IOVS Annual Reviews: Physical Science Suite
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2003;44:2959-2965.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0776
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Galindo, E. E. H.
Right arrow Articles by Meller, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Galindo, E. E. H.
Right arrow Articles by Meller, D.

Expression of {Delta}Np63 in Response to Phorbol Ester in Human Limbal Epithelial Cells Expanded on Intact Human Amniotic Membrane

Elias E. Hernandez Galindo,1 Carsten Theiss,2 Klaus-P. Steuhl,1 and Daniel Meller1

1From the Department of Ophthalmology, University of Essen, Essen, Germany; and the 2Department of Cytology, Institute of Anatomy, University of Bochum, Bochum, Germany.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To evaluate the effect of phorbol 12-myristate 13-acetate (PMA) on the expression of {Delta}Np63 in human limbal epithelial cells (HLECs) during ex vivo expansion on amniotic membrane (AM).

METHODS. Primary HLECs were cultured either on AM or plastic surfaces and were treated with 1 µg/mL PMA for 24 hours. Expression of {Delta}Np63 and the differentiation-associated gap junctional protein connexin 43 (Cx43) were studied by laser scanning microscopy.

RESULTS. The labeling index (LI) of {Delta}Np63 was higher in HLECs cultured on AM than in HLECs grown on plastic (81.4% ± 12.2% and 66.6% ± 16.5%, respectively; P < 0.001). After PMA treatment, {Delta}Np63 expression in HLECs on plastic dramatically decreased to 20.4% ± 11.4%. However, HLECs cultured on AM showed only a moderate decrease in {Delta}Np63 expression (56.4% ± 10.9%, P < 0.001) after PMA treatment. It was also observed that 72.8% ± 17.5% of the {Delta}Np63-positive cells in untreated HLECs cultured on plastic coexpressed Cx43, in contrast to only 21.9% ± 3.7% of the {Delta}Np63-positive cells in HLECs cultured on AM (P < 0.001). The latter indicates that growth over AM preserves limbal phenotype, whereas growth over plastic surface induces or allows transition toward corneal peripheral phenotype.

CONCLUSIONS. {Delta}Np63 protein is typically detected in human corneal epithelial cells with high proliferative capacity including, limbal epithelial stem cells (SCs) and probably also transient amplifying cells (TACs). AM supports {Delta}Np63 protein expression in HLECs and maintains a higher resistance against phorbol ester-induced differentiation, indicating that characteristic signs of limbal epithelial progenitor cells may be preserved during ex vivo expansion on AM.

The protein p63, which has been recently identified as a very close homologue of the prototypical tumor suppressor p531 2 has been shown to be transcribed into at least six different protein isotypes with various functions. In contrast to p53, p63 exhibits a rather tissue-specific distribution.1 2 These transcripts are divided into two groups that are distinguished by the presence or absence of an acidic NH2-terminal transactivation domain. Protein isotypes containing an acidic NH2-terminal transactivation domain (TAp63) are capable of inducing apoptosis,3 4 whereas those without the NH2-terminal transactivation domain may function as dominant-negative p63 isotypes ({Delta}Np63). In addition, alternative splicing of the p63 gene leads to three different C-terminal forms, {alpha}, ß, and {gamma}, for both TAp63 and {Delta}Np63.1 2 {Delta}Np63{alpha} has been reported to be strongly expressed in the epidermis5 6 7 and is selectively expressed in the basal cell compartment of a variety of epithelial tissues (e.g., cervix, urogenital tract, prostate, breast,5 6 7 8 9 10 and cornea).11 Therefore, it has been suggested that {Delta}Np63{alpha} may be essential in the maintenance of a stem cell (SC) population in various epithelial tissues. Furthermore, {Delta}Np63 has been also implicated in the control of epithelial cell proliferation and migration during wound healing.7 9

