October 2004
Volume 45, Issue 10
Free
Cornea  |   October 2004
Calcium-Induced Abnormal Epidermal-like Differentiation in Cultures of Mouse Corneal–Limbal Epithelial Cells
Author Affiliations
  • Tetsuya Kawakita
    From TissueTech, Inc., Miami, Florida; the
    Ocular Surface Center, Miami, Florida; and the
  • Edgar M. Espana
    From TissueTech, Inc., Miami, Florida; the
    Ocular Surface Center, Miami, Florida; and the
  • Hua He
    From TissueTech, Inc., Miami, Florida; the
    Ocular Surface Center, Miami, Florida; and the
  • Lung-Kun Yeh
    Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida.
  • Chia-Yang Liu
    Bascom Palmer Eye Institute, University of Miami School of Medicine, Miami, Florida.
  • Scheffer C. G. Tseng
    From TissueTech, Inc., Miami, Florida; the
    Ocular Surface Center, Miami, Florida; and the
Investigative Ophthalmology & Visual Science October 2004, Vol.45, 3507-3512. doi:10.1167/iovs.04-0266
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      Tetsuya Kawakita, Edgar M. Espana, Hua He, Lung-Kun Yeh, Chia-Yang Liu, Scheffer C. G. Tseng; Calcium-Induced Abnormal Epidermal-like Differentiation in Cultures of Mouse Corneal–Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2004;45(10):3507-3512. doi: 10.1167/iovs.04-0266.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To develop a reproducible method for expanding mouse corneal–limbal epithelial cells and determine the role of extracellular Ca2+ concentration and serum in modulating their growth and differentiation.

methods. Intact and viable corneal epithelial sheets were isolated from CD-1 albino mouse eyeballs by incubating for 18 hours at 4°C in 15 mg/mL dispase II with sorbitol in defined keratinocyte serum-free medium (KSFM) or supplementary hormonal epithelial medium (SHEM). These sheets were trypsinized into single cells and cultured on plastic in KSFM or SHEM. Cultures in KSFM were further manipulated by increasing Ca2+ concentration to 0.9 mM, with or without 5% FBS. Epithelial growth was compared in KSFM/KSFM (digestion medium/culture medium) and SHEM/SHEM by continuous passaging at a 1:3 split and by crystal violet staining of confluent dishes. Epithelial differentiation was assessed by immunostaining and/or immunoblotting to ZO-1, cytokeratin K12 (K12), connexin 43 (Cx43), cytokeratin K10 (K10), and involucrin.

results. Intact and viable corneal–limbal epithelial sheets were consistently isolated from more than 200 mouse eyes. Gradual increases in cell sizes and expression of ZO-1, K12, and Cx43 were noted from KSFM/KSFM to SHEM/KSFM, KSFM/SHEM, and SHEM/SHEM at passage 0. Epithelial growth ended at passage 1 in SHEM/SHEM but continued until passage 3 in KSFM/KSFM. Immunoblot analysis revealed that K12 expression was the highest in SHEM/SHEM, decreased from passages 0 to 1, and disappeared in passage 2 in KSFM/KSFM, with complete replacement of K10 and increasing expression of involucrin. Appearance of K10 was facilitated by 0.9 mM Ca2+ but suppressed by 5% FBS in KSFM at passage 0.

conclusions. Mouse corneal–limbal epithelial sheets can be used for initiating primary cultures, and their differentiation is promoted, whereas growth is suppressed, by a high Ca2+ concentration, even during enzymatic digestion. In serum-free medium, abnormal epidermal-like differentiation is promoted by increasing Ca2+ concentrations but prevented by serum. These results provide the ability to devise a medium to promote growth while maintaining normal differentiation.

