated with 10 µg/ml modified trypsin solution (Promega) in 50 mM ammonium bicarbonate and 5 mM calcium chloride and then was subjected to in-gel digestion for 16 hours at 37°C. Subsequently, the digested peptides were extracted with 20 mM ammonium bicarbonate and 5% formic acid in 50% acetonitrile, and the extract was dried in an evaporator. Peptides were purified with a ZipTip uC18 micro reverse-phase octadecysilica tip (Millipore, Billerica, MA, http://www.millipore.com) according the manufacturer's protocol and eluted directly onto a target plate. After spotting on matrix solution (10 mg/ml -cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% trifluoroacetic acid), matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was performed with an AXIMA-CFR plus mass spectrometer (Shimadzu Corp., Kyoto, Japan, http://www.shimadzu.com). For identification of proteins by peptide mass fingerprinting analysis, monoisotopic peak data were collected by using Mascot. The database search was done with the Mascot protein identification system (Matrix Science Inc., Boston, MA, http://www.matrixscience.com).
Blocking of Laminin-5
Single cells from corneal limbal tissues were incubated for 20 minutes at 37°C in hydrophilic tubes filled with basal medium containing B27 and EGF with anti-laminin-5 mAb (1:200, P3H9-2) or nonimmune IgG. After being washed three times with PBS, the cells were cultured overnight on non-tissue culture dishes. Dishes were washed twice with PBS and basal medium containing B27 to remove nonadherent cells, and then culture was done in basal medium containing B27, EGF, and anti-laminin-5-blocking mAb or control IgG.
Statistical Analysis
Results are presented as mean ± SD. Statistical analysis was performed by the Mann-Whitney test, and p < .05 was considered significant.
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RESULTS
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Culture of Disaggregated Single Cells from Corneal Limbal Epithelium
Adherent single cells obtained from the limbal tissues showed proliferation (Fig. 1A), whereas cells from the central corneal epithelium and Tenon's capsule failed to adhere to the non-tissue culture dishes and did not proliferate. The proliferating adherent cells gradually formed round colonies (Fig. 1B, 1C). After at least 10 days of culture, the cells exhibited a cobblestone-like morphology without exception and formed a double layer (Fig. 1D). Fibroblast-like cells or other types of cells were not observed in the non-tissue culture dishes. Labeling with BrdU for 24 hours resulted in more than 95% positive cells in each colony on day 10 (Fig. 1E), indicating that these were highly proliferative cells. Interestingly, these cells did not proliferate in basal medium containing B27 without EGF. Thus, the adherent colonies were derived from single cells isolated from the limbal epithelium, and these colony-forming cells showed a vigorous proliferative response to EGF. The number of colonies observed after 10 days of culture was 5.1 ± 3.4 (mean ± SD; n = 24) per 1,000 cells, and the average number of colony-forming cells per adherent cell was 27.2 ± 17.4 (n = 24).
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Figure 1. Characteristics of a colony that developed from a single cell harvested from corneal limbal epithelium. (A): An adherent single cell starts to proliferate on a non-tissue culture dish. (B): Adherent cobblestone-like cells are proliferating. (C): The cells form a round colony. (D): Some double layers of cells are seen. (E): Merged image of bright-field and fluorescence-positive cells. More than 95% of the cells within the colony incorporate bromodeoxyuridine on day 10. (F): Merged image of bright-field and fluorescence-positive cells. All cells in the adherent colony show staining for p63. (G): Nuclei were stained with Hoechst 33342. All cells in the colony also show immunoreactivity for nestin. (H): Less than 1% of the cells express a neural marker, MAP2. (I): No staining is observed with control IgG. (J): P63, nestin, and MAP2 gene transcripts are detected in adherent cells from colonies by reverse transcription (RT)-polymerase chain reaction, but not in the control assay without RT. Cells growing over the monolayers express cytokeratin-3 (K) and cytokeratin-12 (L), which are markers of differentiated corneal epithelium. Scale bars = 50 µm. Abbreviations: bp, base pairs; C, control; D, days after the culture; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, markers; MAP2, microtubule-associated protein 2; S, sample.
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Marker Expression in Single-Cell-Derived Colonies
The cells were immunostained to detect p63 (a marker of corneal limbal basal cells) [12], nestin (a marker of undifferentiated cells) [13], neural cell markers, and markers of differentiated corneal epithelium. All of the cells in the colonies showed immunoreactivity for p63 (Fig. 1F) and nestin (Fig. 1G), whereas less than 1% of the cells expressed the neural marker MAP-2 (Fig. 1H). The cells did not express GFAP, β-III tubulin, SMA, or the differentiated corneal epithelial markers CK-3 and CK-12 (data not shown) (Fig. 1I). Expression of the genes for P63, nestin, and MAP-2 was detected in cells from the colonies by RT-PCR but was not seen in the control assay that omitted RT (Fig. 1J). When the colonies were cultured for an additional 4–7 days, elongated cells grew over the adherent monolayers. These cells expressed CK-3 and CK-12, which are markers of differentiated corneal epithelium [14–16] (Fig. 1K, 1L), as confirmed by RT-PCR (Fig. 1M), but not markers of conjunctival epithelium, such as CK-13 (data not shown).