SCs maintain tissue integrity by continuous generation of cells destined to self-renewal of the target tissue. SCs are long-living, relatively undifferentiated cells that exhibit a high potential for cell division (i.e., they are clonogenic cells). SCs exhibit a very low mitotic activity and thus are slow cycling. Replication of SCs gives rise to, on average, one SC that remains in the SC environment and one transit amplifying cell (TAC) that replicates relatively rapidly but has only a limited proliferative potential and therefore has a short life span.12 13 14 Recent cumulative evidence indicates that SCs of the corneal epithelium are localized in the limbus, the narrow transitional zone between the cornea and the bulbar conjunctiva.12 15 16 17 18 19 20 Three subpopulations of limbal epithelial cells have been distinguished in vitro21 22 : holoclones, which correspond to SCs; paraclones, which contain most of the TACs; and meroclones, which are considered to represent early, young TACs with a greater proliferative capacity than TACs found in paraclones.21 22 Pellegrini et al.11 observed that the {Delta}Np63 transcription factor is expressed strongly in holoclones (SC) and weakly in meroclones (early TACs), but is absent in paraclones (TACs), indicating that {Delta}Np63 may be essential in the maintenance of corneal epithelial SC population. Therefore, the level of {Delta}Np63, but not the presence, can serve as an indicator to cell stemness.

Previous studies23 24 have shown that cultured epidermal cells have divergent responses toward tumor promoter phorbol esters characterized by proliferation of less differentiated cells, whereas more mature cells are induced to accelerated terminal differentiation. Moreover, in vitro studies25 26 have identified that limbal epithelial SCs are also more resistant to tumor promoter phorbol esters than TACs. Furthermore, phorbol esters induce corneal epithelial cells to proliferate and thereby exhaust the proliferative capacity of TACs that finally undergo terminal differentiation. In contrast, SCs preserve their proliferation capacity after withdrawal of phorbol ester.25 26 This effect is commonly used to select SCs in vitro by means of reduction or elimination of the TAC subpopulation.

Various ocular surface disorders are characterized by limbal SC deficiency (for review see also Refs. 17 19 27 ) and the renewal of the limbal epithelial SC population is required for restoration of vision and normal corneal surface (for reviews see also Refs. 28 29 30 ). Recent advances in tissue engineering techniques have facilitated the ex vivo expansion of HLECs using different culture substrates or adjuncts such as amniotic membrane (AM), fibrin, or 3T3 feeder layer systems.31 32 33 34 35 36 37 38 39 40 41 Ex vivo expansion of autologous and allogenic HLECs on AM has been considered an attractive technology for its potency in a variety of clinical applications.32 33 35 36 Moreover, experimental studies have shown that outgrowth rate, cell-cycle kinetics, and cell phenotype characteristic of limbal and conjunctival epithelial progenitor cells are preserved during ex vivo expansion on AM,38 39 42 43 supporting the assumption that this tissue engineering technique might be useful to renew SC populations of the entire ocular surface.

In the present study we sought to study whether characteristic features of the limbal epithelial progenitor cells are preserved using this tissue engineering technique. To this end, we determined the expression of {Delta}Np63 protein in HLECs during ex vivo expansion on preserved human AM and the effect of phorbol 12-myristate 13-acetate (PMA) treatment on {Delta}Np63 expression. The data indicates that AM supports {Delta}Np63 protein expression in HLECs and maintains a higher resistance against phorbol ester-induced differentiation indicating that characteristic signs of limbal epithelial progenitor cells may be preserved during ex vivo expansion on AM.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Materials
Dulbecco’s modified Eagle medium (DMEM), Ham’s F-12 nutrient medium, HEPES-buffer solution, amphotericin B, Hank’s balanced salt solution (HBSS), and fetal bovine serum (FBS) were purchased from Invitrogen GmbH (Karlsruhe, Germany). Dimethyl sulfoxide (DMSO), cholera toxin (subunit A), hydrocortisone, epidermal growth factor (EGF), Dispase II, gentamicin, insulin-transferrin-sodium selenite media supplement, poly-L-lysine, bovine serum albumin (BSA), RNase A, glycerol, saline sodium citrate (SSC) buffer, PMA, and embedding medium (Moviol) were obtained from Sigma-Aldrich Chemie GmbH (Munich, Germany). The nylon transfer membrane (Hybond-N+, pore size: 0.45 µm) was obtained from Amersham Biosciences (Freiburg, Germany). The tissue culture plastic plates (six-well) and plastic culture dishes were from Falcon (BD Biosciences, Bedford, MA). Culture plate inserts used to construct the culture system were from Millipore (Eschborn, Germany). Mouse monoclonal antibody against {Delta}Np63 protein,8 9 clone 4A4, was purchased from BD Biosciences-PharMingen (Heidelberg, Germany) and an affinity purified rabbit polyclonal antibody to Cx43 was obtained from Alpha Diagnostic International (San Antonio, TX). Fluorescence-conjugated (Alexa Fluor 546) goat anti-mouse exhibiting a orange fluorescence, fluorescence-conjugated (Alexa Fluor 633) goat anti-rabbit (red fluorescence), and green nucleic acid stain (SYTO 16) were obtained from Molecular Probes, Leiden, The Netherlands.