Ocular surface epithelial cells are similar to epidermal keratinocytes in that they form stratified cell layers with superficial squamous cells and basal cuboidal cells. To study these cells in vitro, it is necessary to establish a culture system that permits proper growth and differentiation. Human epidermal keratinocytes have been successfully cultured in a low-Ca2+, serum-free medium 1 or a high-Ca2+, serum-containing medium with 3T3 fibroblasts feeder layers. 2 Ocular surface epithelia include corneal and conjunctival epithelium, and between them lies the limbal epithelium, in which the basal cells constitute corneal epithelial stem cells (SCs). 3 Using culturing techniques similar to the aforementioned, human corneal 4 5 6 7 and conjunctival 8 9 epithelial cells have also been successfully cultured. 
Unlike human epidermal keratinocytes, mouse keratinocytes have been known to be difficult to culture until recently. 10 Primary cultures of mouse keratinocytes are highly susceptible to extracellular Ca2+ concentrations; a medium containing less than 0.09 mM Ca2+ must be used to maintain proliferation. 11 In a low extracellular Ca2+ concentration, mouse keratinocytes have successfully been cultured in serum-free 12 and serum-containing medium supplemented with fibroblast-conditioned medium. 13 14 Furthermore, elevation of extracellular Ca2+ induces mouse 11 12 and human 15 keratinocytes to terminal differentiation, in which the cells express such markers as the cytokeratin K1/K10 pair 16 and involucrin. 17 Except for one attempt by Hazlett et al., 18 who cultured mouse corneal epithelial cells directly from corneal explants, there has not been any report showing successful isolation and culturing of mouse ocular surface epithelial cells. 
Recently we have reported a method of isolating intact and viable human 19 and rabbit 20 limbal epithelial sheets, and successfully cultured them, as well single cells, in a medium termed supplementary hormonal epithelial medium (SHEM) containing a high concentration of Ca2+ (0.9 mM) and supplemented with serum and growth factors. These accomplishments prompted us to apply this method to isolating mouse ocular surface epithelia and establishing a culture system for proper growth and differentiation. During the course of investigation, we discovered that unlike human and rabbit corneal epithelial cells, in vitro growth and differentiation of mouse corneal epithelial cells on plastic substrate are highly dependent on extracellular Ca2+ and serum. The significance of this finding is further discussed. 
Materials and Methods
The tissue culture plastic ware was purchased from BD Biosciences (Lincoln Park, NJ). Amphotericin B, Dulbecco’s modified Eagle’s medium (DMEM), F-12 nutrient mixture (F12), defined keratinocyte-SFM (KSFM), fetal bovine serum (FBS), gentamicin, Hanks’ balanced salt solution (HBSS), HEPES-buffer, phosphate-buffered saline (PBS), soybean trypsin inhibitor, and 0.25% trypsin/1 mM EDTA were purchased from Invitrogen-Gibco (Grand Island, NY). A cell viability–cytotoxicity assay kit (Live/Dead) was purchased from Molecular Probes (Eugene, OR) and dispase II powder from Roche Diagnostics. (Indianapolis, IN). Other reagents and chemicals including bovine serum albumin (BSA), cholera-toxin (subunit A), dimethyl sulfoxide, hydrocortisone, insulin-transferrin-sodium selenite (ITS) media supplement, mouse-derived epidermal growth factor (EGF), sorbitol, propidium iodide (PI) and fluorescein-conjugated (FITC) secondary antibodies were from Sigma-Aldrich (St. Louis, MO). Optimal cutting temperature (OCT) compound was purchased from Sakura Finetek (Torrance, CA), polyclonal antibody to ZO-1 from Chemicon (Temecula, CA), goat polyclonal antibody to K12 from Santa Cruz Biotechnology (Santa Cruz, CA), rabbit polyclonal antibody to connexin 43 (Cx43) from Zymed (San Francisco, CA), and rabbit polyclonal antibody to involucrin from Covance (Berkley, CA). Monoclonal antibody to K10 was kindly provided by Tung-Tien Sun (New York University, New York, NY). The horseradish peroxidase–conjugated antibodies were from Bio-Rad (Hercules, CA), excluding rabbit anti-goat antibody (Pierce, Rockford, IL). 
Isolation of Mouse Corneal Epithelial Sheets
CD-1 albino mice more than 1 month old (Charles River, Boston, MA) were handled according to the guidelines in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Two hundred eye globes were enucleated from the mice with forceps after death, washed profusely in PBS, stored in KSFM, and then transported at 4°C within 24 hours to the laboratory. These eyes were enzymatically digested at 4°C for 18 hours in KSFM or SHEM containing 15 mg/mL dispase II and 100 mM sorbitol. KSFM was supplemented with 10 ng/mL mouse-derived EGF and 10−10 M cholera toxin A subunit, as described before. 14 SHEM consisted of an equal volume of HEPES-buffered DMEM and F12, containing bicarbonate, 0.5% dimethyl sulfoxide, 10 ng/mL mouse-derived EGF, 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL sodium selenite, 0.5 μg/mL hydrocortisone, 10−10 M cholera toxin A subunit, 5% FBS, 50 μg/mL gentamicin, and 1.25 μg/mL amphotericin B. The Ca2+ concentration of KSFM and SHEM were 0.07 and 0.9 mM, respectively. Subsequently, each mouse eye was fixed by suction applied to the posterior pole, by using a transfer pipette, and was gently shaken in the respective medium to loosen the ocular surface epithelial sheet (see Fig. 1 ). All epithelial sheets were then rendered into single cells by 0.25% trypsin/1mM EDTA in HBSS. The Trypsin reaction was blocked with soybean trypsin inhibitor. 
Cell Viability
To assess viability of isolated cells, an assay (Live/Dead; Molecular Probes) was performed as described previously. 21 Briefly, after the removal of the culture medium, five isolated corneal epithelial sheets from different mice were washed twice with HBSS and incubated for 40 minutes at room temperature in the dark with 0.5 mL assay reagent consisting of 2 mM calcein-AM and 4 mM ethidium homodimer in PBS. Images were photographed by epifluorescence microscope (Te-2000u Eclipse; Nikon, Tokyo, Japan). 