Self-Renewal and Reconstitution of a Three-Dimensional Human Corneal Epithelial Equivalent by Single Cells
To test self-renewal capacity, disaggregated single cells obtained from adherent colonies were passaged every 5–7 days. Colonies developed from single cells after the first (Fig. 2A) and second (Fig. 2B) passages, indicating that these cells had the capacity for self-renewal. In a typical case, approximately 2 x 102 colonies were harvested from 4 x 104 disaggregated single cells, and 35 and 7 colonies were obtained as colonies of first and second passages, respectively. As limbal progenitor cells were propagated further, however, their morphology changed dramatically, along with a progressive decrease of proliferation, clonogenicity, and differentiation potential. Each adherent colony that grew from a single cell on non-tissue culture dishes was transferred to six-well insert culture dishes and was grown in serum-free medium containing B27 and EGF without feeder cells. After 3 weeks, approximately 20% of these colonies formed stratified epithelial sheets that resembled normal corneal epithelium. The cell sheets had a cobblestone-like morphology (Fig. 2C); were composed of five or six stratified layers, from cuboidal basal cells to elongated superficial cells, when examined after hematoxylin and eosin staining (Fig. 2D); and were cytokeratin 3-positive (Fig. 2E) and cytokeratin 12-positive (Fig. 2F). These observations indicated that single cells from the corneal limbal epithelium were able to reconstitute a three-dimensional human corneal epithelial equivalent tissue that had a structure consistent with the normal structure of the corneal epithelium and was large enough to cover the entire human corneal surface. The average number of cells in each cell sheet was 5.5 ± 1.8 x 105 (mean ± SD) (n = 6). In a typical case, 1.2 x 106 cells were isolated from the limbal tissue of a donor cornea, and these cells generated approximately 6 x 103 colonies and 1.2 x 103 complete stratified cell sheets.
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Figure 2. Passage culture and reconstitution of a corneal epithelial equivalent from a single cell. The passaged single cells form colonies. First (A) and second (B) passage colonies are shown. (C): The cell sheet shows small cobblestone-like cells on the phase-contrast image, whereas complete multilayered corneal epithelium composed of cuboidal basal cells and elongated surface cells is seen on H&E staining (D). Corneal epithelial equivalent from a single cell expresses cytokeratin-3 (E) and cytokeratin-12 (F). Scale bars = 50 µm. Abbreviation: D, days after the culture.
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Extracellular Matrix Analysis by Mass Spectrometry
Extracellular matrix (ECM) proteins are well-known regulators of multiple cellular functions, including differentiation. We hypothesized that certain ECM proteins selectively expressed on the surface of limbal progenitor cells might promote their adhesion to non-tissue culture dishes. After adherent cells derived from corneal limbal tissues were removed, the proteins on the non-tissue culture dishes were harvested, diluted, and subjected to SDS-PAGE (Fig. 3A, lane A). Diluted nonadherent cells were used as the control (Fig. 3A, lane B). Protein bands were analyzed by MALDI-TOF MS. Table 2 shows the proteins identified by this analysis in lane A. Laminin-5 was a candidate for promoting adhesion to non-tissue culture dishes on the basis of the differences between the proteins in lanes A and B (Fig. 3A, arrow 2). Pretreatment of dissociated single cells from corneal limbal tissues with an anti-laminin-5-blocking Ab completely prevented the formation of round colonies (Fig. 3B). During all experimental procedures, hydrophilic tubes (Sumilon Stem Full) were used for collection of cells to prevent adhesion to the inside of the tube. As shown in Figure 3C, the number of colonies obtained after collection with normal tubes (BD Falcon 15-ml conical tubes) was significantly lower, indicating that hydrophilic tubes are essential for efficient isolation of laminin-5-positive undifferentiated cells.
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Figure 3. Extracellular matrix promoting adhesion to uncoated culture dishes and isolation of progenitor cells. (A): Lane A contained the proteins derived from adherent cells that grew on an uncoated dish. Lane B shows the diluted proteins obtained from nonadherent cells. Proteins are identified from 1–10 in lane A on the basis of the MWs determined by mass spectrometry. An MW band corresponding to number 2 in lane A was not detected in control lane B. Number 2 represents laminin-5. (B): Colony number formed on the non-tissue culture dishes per 1 x 103 cells were compared between dissociated single cells treated with anti-laminin-5-blocking antibody and control IgG. In contrast to the normally formed colonies in the cells treated with control IgG, no colony formation was observed in the cells treated with anti-laminin-5-blocking antibody. *, p < .001. (C): The colony isolation number was compared between hydrophilic and normal tubes. The colony isolation rate with normal tubes was reduced to one-fourth of that achieved with hydrophilic tubes, and the difference was statistically significant (*, p = .018). Abbreviations: Ab, antibody; MW, molecular weight.
