Differentiation in Cultured Limbal Epithelium as Defined by Keratin Expression

Transcription

1 Investigative Ophthalmology & Visual Science, Vol. 32, No. 12, November 1991 Copyright Association for Research in Vision and Ophthalmology Differentiation in Cultured Limbal Epithelium as Defined by Keratin Expression Akira Kiritoshi, Nirmala SundarRaj, and Richard A. Thoft The authors investigated differentiation of cultured corneal and limbal epithelial cells by immunochemically evaluating the changes in the profiles of keratins recognized by two monoclonal antibodies: AE5, which recognizes K3, and AEl, which recognizes a group of acidic keratins including K16, which is present in the hyperproliferative cells. After 1 and 2 weeks in culture, the human epithelial cells did not react with AE5 but did react strongly with AEl. At 3 weeks, only suprabasal cells exhibited a moderate reactivity with AE5, whereas AEl binding was seen in all of the cells. After 5 to 6 weeks in culture, all of the cells reacted moderately with AE5 and AEl. Treatment of 2-week-old limbally derived cultures with mitomycin C (mitosis inhibitor) did not inhibit subsequent K3 expression. Thus, K3 expression was associated with maturation or a later stage of differentiation that did not require an additional cell division. Unlike human epithelial cells, rabbit suprabasal epithelial cells expressed K3 (reactivity to AE5) after only ten days in culture. The epithelium derived from central human cornea lost K3 by 1 week in tissue culture but expressed keratin(s) recognized by AEl. Even after 4-6 weeks, cells derived from the central cornea did not become confluent and did not react with AE5. Thus, limbally derived human and rabbit epithelial cells undergo chronological changes in K3 expression similar to that seen in rabbit epithelial cells derived from central cornea. However, cultured human limbal epithelial cells take a significantly longer time to express K3 (a phenotypic characteristic of differentiated corneal epithelium) than do rabbit epithelial cells. Invest Ophthalmol Vis Sci 32: ,1991 Human comeal epithelium consists of from four to five layers of stratified cells with the apical layers continuously desquamated from the cornea. Thoft and Friend 1 proposed an "X,Y,Z hypothesis of corneal epithelial maintenance" in which the desquamated cells (Z) are replaced not only by the dividing basal cells (X) but also by the cells migrating in from the periphery (Y). The source of "Y" is believed to be the stem cells located in the basal epithelial layer in the limbus. Cotsarelis et al 2 recently demonstrated that there is a population of limbal basal cells (presumably the stem cells) that are normally slow cycling and can be preferentially stimulated to proliferate. Schermer et al 3 showed that a basic keratin (K3, Mr = 64 K) recognized by a monoclonal antibody designated AE5 and an acidic keratin (K12, Mr = 55 K) were located in the suprabasal layer in the limbus, as well as in all of From the Department of Ophthalmology, The Eye and Ear Institute of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Supported by National Institutes of Health grants EY and core grant EY08098 (R.A.T.), and EY (N.S), and grants from Research to Prevent Blindness Inc., New York, and The Eye and Ear Institute of Pittsburgh. Submitted for publication: July 6, 1990; accepted June 26,1991. Reprint requests: Richard A. Thoft, MD, The Eye And Ear Institute of Pittsburgh, 203 Lothrop Street, Pittsburgh, PA the layers in the central corneal epithelium in rabbit. Expression of these keratins was shown to be a characteristic of the suprabasal cells in rabbit epithelial cells growing in tissue culture. Thus, these two keratins have been regarded as markers of differentiation of the corneal epithelial cells. Another monoclonal antibody, AEl, which recognizes a group of acidic keratins, does not react with the basal cells but reacts with apical cells of the central cornea and with basal and suprabasal cells in the limbus. 4 ' 5 It has been demonstrated that AE1 recognizes a 48 K keratin present in a variety of hyperproliferative human diseases, 6 and this keratin also is found in cultured corneal epithelial cells, 7 indicating that AEl reactivity of corneal epithelial cells may be related to the hyperproliferative state of the cultured cells. We are investigating the use of limbally derived, cultured epithelial cells for transplantation to treat human ocular surface disease. Ebato et al 8 showed that under explant culture conditions, human limbal epithelial cells grow much better than the peripheral and central corneal epithelium. The adhesive properties of cultured cells possibly are altered as the cells undergo division and maturation in culture. In the present investigation, the time course of differentiation (maturation) of the limbal explant cultures was evaluated by analyzing the expression of K3 keratin (recognized by AE5).