Primary Cultures and Subcultures
Single cells derived from isolated corneal–limbal epithelial sheets were seeded as a density of 2.0 × 104 cells/cm2 and cultured for 7 days in 24-well plates (n = 5) in one of the four experimental conditions: KSFM/KSFM (digestion medium/culture medium), SHEM/KSFM, KSFM/SHEM, and SHEM/SHEM for immunohistochemical analysis, or in 35-mm dishes (n = 3) in either KSFM/KSFM or SHEM/SHEM for subculturing and immunoblot analysis. Confluent cultures were subcultured in 1:3 trypsin/EDTA. The growth potential was assessed by staining confluent dishes with crystal violet. In another experiment, cells in primary cultures with the same seeding density were grown in KSFM, with or without adjusting the Ca2+ concentration to 0.9 mM by adding CaCl2 and with or without adding 5% FBS. 
Immunostaining
Normal mouse corneas and tails were embedded in OCT and snap frozen in liquid nitrogen. All sections (5 μm thick) were fixed with cold methanol for 5 minutes at −20°C, blocked, and permeabilized as previously described. 22 Cells cultured on plastic were washed three times with PBS after removing the culture medium and then fixed with cold methanol. After they were blocked with 1% BSA for 30 minutes, cultured cells and tissues were incubated for 1 hour with primary antibodies against ZO-1 (1:100), 23 24 25 26 K12 (1:50), 27 28 Cx43 (1:100), 29 30 31 K10 (1:200), 16 and involucrin (1:200). 17 Specific binding was detected by an FITC-conjugated anti-mouse secondary antibody (1:200), counterstained with PI (0.1 μg/mL), and mounted in anti-fade solution (Vector Laboratories, Burlingame, CA). Images were photographed with an epifluorescence microscope. 
Immunoblot Analysis
Confluent cultured cells in 35-mm dishes in each experimental condition were washed three times with PBS, after the culture medium was removed, and were lysed by RIPA buffer. The protein concentration was determined by bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Proteins were loaded according to a β-actin control, electrophoresed in a polyacrylamide gradient gel (4%–15%), and transferred to nitrocellulose membranes. These membranes were blotted with 5% low-fat dry milk; incubated overnight at 4°C with primary antibody against K12 (1:500), involucrin (1:1000), K10 (1:1000), and β-actin (1:3000); and transferred to a 1:2000 solution of the appropriate secondary antibody. Immunoreactivity was then visualized (Opti-4CN reagent; Bio-Rad, Hercules, CA; or Western Lightning chemiluminescence; PerkinElmer, Boston, MA). 
Results
Viability and Culturability of Isolated Mouse Corneal–Limbal Epithelial Sheets
Using the same method as described, 19 20 we isolated mouse ocular surface epithelial sheets. As shown in Figure 1 , the isolated mouse sheet appeared to be dome shaped, conforming to the anterior surface of the globe (Fig. 1A) . A cell-viability assay was used to determine the percentage of living cells in isolated corneal epithelial sheets. All cells were stained green (live; Fig. 1B ). This result showed that cells in the isolated corneal–limbal epithelial sheets remained viable, a finding consistent with our earlier reports. 19 20  
We noted that there were no epithelial cells left on the corneal stroma after sheet removal. To determine whether the isolated sheet contained limbal or conjunctival epithelium, frozen sections of a mouse eye globe, immediately at the conclusion of dispase digestion, were stained with hematoxylin, which showed that the basal epithelium had already been separated from the corneal stroma and that these sheet contained both corneal and limbal epithelia (Fig. 1C) . The edge of the sheet showed that there were no goblet cells (Fig 1D) . We successfully applied this method to isolate intact epithelial sheets from more than 200 enucleated mouse eyeballs. 
We demonstrated that such isolated human 19 and rabbit 20 limbal epithelial sheets could be cultured on plastic in SHEM to generate outgrowth. Nevertheless, we failed to do so, because mouse corneal–limbal sheets quickly degenerated. Because mouse epidermal keratinocytes can be cultured on plastic in a medium containing a low Ca2+ concentration (see the introduction), we thus wondered whether the aforementioned failure might be due to a higher Ca2+ concentration (0.9 mM) in SHEM during dispase digestion and subsequent culturing. To examine this hypothesis, we performed dispase II digestion in SHEM or serum-free KSFM, which contains low Ca2+ (0.07 mM). The resultant corneal–limbal epithelial sheets were rendered into single cells by trypsin/EDTA, yielding an average of 5.2 ± 1.8 × 104 cells per sheet (n = 5). 
Epithelial Phenotype in Primary Cultures Containing KSFM or SHEM
Within 24 hours after seeding, some cells attached to plastic and started spreading. In all four conditions, more than 70% of seeded cells did not attach, presumably because of their being suprabasal cells. After they were transferred to a new dish, these floating cells did not show any attachment in the ensuing 2 days (not shown). Cells in KSFM/KSFM were uniformly small and formed a monolayer on confluence (Fig. 2A 2C) . In contrast, cells in SHEM/SHEM were large and squamous, with many vacuoles and became stratified at the center of the dish after confluence (Figs. 2B 2D) . Cells were mixed with small cells and large squamous cells in SHEM/KSFM (Fig. 3B1) and KSFM/SHEM (Fig. 3C1)