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DISCUSSION
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Stem cells proliferate through a process known as asymmetric cell division as well as undergoing symmetric cell division. The decision of each daughter cell to maintain its original nature or to differentiate into a lineage of mature, nondividing functional cells is a stochastic process [17, 18], and the "stemness" of a cell can vary according to its environment. The cells that we isolated from human corneal limbal tissues on the non-tissue culture dishes in the present study showed limited self-renewal, original tissue-committed differentiation, and a high proliferative capacity, as evidenced by BrdU incorporation and by reconstitution of a three-dimensional tissue equivalent that displayed normal in vivo tissue architecture from single cells. The finding that cells isolated from the central corneal epithelium and sublimbal Tenon's capsule did not adhere to the dishes also suggests that these limbal tissue-derived single cells forming highly proliferative colonies are progenitor cells from the corneal limbal epithelium, because corneal epithelial progenitor cells are postulated to be localized exclusively to the corneal limbal epithelium. Moreover, the existence of neuronal marker MAP-2-positive cells from single cells suggests that these cells could be classified into lineage-uncommitted bipotent progenitor cells. As for the self-renewal capacity of the these cells, we could not deny the possibilities that these progenitor cells are not uniform in their self-renewal properties and that all of the replating capacity does not come from a rare subset of the colony initiating cells. Therefore, additional studies are required to determine whether these progenitor cells are adult stem cells or not.
This is a novel method for enriching progenitor cells from human corneal limbal epithelium, which consists of self-renewing squamous cells. Our technique used non-tissue culture dishes for floating culture in serum-, feeder cell-, and bovine pituitary extract-free medium. It may not be limited to use with the human corneal epithelium and may have wider applications in the field of squamous cell biology, because we found that single cells obtained from conjunctival and oral mucosal epithelia using this technique also formed round colonies and stratified tissues (unpublished observation). Further study is ongoing in our laboratory.
The laminin family of ECM proteins is expressed ubiquitously but is especially abundant in the basement membrane of various epithelial tissues [19, 20]. Expression of laminin-5 by our cultured cells can probably be attributed to the presence of EGF in the medium, because EGF promotes laminin-5 synthesis [21]. Laminin-5 shows distinct temporal and spatial patterns of expression in various epithelial tissues during development and influences cellular phenotypes from the early embryonic period onward [22, 23]. Laminin-5 has also been reported to induce osteogenic differentiation of human mesenchymal stem cells via a process mediated through focal adhesion kinase [24]. In the present study, laminin-5 (detected by mass spectrometry) was found to be a key factor in allowing progenitor cells to adhere to non-tissue culture dishes. This conclusion was supported by the finding that blocking of laminin-5 prevented the formation of round colonies by dissociated single cells from corneal limbal tissues. Laminin-5 may be a useful marker for detecting progenitor cells derived from the corneal epithelium and other squamous tissues. Moreover, our technique achieved the isolation of purified corneal epithelial progenitor cells without contamination by fibroblasts, which often interfere with primary culture of cells isolated from tissues. Considering that corneal and conjunctival differentiated cells, as well as progenitor cells, may produce laminin-5, progenitor cells from epithelium may produce a high amount of laminin-5 during culture in the basal medium containing B27 and EGF. Interestingly, laminin-5 has been reported to have a critical role in tumor growth and invasion [25–28]. Accordingly, our technique may also be useful for the isolation of cancer stem cells from tumors, considering that extracellular codeposition of laminin-5 and large unspliced tenascin-C has been detected in oral squamous cell carcinoma [29].
We should also emphasize the critical role of the hydrophilic test tubes (Sumilon Stem Full) used throughout these experiments for efficient isolation of progenitor cells. If hydrophilic tubes are not used during cell collection, the colony isolation rate is reduced to one-fourth of that achieved with such tubes (Fig. 3C). Therefore, hydrophilic tubes are crucial for processing of progenitor cells expressing adhesive ECM proteins to minimize cell loss, and the results obtained with such tubes may contribute to the development of stem cell biology.
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CONCLUSION
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Our new technique using non-tissue culture dishes makes it possible to isolate progenitor cells from the human corneal limbal epithelium, and laminin-5 has a critical role in the adhesion of these cells. This technique may also deserve wider attention in the field of squamous cell biology.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We thank Drs. Tsuyoshi Takato and Kazuhiko Yamada for providing samples and Kayo Aoyama for technical support.
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FOOTNOTES
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Author contributions: S. Yokoo: conception and design, collection and/or assembly of data; S. Yamagami: conception and design, financial support, manuscript writing; T.S.: collection and/or assembly of data; T.U.: data analysis and interpretation; T.-A.S.: administrative support; S.A. and M.A.: financial support, administrative support; J.H.: conception and design, manuscript writing.
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Abstract
Introduction