Tissue Procurements
Human tissue was handled according to the tenets of the Declaration of Helsinki. Corneoscleral tissue from human donor eyes, aged between 30 to 50 years, was obtained from the Cornea Bank of Essen (Germany). After obtaining proper informed consent, AM was processed and preserved according to Tseng et al.44 Briefly, the human placenta was collected shortly after elective cesarean delivery. Human immunodeficiency virus, human hepatitis virus types B and C, and syphilis were excluded by serologic tests. In addition, hepatitis virus type C was excluded by PCR. Under a lamellar-flow hood, the placenta was cleaned of blood clots with sterile phosphate-buffered saline (PBS) solution (pH 7.4) containing 50 µg/mL penicillin, 50 µg/mL streptomycin, 100 µg/mL neomycin, and 2.5 µg/mL amphotericin B (Invitrogen GmbH). The amnion was separated from the chorion by blunt dissection and flattened onto a positively charged nylon transfer membrane (Hybond-N+; Amersham Biosciences) with the epithelium-basement membrane surface up. The nylon membrane with the adherent AM was stored at -80°C in a sterile vial containing DMEM (Invitrogen) and sterile glycerol (Sigma-Aldrich Chemie GmbH) at the ratio of 1:1 (vol/vol).

Three-Dimensional Cultures on AM
After careful removal of excessive sclera, iris, corneal endothelium, conjunctiva, and Tenon’s capsule, the remaining tissue was placed in a culture dish, and limbus explants of approximately 1 x 1.5 x 2.5 mm were cut with a scalpel. Preserved human intact AM was fastened onto a culture insert as recently reported.38 On the center of the AM a limbal explant was placed and cultured in supplemental hormonal epithelial medium (SHEM) consisting of an equal volume of HEPES-buffered DMEM containing bicarbonate and Ham’s F12. The medium was supplemented with 5% FBS, 0.5% DMSO, 2 ng/mL mouse EGF, 5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL selenium, 0.5 µg/mL hydrocortisone, 30 ng/mL cholera toxin, 5% FBS, 50 µg/mL gentamicin, and 1.25 µg/mL amphotericin B.38 43 45 Cultures were incubated at 37°C, 5% CO2, and 95% humidity for 4 to 5 weeks. A set of cultures was treated with medium containing 1 µg/mL PMA for 24 hours and afterward switched to PMA-free medium for 3 days. All cell cultures were terminated and fixed in 4% paraformaldehyde PBS for 30 minutes. Four cultures per group were included in the present study. Air lifting of cell cultures, as described by several groups,34 45 was avoided to minimize the promotion of differentiation processes in vitro.

HLECs cultured on plastic dishes were used as the control group. Although the interactions of HLECs with the substrate by adhesion proteins and the effect of these on development, growth, and differentiation of epithelial cells are important elements to consider, conventional cultures on plastic were chosen to demonstrate the suitability of AM as a substrate and carrier for transplantation in ocular surface tissue engineering techniques.