Immunostaining was performed to assess the expression of several cell markers in confluent primary cultures. ZO-1, a component of the tight-junction complex present between superficial epithelial cells of stratified epithelia undergoing terminal differentiation, 32 is expressed by mouse corneal superficial cells in vivo. 33 Immunostaining for ZO-I was negative in KSFM/KSFM (Fig. 3A2) , but was positive in the intercellular junction between large squamous cells with increasing intensity from SHEM/KSFM (Fig. 3B2) , to KSFM/SHEM (Fig. 3C2) , and SHEM/SHEM (Fig. 3D2) . The extent of positive staining for K12 correlated with the cell size. K12 staining was negative in most small epithelial cells, positive in some intermediate cells, and strongly positive in nearly all squamous epithelial cells. Therefore, K12 expression in KSFM/KSFM was the least and limited only to occasional intermediate cells (Fig. 3A3) . Positive staining for K12 was found in nearly 50% of the cells in both SHEM/KSFM (Fig. 3B3) and KSFM/SHEM (Fig. 3C3) , and became pronounced in SHEM/SHEM (Fig. 3D3) , with intense cytoplasmic staining. To substantiate this finding, we also analyzed Cx43 expression, which forms intercellular gap junctions in the corneal basal epithelium but is absent in the limbal basal epithelium. 29 Immunostaining for Cx43 was negative in KSFM/KSFM (Fig. 3A4) and SHEM/KSFM (Fig. 3B4) , but was positive as a punctate pattern scattered between some superficial cells in KSFM/SHEM and SHEM/SHEM (Figs. 3C4 3D4 , respectively). This result showed that Cx43 expression was also downregulated in KSFM in the direction of the limbal basal epithelium, but was upregulated in SHEM in the direction of the corneal suprabasal epithelium. Nuclear counterstaining with PI revealed that the nuclear size was the smallest in KSFM/KSFM and was gradually enlarged from SHEM/KSFM, to KSFM/SHEM, to SHEM/SHEM. Collectively, these data show that epithelial differentiation was promoted from KSFM/KSFM to SHEM/SHEM. 
Growth Potential and Differentiation for Subcultures in KSFM and SHEM
On confluence, cells in KSFM/KSFM and SHEM/SHEM were subcultured in respective medium to passage 1 and cultured until the growth potential was exhausted. Cells cultured in KSFM/KSFM also reached confluence on day 4 in passage 0, but on day 7 in passage 1 and never achieved confluence in passage 2. Cells in SHEM/SHEM reached confluence at day 4 in primary cultures (i.e., passage 0) and never achieved confluence in passage 1. The total number of cells harvested in KSFM in passages 0, 1, and 2 was approximately 5.0 × 105, 3.0 × 105, and 1.5 × 105, respectively, and the number in SHEM at passage 0 was approximately 1.5 × 105 (n = 3). Such a difference in the growth potential of these two media was also illustrated by crystal violet staining of the confluent dish at each passage (Fig. 4A) . The intensity of cresyl violet staining correlated with the cell size and morphology. Cells were more uniformly small when passaged in KSFM in passages 0 and 1, but became enlarged at passage 2, yielding only small cells clustered in some areas (Fig. 4B) . Nevertheless, all cells in SHEM became enlarged, with much fewer cells remaining adherent after subculturing to passage 1. Such a dramatic change in cell morphology between KSFM/KSFM and SHEM/SHEM was consistently observed in three different experiments (not shown). These results collectively indicate that growth potential was maintained better in KSFM, whereas senescence was markedly promoted in SHEM. 
Immunoblot analysis of protein extracts from cells cultured in KSFM/KSFM and SHEM/SHEM at passage 0 confirmed the expression of K12 (Fig. 5A) , consistent with the immunostaining result (Fig. 3) . Nevertheless, K12 expression decreased in KSFM/KSFM when cells were subcultured to passage 1 and disappeared at passage 2 (Fig. 5A) . This result suggests that cells continuously subcultured in KSFM/KSFM began to lose normal terminal differentiation. This notion was confirmed by the appearance of K10 expression in passage 2 and increased of involucrin from passages 0 to 1 and 2 in KSFM/KSFM (Fig. 5A) . Immunostaining for K10 was negative in normal mouse cornea (Fig. 5B) , but strongly positive in suprabasal layers of mouse epidermis (Fig. 5C) . Wang et al. 34 reported a basal staining pattern of involucrin in the mouse corneal epithelium. We used the same antibody to show that immunostaining for involucrin was weakly positive in the suprabasal cells of the mouse corneal epithelium in vivo (Fig. 5D) . As a contrast, intense staining was noted in suprabasal cells of the mouse epidermis (Fig. 5E)
Regulation of Abnormal Epithelial Cell Differentiation
One major difference between KSFM and SHEM is extracellular Ca2+ concentration. To verify that extracellular Ca2+ concentration was an important modulating factor in the aforementioned differences between these two media, we added CaCl2 to increase Ca2+ to 0.9 mM in KSFM in primary cultures. We noted that growth potential diminished with an increase of cell size when extracellular Ca2+ concentration was increased from 0.07 to 0.9 mM (not shown). Immunoblot analysis showed that the expression of cornea-specific K12 was similar in these two conditions with different Ca2+ concentrations (Fig. 4) . However, the expression of K10 appeared only in KSFM with a high Ca2+ concentration. Another major difference between KSFM and SHEM is the presence of 5% FBS in SHEM. We thus added 5% FBS to KSFM with either a low- or high-Ca2+ concentration. Taking account of the fact that FBS also contains Ca2+, we calculated that KSFM with 5% FBS did not contain more than 0.12 mM. Immunoblot analysis showed that the expression of K12 was not affected by addition of FBS in either Ca2+ concentration, but the expression of K10 in KSFM containing a high Ca2+ concentration was dramatically decreased when FBS was added (Fig. 6)
Discussion