2 0074 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / November 1991 Vol. 32 Materials and Methods Primary Cultures of Rabbit and Human Corneal Epithelium All of the experiments done with rabbit conform to the ARVO Resolution on the Use of Animals in Research. Adult New Zealand white rabbits were killed by an intravenous injection of Nembutal. The eyes were proptosed, and the corneas were excised, including the surrounding limbus. Donor human eyes deemed unsuitable for grafting were obtained from the Medical Eye Bank of Western Pennsylvania (Pittsburgh, PA). Using sterile techniques, corneal explants of 2-4 mm 2 were prepared from the limbal and central zones of the human eyes and the limbal zone of the rabbit eyes as described by Ebato et al. 8 Each explant was placed with the epithelial side up in a well of a 4-well Lab-Tek Chamber slide (Nunc Inc., Naperville, IL). The rest of the procedure was the same as that described by Ebato et al. For both rabbit and human epithelial cultures, modified SHEM medium that contained Hams F12 and Dulbecco's modified eagles medium (1:1) supplemented with mouse epidermal growth factor (10 ng/ml), insulin (5 Mg/ml), cholera toxin (0.1 tig/ml), glutamine (1/ig/ml), dimethyl sulfoxide (0.5%), and 15% fetal bovine serum was used. The cultures were incubated at 37 C in a 5% CO 2 :95% air atmosphere in a water-jacketed incubator. Medium was replaced with fresh medium once every 3 days. The explants were removed from the dishes after days. After 2 weeks, some of the cultures from limbal explants were treated for 30 min at 37 C with mitomycin C (Sigma, St. Louis, MO) at a concentration of 20 Mg/ml of culture medium. 9 Medium was then replaced with SHEM, and after an additional 3-14 days in culture, the cells were processed for immunostaining. Mitomycin C covalently binds with two sites, one on each of the complementary strands of DNA, inhibiting mitosis yet allowing metabolic activity. Immunofluorescence Staining of the Cultured Epithelial Cells At different times in culture, the cells were processed as follows for an indirect immunofluorescence staining. The medium from the cultures was removed, and the cells were gently rinsed with phosphate-buffered saline. The cells were fixed and permeabilized by treating them for 10 min with cold (-20 C) ethyl alcohol and acetone (1:1). The fixed cells were immunostained with hybridoma culture supernatants containing AE5 or AE1 3>4 (a gift from Dr. T-T. Sun), using a previously described procedure. 10 Frozen sections of human and rabbit cornea and limbus were included as positive controls for staining for AE5andAEl. The stained cells were viewed with an Olympus photomicroscope using epifluorescence objectives. Photomicrographs were taken at a fixed time exposure of 10 sec and developed and printed identically, allowing us to judge relative intensities of staining. Results Human Epithelium from Limbal Explants At 1 week in culture, the outgrowth of the cells from the human limbal explants had not reached confluence. These cells immunoreacted strongly with AE1 but did not immunostain with AE5 (Fig. 1, A and B). After 2 weeks in culture, outgrowths were confluent, but the pattern of reactivity with AE1 and AE5 had not changed. After 3 to 4 weeks in culture, while all of the cells continued to exhibit staining reactivity with AE1, only the suprabasal cells reacted moderately with AE5, as seen in Figures 1C and ID, respectively. After 5-6 weeks in culture, all of the cells reacted moderately with AE1 and AE5 (Fig. 1, E and F). Rabbit Epithelium from the Limbal Explants Although different culture conditions were used in this study, rabbit epithelial cells exhibited the chronological pattern of staining that has been reported in the literature. 