Immunofluorescence Studies and Confocal Laser Scanning Microscopy
Ten-micrometer-thick sections of cornea were cut at -20°C with a cryotome (Kryostat 1720; Leica, Heidelberg, Germany), collected on poly-L-lysine-coated glass slides and fixed in 4% paraformaldehyde in PBS for 30 minutes. Corneal sections and cell cultures were pretreated with 1% Triton X-100 for 10 minutes. In addition, permeabilization was performed with 10% methanol for 10 minutes. BSA (3%) was applied for 1 hour at room temperature to block nonspecific binding sites. Afterward, the specimens were incubated overnight with mouse monoclonal antibody against {Delta}Np63 at 1:100 in PBS. After extensive washing in PBS, the specimens were incubated for 1 hour with a secondary fluorescence-conjugated goat anti-mouse antibody (1:400; Alexa Fluor 546; Molecular Probes) that exhibits an orange to red fluorescence. Thereafter, specimens were treated with RNase A in 2x SSC buffer for 30 minutes at 37°C. Green fluorescent nucleic acid stain (SYTO; Molecular Probes) was used as a nuclear counterstain. Finally, sections were mounted with in embedding medium (Moviol; Sigma-Aldrich Chemie, GmbH) and images were obtained by means of a confocal laser scanning microscope (LSM 510; Carl Zeiss Meditech, Oberkochen, Germany). The {Delta}Np63 expression was calculated by counting positive nuclei in at least 10 200 x 200-µm2 areas of each HLEC culture (n = 4), and the labeling index (LI) of {Delta}Np63 was displayed as the number of {Delta}Np63 positive cells/total number of cells x 100%.

Coexpression of {Delta}Np63 and Cx43 was shown by incubating HLEC cultures with mouse anti-p63 and a rabbit affinity purified polyclonal antibody against Cx43 (both at 1:100 in PBS, overnight). Afterward, specimen were incubated with fluorescence-conjugated goat anti-mouse (1:400; Alexa Fluor 546; Molecular Probes) for 1 hour, followed by an incubation with fluorescence-conjugated goat anti-rabbit (Alexa Fluor 633; Molecular Probes; 1:400 for 1 hour; dark red fluorescence). Quantitative analysis of {Delta}Np63 and Cx43 expression between the described experimental groups was performed using the same recording parameters (objective, stack size, pinhole, gain amplitude, and contrast) on the laser scanning microscope. LI of Cx43 was displayed as number of Cx43-positive cells/total number of cells x 100%. In addition, the number of {Delta}Np63-positive cells coexpressing Cx43 (Cx43 and {Delta}Np63 positive cells/total number of {Delta}Np63-positive cells x 100) was calculated.

Statistical Methods
The statistical analysis was performed by an ANOVA (analysis of variance) with an additional post hoc analysis (the Scheffé test).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Distribution of {Delta}Np63 and Cx43 in Human Limbus and Cornea
Analysis of the {Delta}Np63 expression pattern revealed a strong nuclear labeling in basal epithelial cells of the limbus (Fig. 1A) , whereas {Delta}Np63 was almost absent in suprabasally located limbal epithelial cells. Labeling was occasionally detected in basal epithelial cells of the peripheral cornea (Figs. 1A 1B) and absent in the central cornea.



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 1. Expression of {Delta}Np63 protein (A, B) and Cx43 (C, D) in human limbal and corneal epithelial cells. (A) In vivo, nuclear {Delta}Np63 staining was strongly detected in the basal layer of limbal epithelium and weakly in basal epithelial cells of the peripheral cornea. Arrow: border between limbus (left) and cornea (right). Inset: A counterstain of the presented area. (B) {Delta}Np63 was weakly detected in basal epithelial cells of peripheral cornea. A counterstain of the presented area is illustrated in the inset. (C, D) Subpopulations (arrows) of the basal limbal epithelium did not express Cx43; however, strong punctate labeling of Cx43 (red) was observed in basal and suprabasal epithelial cells of the peripheral cornea (D). Cell nuclei were counterstained with SYTO-16 (green). Scale bar, 50 µm.

 
Subpopulations of basal limbal epithelial cells were devoid of Cx43, with isolated and faint Cx43-positive labeling of suprabasal epithelial cells (Fig. 1C) . However, in the cornea, a strong and punctate membrane-bounded pattern for Cx43 was predominantly observed in the basal epithelial cell layer and to lesser extent in the suprabasal epithelial cells (Fig. 1D) .