In this study, we demonstrated the possibility of initiating primary cultures from isolated mouse corneal–limbal epithelial sheets. Nevertheless, a brief exposure to SHEM during 18 hours of digestion was sufficient to induce more epithelial differentiation, as evidenced by increasing staining for ZO-1 (Fig. 4A3) and K12 (Fig. 4B3) , when compared with KSFM. Continuous exposure to SHEM during cultivation clearly promoted more epithelial differentiation, as evidenced by increasing cell size and nuclear size and positive staining for ZO-1 (Fig. 4D2) and Cx43 (Fig. 4D4) . On the contrary, cells isolated and cultured in KSFM maintained the least differentiation status with the smallest cell size, the smallest nuclear size, the least staining for K12 (Fig. 4A3) , and negative staining for ZO-1 (Fig. 4A2) and Cx43 (Fig. 4A4) . K12 is not expressed in the limbal basal epithelium, but is expressed in the full thickness of the corneal epithelium in the mouse. 27 Similarly, Cx43 is not expressed in the limbal basal epithelium, but is expressed in the corneal basal epithelium, which contains transient amplifying cells, in the mouse. 29 ZO-1 is known to be expressed only by superficial cells in a stratified epithelium. 24 25 Using a combination of in vivo confocal microscopy and flow cytometry, we have reported recently that the limbal basal epithelium has the smallest cell size in the corneal epithelial differentiation scheme. 35 Because such cells cultured in KSFM share a number of features normally found in the limbal basal epithelium, it is tempting to speculate that KSFM helps to maintain limbal epithelial SCs, and that SHEM promotes more corneal differentiation. KSFM also maintains the basal cell phenotype of mouse epidermal keratinocytes. 14 For these reasons, it may be that cells should be subcultured in KSFM but not in SHEM (Fig. 5A) . Future studies using single cell clonal assays are needed to substantiate this hypothesis. 
KSFM differs from SHEM in many ways, including, but not limited to, a lower Ca2+ concentration (0.07 mM) and the lack of 5% FBS. As a first step in identifying components in the medium that may be responsible for these differences, we increased Ca2+ concentration in KSFM to 0.9 mM—that is the same level as SHEM. In such conditions, cells in primary cultures dramatically increased in cell size and nuclear size in 7 days (not shown), yielding morphology similar to that observed in SHEM. Nevertheless, unlike cells cultured in SHEM, where K12 but not K10 was expressed (Fig. 5A) , cells cultured in KSFM with 0.9 mM Ca2+ expressed K12 and K10 (Fig. 6) . These results indicate that differentiation of mouse corneal–limbal epithelial cells is sensitive to extracellular Ca2+ concentrations, a finding that has been reported in several other types of epithelial cells. 7 11 36 37  
We were particularly intrigued by the finding that 0.9 mM Ca2+ actually promotes abnormal differentiation in primary cultures of mouse corneal–limbal epithelial cells cultured in KSFM for 7 days. It should be noted that even in KSFM with 0.07 mM Ca2+, cells eventually turned off K12 expression and switched on K10 expression if continuously subcultured to passage 2 (Fig. 5A) . The emergence of K10 expression signifying abnormal epidermal-like differentiation was also supported by an increasing amount of involucrin, a marker of epidermal differentiation. 17 Such an abnormal (epidermal-like) terminal differentiation coincided with the time when cells also lost growth potential (Fig. 4A) . In mouse epidermal keratinocytes, the simple lowering of extracellular Ca2+ concentrations alone can prevent differentiation and effectively preserve SC growth potential. 17 Nevertheless, our study showed that this maneuver was not sufficient to prevent differentiation of mouse corneal–limbal epithelial cells in KSFM. It remains to be determined whether the eventual loss of growth potential of mouse corneal–limbal epithelial cells in KSFM is due to the emergence of abnormal epidermal-like differentiation. 
For epidermal keratinocytes, expression of K10 and involucrin is a part of normal terminal differentiation, 16 17 and hence it is appropriate to culture them in a serum-free medium and stimulate their differentiation by increasing extracellular Ca2+ concentrations. 17 Nevertheless, expression of K10 and involucrin is abnormal for all nonepidermal stratified epithelia, including ocular surface epithelia. For this reason, we question the wisdom of culturing ocular surface epithelial cells in a serum-free medium, especially for stimulating their differentiation by increasing extracellular Ca2+ concentrations. 
We have reported that rabbit corneal epithelial cells undergo abnormal epidermal differentiation, as judged by the formation of cornified envelopes when cultured in a serum-free medium with a high Ca2+ concentration. 7 The abnormal epidermal-like differentiation resembles the squamous metaplasia in several ocular surface diseases. We have reported that the expression of the K3/K12 pair is replaced by the K1/K10 pair when nonkeratinized corneal and conjunctival epithelia ultrastructurally transform to a keratinized epidermis-like epithelium in a rabbit model of vitamin A deficiency. 38 Therefore, we speculate that the expression of K10 and involucrin by mouse corneal–limbal epithelial cells cultured in KSFM is caused by vitamin A deficiency. This hypothesis is in part suggested by the findings that K10 expression in KSFM with 0.9 mM Ca2+ was suppressed by 5% FBS (Fig. 6) and that K10 was not expressed by cells cultured in SHEM (Fig. 5A) . Although additional studies are necessary to substantiate this hypothesis, the findings presented herein strongly suggest that it is important to guard against such abnormal epidermal-like differentiation when ocular surface epithelial cells are cultured in a serum-free medium. Supplementation with serum factors such as vitamin A may be critical in maintaining normal terminal differentiation of corneal–limbal epithelial cells. Further refinement may lead to a new culturing system that can be used to expand mouse corneal–limbal epithelial cells in vitro so that one day we can investigate molecular mechanisms governing corneal epithelial growth and differentiation by taking advantage of the variety of transgenic mice available. 
 