3 At 7 days in culture, rabbit limbal epithelial cell outgrowths did not react with AE5 but did react strongly with AE 1. At 10 days, only the suprabasal cells in the outgrowth reacted with AE5, whereas reactivity with AE1 was present in all of the cells. After 2 weeks in culture, all of the cells continued to react with AE1 and also reacted strongly with AE5 as shown in Figure 2, A and B. The chronological changes in the immunostaining profiles of keratins in cultured limbal epithelial cells are summarized in Table 1. Limbal Epithelial Cultures: Effect of Mitomycin C Treatment Mitomycin C treatment of epithelial outgrowth of human limbal explants at 2 weeks in culture was used to inhibit further mitosis while allowing the cells to remain metabolically active. As described, at 2 weeks in culture, the outgrowths were confluent and did not react with AE5 but did react with AE1. Three days after mitomycin C treatment, AE5 staining was not evident, but moderate staining was seen with AE1. However, 7-10 days after the treatment, the suprabasal cells exhibited reactivity with AE5. After 2 weeks,

3 No. 12 KERATINS IN DIFFERENTIATION OF CULTURED LIMDAL EPITHELIUM / Kiriroshi er QI 3075 Fig. 1. Immunofluorescence staining of human cornea! epithelial cultures derived from limbal explants. The outgrowths of epithelial cells after the following different times in culture were immunostained with AEl (A,C,E) and AE5 (B,D,F) using an indirect immunofluorescence technique: A and B, 1 week; C and D, 4 weeks; and E and F, 6 weeks. Note a strong immunostaining with AE 1 of cells in culture at 1 week and relatively moderate level of staining at 4 and 6 weeks. Bar = 75 f±m. all of the cells in the outgrowth reacted moderately with AE 1 and AE5, as judged from the staining shown in Figure 3, A and B. during this time. Outgrowths from central cornea did not reach confluence even after 6 weeks in culture. Discussion Human Epithelial Cultures from Central Cornea Central corneal cells in vivo react with AE5. However, the reactivity with AE5 was lost in the outgrowths of these cells in explant culture, even after 4-6 weeks in culture. Reaction with AEl continued In this study, we evaluated the state of differentiation of cultured rabbit and human corneal epithelial cells using as markers, keratin K3 (recognized by monoclonal antibody AE5)3 and other keratin(s) recognized by another monoclonal antibody, AEl.4 The

4 3076 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / November 1991 Fig. 2. Immunofluorescence staining of rabbit corneal epithelial cultures derived from limbal explants. The outgrowths of epithelial cells after 2 weeks in culture were immunostained with AE1 (A) and AE5 (B) using an indirect immunofluorescence technique. Bar = 75 /im. absence of K3 in the basal cells in the limbus but its presence in all of the epithelial cells in the central cornea make this keratin a good marker for an advanced state of corneal epithelial differentiation, both in vivo and in cultured rabbit epithelial cells.3 However, AE1 reacts with basal and suprabasal cells in the limbus and not in the central cornea.4'5 Thus, we speculated that an inverse pattern of distribution of keratins rectable 1. Keratin immunostaining of cultured human and rabbit epithelial cells derived from limbal explants Time of incubation Monoclonal antibody 7 days 10 days 2 wk 3 wk 4 wk 5 wk 6 wk Human epithelium AE1 AE5 ND ND - * * ND ND ND ND ND ND ND ND * Rabbit epithelium AE1 AE5, strong immunoreactivity;, moderate reactivity compared with ; -, no immunostaining; ND, not determined. * Immunostaining of only suprabasal cells. Vol. 3 2 Fig. 3. Immunofluorescence staining of limbally derived human epithelial cells after mitomycin C treatment. The outgrowths were treated with mitomycin C at 2 weeks in culture, incubated for an additional 2 weeks, and then immunoreacted with AE1 (A) and AE5 (B) using an indirect immunofluorescence technique. Note that the cells react with both AE1 (A) and AE5 (B). Bar = 75 *im. ognized by AE5 and AE1 may exist in corneal epithelial cells at different stages of epithelial differentiation, with AE5 staining representing cells at a more advanced stage of differentiation. Schermer et al3 studied K3 expression by cultured rabbit epithelial cells derived from central cornea using feeder cells to support their growth. In our study, a similar pattern of sequential changes in K3 expression was seen in the rabbit epithelial cells of limbal origin grown without feeder layers. By 10 days in culture, the suprabasal cells had started expressing K3 (AE5 staining), and all of the cells reacted strongly with AE1. After 14 days, all of the cells reacted strongly with AE5, but AE1 staining was reduced. Limbally derived human corneal epithelial cells also showed similar sequential changes. However, a significantly longer time was required for them to reach an advanced stage of differentiation as determined by comparing the staining pattern with AE5 and AE1. Based on these observations it was concluded that the sequential changes that occur in keratin phenotype during the in vivo differentiation of TV -iv -