Expression of {Delta}Np63 Protein in Ex Vivo Expanded HLECs before and after PMA Treatment
Next, we investigated the influence of PMA-induced differentiation on {Delta}Np63 expression. The data showed a high and homogeneous expression of {Delta}Np63 protein in HLECs expanded on AM prior and after PMA exposure (Table 1) . The LI for {Delta}Np63 in the untreated group (Fig. 2A 2B ; PMA-) was higher in HLECs cultured on AM than in HLECs cultured on plastic (81.4% ± 12.1% and 66.6% ± 16.5%, respectively, P < 0.001). After a 24-hour treatment with PMA, decreased {Delta}Np63 expression was evidenced in both groups (Figs. 2C 2D , Table 1 ). HLECs cultured on AM showed a higher resistance to phorbol ester than HLECs grown on plastic, which was reflected by a significantly higher LI for {Delta}Np63 in HLECs cultured on AM (56.4% ± 10.9%) compared with HLECs grown on plastic (20.42% ± 11.4%, P < 0.001, Table 1 ).


View this table:
[in this window]
[in a new window]
  		TABLE 1. The Effect of PMA on the LI of {Delta}Np63 in HLECs

 


View larger version (62K):
[in this window]
[in a new window]
  		FIGURE 2. Confocal laser scanning microscopy of {Delta}Np63 expression in HLECs on AM or plastic. Immunoreactivity of {Delta}Np63 protein (orange-red) is higher in HLECs cultured on AM (A) compared with HLECs cultured on plastic (B). HLECs on AM (C) showed a higher resistance against the phorbol ester PMA compared with HLECs on plastic (D). Cell nuclei were counterstained with nucleic acid stain (green). Scale bar, 50 µm.

 
We also noted some topographic differences of the LI for {Delta}Np63 in the different cell populations. {Delta}Np63 expression in HLECs on AM was homogenous, whereas {Delta}Np63 labeling of HLECs cultured on plastic was reduced from the center of the organ culture to the periphery of the culture (Table 1) . LI of {Delta}Np63 in the central and peripheral areas of untreated HLEC cultures grown on plastic was 72.8% ± 18.1% and 60.4% ± 12.2%, respectively (P < 0.05) indicating increased differentiation processes of HLECs toward peripheral culture compartments. After PMA treatment, this difference remained consistent (LI = 26.9% ± 8.6% for central and 13.9% ± 10% for peripheral areas; P < 0.05, Table 1 ).

Colocalization Studies of {Delta}Np63 and Cx43 in HLECs
The observed response of HLECs to PMA suggested the existence of a subpopulation of {Delta}Np63-positive cells that are sensitive for PMA. Therefore, these cells might represent early TAC, which also express {Delta}Np63 as previously suggested by Pellegrini et al.11 To identify this subpopulation of cells in culture, we examined coexpression of {Delta}Np63 with the differentiation-associated marker Cx43 (Table 2) . In the untreated groups, a higher expression of Cx43 was observed in HLECs cultured on plastic (63.1% ± 21.5%) than in HLECs on AM (20.0% ± 2.9%, P < 0.001, Table 1 ) confirming our previous data.42 Moreover, Cx43 appeared as small, immunoreactive dots in HLECs cultured on AM that rarely encircled cells, whereas HLECs cultured on plastic exhibited Cx43-immunoreactive signals that had an elongated appearance and clearly encircled the cells. Of note, 72.8% ± 17.5% of {Delta}Np63-positive cells in untreated HLECs cultured on plastic were labeled by Cx43, in contrast to only 21.9% ± 3.7% in HLECs grown on AM (Figs. 3A 3B ; P < 0.001). This indicates that growth over AM preserves limbal phenotype, whereas growth over plastic surface induces or allows transition toward corneal peripheral phenotype.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Colocalization LI of {Delta}Np63 and Cx43

 