Figure 1.
 
Characterization of isolated epithelial sheets. (A) Under low magnification, an isolated mouse corneal–limbal epithelial sheet appeared to be dome shaped, conforming to the anterior surface of the mouse eye. (B) Almost all the cells were living (green), according to a cell-viability assay. (C) The edge of the epithelial sheet (D, inset) showed that there were no goblet cells. (D) Hematoxylin staining of a mouse cornea immediately after dispase digestion showed a clear separation of ocular surface epithelial sheet from the stroma. Bar, 100 μm, except (C).
Figure 1.
 
Characterization of isolated epithelial sheets. (A) Under low magnification, an isolated mouse corneal–limbal epithelial sheet appeared to be dome shaped, conforming to the anterior surface of the mouse eye. (B) Almost all the cells were living (green), according to a cell-viability assay. (C) The edge of the epithelial sheet (D, inset) showed that there were no goblet cells. (D) Hematoxylin staining of a mouse cornea immediately after dispase digestion showed a clear separation of ocular surface epithelial sheet from the stroma. Bar, 100 μm, except (C).
Figure 2.
 
Different cellular morphology in KSFM and SHEM. Confluent cultures in KSFM/KSFM showed a monolayer of compact and small cells (A, C), whereas those in SHEM/SHEM showed a monolayer of large squamous cells (B, D) under low and high magnification, respectively. Bar, 100 μm.
Figure 2.
 
Different cellular morphology in KSFM and SHEM. Confluent cultures in KSFM/KSFM showed a monolayer of compact and small cells (A, C), whereas those in SHEM/SHEM showed a monolayer of large squamous cells (B, D) under low and high magnification, respectively. Bar, 100 μm.
Figure 3.
 
Epithelial phenotypic changes by combination of different digestion and culturing media in passage 0. Cells increased in size from KSFM/KSFM, to SHEM/KSFM, to KSFM/SHEM, to SHEM/SHEM (A1, B1, C1, D1, respectively). Immunostaining for ZO-I was negative in KSFM/KSFM, showing intercellular punctate staining in SHEM/KSFM and pronounced intercellular linear staining outlining the large squamous cells in KSFM/SHEM and SHEM/SHEM (A2, B2, C2, D2, respectively). Immunostaining for K12 was the least and was limited to some cells in KSFM/KSFM, became more pronounced in SHEM/KSFM and KSFM/SHEM, and was the most intense in SHEM/SHEM (A3, B3, C3, D3, respectively). Immunostaining for Cx43 was negative in KSFM/KSFM and KSFM/SHEM, but scattered positive punctuate staining between cells in KSFM/SHEM and SHEM/SHEM was noted (arrows, A4, B4, C4, D4, respectively). Bar, 100 μm.
Figure 3.
 
Epithelial phenotypic changes by combination of different digestion and culturing media in passage 0. Cells increased in size from KSFM/KSFM, to SHEM/KSFM, to KSFM/SHEM, to SHEM/SHEM (A1, B1, C1, D1, respectively). Immunostaining for ZO-I was negative in KSFM/KSFM, showing intercellular punctate staining in SHEM/KSFM and pronounced intercellular linear staining outlining the large squamous cells in KSFM/SHEM and SHEM/SHEM (A2, B2, C2, D2, respectively). Immunostaining for K12 was the least and was limited to some cells in KSFM/KSFM, became more pronounced in SHEM/KSFM and KSFM/SHEM, and was the most intense in SHEM/SHEM (A3, B3, C3, D3, respectively). Immunostaining for Cx43 was negative in KSFM/KSFM and KSFM/SHEM, but scattered positive punctuate staining between cells in KSFM/SHEM and SHEM/SHEM was noted (arrows, A4, B4, C4, D4, respectively). Bar, 100 μm.
Figure 4.
 
Difference in growth and morphology in KSFM/KSFM and SHEM/SHEM. (A) Crystal violet staining of confluent culture dishes showed that cells cultured in KSFM/KSFM continued to be successfully subcultured to passage 2, whereas cells in SHEM/SHEM could not be subcultured after passage 0. (B) Cells in passages 1 and 2 in KSFM/KSFM were small, but some cells increased in size in passage 2. In contrast, cells in SHEM/SHEM in passage 1 were already enlarged. Cells in passage 0 in both conditions are shown in Figure 3 . Bar, 100 μm.
Figure 4.
 
Difference in growth and morphology in KSFM/KSFM and SHEM/SHEM. (A) Crystal violet staining of confluent culture dishes showed that cells cultured in KSFM/KSFM continued to be successfully subcultured to passage 2, whereas cells in SHEM/SHEM could not be subcultured after passage 0. (B) Cells in passages 1 and 2 in KSFM/KSFM were small, but some cells increased in size in passage 2. In contrast, cells in SHEM/SHEM in passage 1 were already enlarged. Cells in passage 0 in both conditions are shown in Figure 3 . Bar, 100 μm.
Figure 5.
 