5 No. 12 KERATINS IN DIFFERENTIATION OF CULTURED LIMDAL EPITHELIUM / Kiriroshi er ol 3077 limbal cells to corneal epithelial cells are mimicked by the cells grown in culture from limbal explants of rabbit, as well as human, eyes. The human epithelial cultures from the central cornea behaved differently than cultures derived from limbus. K3 expression was lost once these cells were cultured, and they did not regain the phenotype of mature corneal epithelial cells with respect to K3 expression. One of the reasons for this difference between the reported findings on K3 expression in rabbit central corneal epithelial cells and human corneal epithelial cultures derived from the central cornea may be that the human cultures did not achieve confluence and did not form multiple layers. Another possibility is that the majority of human central corneal epithelial cells are already in an advanced stage of differentiation and have irreversibly lost the ability to transcribe or translate messages for K3 synthesis. If the latter hypothesis is true, the in vivo presence of K3 in all of the layers in the central cornea may be attributable to a slow turnover rate of K3. Inhibition of mitosis by mitomycin C-treatment of limbal cells at 2 weeks in culture did not prevent subsequent expression of K3. This finding indicated that the ability of the differentiated corneal epithelial cells to express K3 is not necessarily acquired immediately after mitosis of nondifferentiated limbal epithelial cells. Thus, the expression of K3 may represent a phenotype of an advanced state of differentiation (maturation). We are investigating the possibility of using cultured epithelial sheets for ocular surface transplantation. Information discussed in this communication will be used to analyze the relationship between the length of time in culture, the state of differentiation, and the adhesive properties of the cultured limbal epithelial cells. Key words: corneal epithelium, limbal epithelium, cell culture, keratins, differentiation Acknowledgments The authors express their gratitude to Tung-Tien Sun, PhD, for providing the monoclonal antibodies to keratins used in this study and also for his encouragement. References 1. Thoft RA and Friend J: The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci 24:1442, Cotsarelis G, Cheng S-Z, Dong G, Sun T-T, and Lavker R: Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells. Cell 57:201, Schermer A, Galvin S, and Sun T-T: Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol 103:49, Tseng SCG, Jarvinen JJ, Nelson WG, Huang JOW, Woodcock-Mitchell J, and Sun T-T: Correlation of specific keratins with different types of epithelial differentiation: Monoclonal antibody studies. Cell 30:361, Wiley L, SundarRaj N, Sun T-T, and Thoft RA: Regional heterogeneity in human corneal and limbal epithelia: An immunohistochemical evaluation. Invest Ophthalmol Vis Sci 32:594, Weiss RA, Eichner R, and Sun T-T: Monoclonal antibody analysis of keratin expression in epidermal diseases: A 48 kd and a 56 kd keratin as molecular markers for keratinocyte hyperproliferation. J Cell Biol 98:1397, Doran TI, Vidrich A, and Sun T-T: Intrinsic and extrinsic regulation of the differentiation of skin, corneal and esophageal epithelial cells. Cell 22:17, Ebato B, Friend J, and Thoft RA: Comparison of central and peripheral human corneal epithelium in tissue culture. Invest Ophthalmol Vis Sci 28:1450, MacPherson I and Bryden A: Mitomycin C treated cells as feeders. Exp Cell Res 69:240, SundarRaj N, Martin J, and SundarRaj CV: Cell-surface associated proteins of cornealfibroblasts:dissection with monoclonal antibodies. J Cell Biochem 21:277, 1983.