Expression of K12, K10, and involucrin in cells in KSFM and SHEM. (A) Immunoblot analysis of K12, K10, and involucrin in cells cultured in KSFM in passages 0, 1, and 2 and in SHEM in passage 0. Based on the β-actin loading control, K12 expression was the highest in passage 0 in SHEM, followed by KSFM in passage 0, decreased in KSFM in passage 1, and disappeared in passage 2. K10 expression appeared in KSFM in passage 2. Involucrin expression was the lowest in SHEM and increased in KSFM from passages 0 to 2. Immunostaining of K10 was negative in mouse cornea (B), but strongly positive in mouse epidermis. (C) Immunostaining for involucrin was weakly positive in suprabasal cells of the mouse corneal epithelium (D), but strongly positive in the suprabasal cells of the mouse epidermis (E). Bars, 100 μm.
Figure 5.
 
Expression of K12, K10, and involucrin in cells in KSFM and SHEM. (A) Immunoblot analysis of K12, K10, and involucrin in cells cultured in KSFM in passages 0, 1, and 2 and in SHEM in passage 0. Based on the β-actin loading control, K12 expression was the highest in passage 0 in SHEM, followed by KSFM in passage 0, decreased in KSFM in passage 1, and disappeared in passage 2. K10 expression appeared in KSFM in passage 2. Involucrin expression was the lowest in SHEM and increased in KSFM from passages 0 to 2. Immunostaining of K10 was negative in mouse cornea (B), but strongly positive in mouse epidermis. (C) Immunostaining for involucrin was weakly positive in suprabasal cells of the mouse corneal epithelium (D), but strongly positive in the suprabasal cells of the mouse epidermis (E). Bars, 100 μm.
Figure 6.
 
Effect of increasing extracellular Ca2+ and serum on epithelial differentiation. Cells were cultured for 7 days in KSFM with 0.07 mM Ca2+ without (KSFM) or with (FBS) 5% FBS or in KSFM with 0.9 mM Ca2+ without (Ca2+) or with (FBS+Ca2+) 5% FBS. Judged by the β-actin loading control, K12 was similarly expressed in these four conditions. In contrast, K10 was expressed only in high-Ca2+ medium without FBS. Such expression was dramatically decreased when serum was applied.
Figure 6.
 
Effect of increasing extracellular Ca2+ and serum on epithelial differentiation. Cells were cultured for 7 days in KSFM with 0.07 mM Ca2+ without (KSFM) or with (FBS) 5% FBS or in KSFM with 0.9 mM Ca2+ without (Ca2+) or with (FBS+Ca2+) 5% FBS. Judged by the β-actin loading control, K12 was similarly expressed in these four conditions. In contrast, K10 was expressed only in high-Ca2+ medium without FBS. Such expression was dramatically decreased when serum was applied.
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Figure 1.
 
Characterization of isolated epithelial sheets. (A) Under low magnification, an isolated mouse corneal–limbal epithelial sheet appeared to be dome shaped, conforming to the anterior surface of the mouse eye. (B) Almost all the cells were living (green), according to a cell-viability assay. (C) The edge of the epithelial sheet (D, inset) showed that there were no goblet cells. (D) Hematoxylin staining of a mouse cornea immediately after dispase digestion showed a clear separation of ocular surface epithelial sheet from the stroma. Bar, 100 μm, except (C).
Figure 1.
 
Characterization of isolated epithelial sheets. (A) Under low magnification, an isolated mouse corneal–limbal epithelial sheet appeared to be dome shaped, conforming to the anterior surface of the mouse eye. (B) Almost all the cells were living (green), according to a cell-viability assay. (C) The edge of the epithelial sheet (D, inset) showed that there were no goblet cells. (D) Hematoxylin staining of a mouse cornea immediately after dispase digestion showed a clear separation of ocular surface epithelial sheet from the stroma. Bar, 100 μm, except (C).
Figure 2.
 
Different cellular morphology in KSFM and SHEM. Confluent cultures in KSFM/KSFM showed a monolayer of compact and small cells (A, C), whereas those in SHEM/SHEM showed a monolayer of large squamous cells (B, D) under low and high magnification, respectively. Bar, 100 μm.
Figure 2.
 
Different cellular morphology in KSFM and SHEM. Confluent cultures in KSFM/KSFM showed a monolayer of compact and small cells (A, C), whereas those in SHEM/SHEM showed a monolayer of large squamous cells (B, D) under low and high magnification, respectively. Bar, 100 μm.
Figure 3.
 
Epithelial phenotypic changes by combination of different digestion and culturing media in passage 0. Cells increased in size from KSFM/KSFM, to SHEM/KSFM, to KSFM/SHEM, to SHEM/SHEM (A1, B1, C1, D1, respectively). Immunostaining for ZO-I was negative in KSFM/KSFM, showing intercellular punctate staining in SHEM/KSFM and pronounced intercellular linear staining outlining the large squamous cells in KSFM/SHEM and SHEM/SHEM (A2, B2, C2, D2, respectively). Immunostaining for K12 was the least and was limited to some cells in KSFM/KSFM, became more pronounced in SHEM/KSFM and KSFM/SHEM, and was the most intense in SHEM/SHEM (A3, B3, C3, D3, respectively). Immunostaining for Cx43 was negative in KSFM/KSFM and KSFM/SHEM, but scattered positive punctuate staining between cells in KSFM/SHEM and SHEM/SHEM was noted (arrows, A4, B4, C4, D4, respectively). Bar, 100 μm.
Figure 3.
 
Epithelial phenotypic changes by combination of different digestion and culturing media in passage 0. Cells increased in size from KSFM/KSFM, to SHEM/KSFM, to KSFM/SHEM, to SHEM/SHEM (A1, B1, C1, D1, respectively). Immunostaining for ZO-I was negative in KSFM/KSFM, showing intercellular punctate staining in SHEM/KSFM and pronounced intercellular linear staining outlining the large squamous cells in KSFM/SHEM and SHEM/SHEM (A2, B2, C2, D2, respectively). Immunostaining for K12 was the least and was limited to some cells in KSFM/KSFM, became more pronounced in SHEM/KSFM and KSFM/SHEM, and was the most intense in SHEM/SHEM (A3, B3, C3, D3, respectively). Immunostaining for Cx43 was negative in KSFM/KSFM and KSFM/SHEM, but scattered positive punctuate staining between cells in KSFM/SHEM and SHEM/SHEM was noted (arrows, A4, B4, C4, D4, respectively). Bar, 100 μm.
Figure 4.
 
Difference in growth and morphology in KSFM/KSFM and SHEM/SHEM. (A) Crystal violet staining of confluent culture dishes showed that cells cultured in KSFM/KSFM continued to be successfully subcultured to passage 2, whereas cells in SHEM/SHEM could not be subcultured after passage 0. (B) Cells in passages 1 and 2 in KSFM/KSFM were small, but some cells increased in size in passage 2. In contrast, cells in SHEM/SHEM in passage 1 were already enlarged. Cells in passage 0 in both conditions are shown in Figure 3 . Bar, 100 μm.
Figure 4.
 
Difference in growth and morphology in KSFM/KSFM and SHEM/SHEM. (A) Crystal violet staining of confluent culture dishes showed that cells cultured in KSFM/KSFM continued to be successfully subcultured to passage 2, whereas cells in SHEM/SHEM could not be subcultured after passage 0. (B) Cells in passages 1 and 2 in KSFM/KSFM were small, but some cells increased in size in passage 2. In contrast, cells in SHEM/SHEM in passage 1 were already enlarged. Cells in passage 0 in both conditions are shown in Figure 3 . Bar, 100 μm.
Figure 5.
 
Expression of K12, K10, and involucrin in cells in KSFM and SHEM. (A) Immunoblot analysis of K12, K10, and involucrin in cells cultured in KSFM in passages 0, 1, and 2 and in SHEM in passage 0. Based on the β-actin loading control, K12 expression was the highest in passage 0 in SHEM, followed by KSFM in passage 0, decreased in KSFM in passage 1, and disappeared in passage 2. K10 expression appeared in KSFM in passage 2. Involucrin expression was the lowest in SHEM and increased in KSFM from passages 0 to 2. Immunostaining of K10 was negative in mouse cornea (B), but strongly positive in mouse epidermis. (C) Immunostaining for involucrin was weakly positive in suprabasal cells of the mouse corneal epithelium (D), but strongly positive in the suprabasal cells of the mouse epidermis (E). Bars, 100 μm.
Figure 5.
 
Expression of K12, K10, and involucrin in cells in KSFM and SHEM. (A) Immunoblot analysis of K12, K10, and involucrin in cells cultured in KSFM in passages 0, 1, and 2 and in SHEM in passage 0. Based on the β-actin loading control, K12 expression was the highest in passage 0 in SHEM, followed by KSFM in passage 0, decreased in KSFM in passage 1, and disappeared in passage 2. K10 expression appeared in KSFM in passage 2. Involucrin expression was the lowest in SHEM and increased in KSFM from passages 0 to 2. Immunostaining of K10 was negative in mouse cornea (B), but strongly positive in mouse epidermis. (C) Immunostaining for involucrin was weakly positive in suprabasal cells of the mouse corneal epithelium (D), but strongly positive in the suprabasal cells of the mouse epidermis (E). Bars, 100 μm.
Figure 6.
 
Effect of increasing extracellular Ca2+ and serum on epithelial differentiation. Cells were cultured for 7 days in KSFM with 0.07 mM Ca2+ without (KSFM) or with (FBS) 5% FBS or in KSFM with 0.9 mM Ca2+ without (Ca2+) or with (FBS+Ca2+) 5% FBS. Judged by the β-actin loading control, K12 was similarly expressed in these four conditions. In contrast, K10 was expressed only in high-Ca2+ medium without FBS. Such expression was dramatically decreased when serum was applied.
Figure 6.
 
Effect of increasing extracellular Ca2+ and serum on epithelial differentiation. Cells were cultured for 7 days in KSFM with 0.07 mM Ca2+ without (KSFM) or with (FBS) 5% FBS or in KSFM with 0.9 mM Ca2+ without (Ca2+) or with (FBS+Ca2+) 5% FBS. Judged by the β-actin loading control, K12 was similarly expressed in these four conditions. In contrast, K10 was expressed only in high-Ca2+ medium without FBS. Such expression was dramatically decreased when serum was applied.
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