View larger version (40K):
[in this window]
[in a new window]
  		FIGURE 3. Coexpression of {Delta}Np63 and Cx43 in HLECs on AM or plastic. (A) The coexpression of Cx43 (red) and {Delta}Np63 (blue, pseudocolor for fluorescent stain [Alexa Fluor 546; Molecular Probes, Leiden, The Netherlands]) was significantly higher in HLECs cultured on plastic (B) than on AM (A). Cell nuclei were counterstained with nucleic acid stain (green). Scale bar, 50 µm.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The p63 gene encodes at least six different protein isotypes resulting from alternative splicing and the use of an alternative promoter. The {Delta}Np63 isotypes that lack the NH2-terminal transactivation domain block some of the activities of the corresponding full-length proteins.1 For instance, {Delta}Np63 isotypes have been shown to exhibit antiapoptotic properties in UV-induced epidermal damage.4 Expression of p63 appears to vary at different stages of growth and differentiation in a variety of epithelial tissues. Moreover, cell differentiation typically triggers an increase of TAp63 levels and a concomitant decrease of dominant-negative {Delta}Np63.1 7 46 In accordance, {Delta}Np63 has been reported to be strongly expressed in basal epithelial cells of different tissues, such as cervix, urogenital tract, prostate, breast,5 6 7 8 9 and cornea.11 Therefore, it has been suggested that {Delta}Np63{alpha} may play an important role in the maintenance of SC populations in various epithelial tissues.

In the present study, we evaluated the expression pattern of {Delta}Np63 in HLECs ex vivo, cultured on different substrates and the effect of PMA on {Delta}Np63 expression. Furthermore, colocalization of {Delta}Np63 and the differentiation-associated antigen Cx43 was examined. The herein presented data on {Delta}Np63 expression in human cornea and limbus confirm previously published observations.11 No {Delta}Np63 expression was observed in the human central cornea, but it was occasionally observed in basal cells of the peripheral cornea. These cells may represent young, or "early", TACs that have been reported also to express {Delta}Np63.11 However, {Delta}Np63 labeling of basal and suprabasal limbal epithelial cells was compared with adjacent cells gradually more dense as recently reported,11 which may be attributed to the higher sensitivity of the microscopy system used in this study.

Based on the notion that {Delta}Np63 does not selectively label SCs but also labels young TACs,11 we used PMA to eliminate {Delta}Np63-positive young TACs. This is reflected by the dramatic decrease of LI for {Delta}Np63 in HLECs cultured on plastic compared with HLECs cultured on AM, which indicates that HLECs cultured on AM are more resistant to phorbol ester, a characteristic feature of limbal epithelial progenitor cells.25 26

A simple classification of the progeny of limbal epithelial cells based on the discrimination between strong or weak labeling for {Delta}Np63 is not sufficient for characterization of these cells. We therefore performed double labeling with {Delta}Np63 and the well-established differentiation associated antigen Cx43. Cx43 is known to be absent in limbal epithelial SCs (Fig. 1C) whereas it is predominantly expressed in TACs localized in limbal suprabasal epithelium and/or corneal basal epithelium.47 48

These studies indicate three subpopulations of HLECs in our cell cultures: cells positive for {Delta}Np63 only, cells positive for Cx43 only, and cells with a simultaneous expression of {Delta}Np63 and Cx43. According to recent publications,11 47 48 HLECs with an exclusive {Delta}Np63 labeling may represent SCs, whereas HLECs labeled with Cx43 only may be mature TACs. The simultaneous labeling for both antigens may indicate young TACs, which still retain a high but limited proliferative capacity and, in contrast to SCs, have already entered into the process of cellular differentiation as indicated by the expression of the differentiation-associated marker Cx43. These observations imply that colocalization of different antigens, as demonstrated by double-labeling and laser confocal microscopy, facilitates the determination of certain cell phenotypes of the spectrum of epithelial cell progeny.

Furthermore, the most {Delta}Np63-positive HLECs cultured on plastic compared with HLECs cultured on AM were also Cx43 positive, indicating that the substrate specifically regulates the state of differentiation. It is important to point out that the {Delta}Np63-positive subpopulation of HLECs cultured on AM was predominantly free of Cx43 expression and therefore presumably contained SCs, in contrast to the apparent predominance of TACs in HLECs on plastic. However, further studies are needed to establish a more reliable method that may specifically identify SCs.

Our data also suggest that conventional plastic cell culture dishes provide a relatively unfavorable environment for the growth of epithelial cells. This dramatic contrast suggests the AM culture model as a superior carrier for transplantation of ex vivo-expanded HLECs. However, other culture substrates, such as the 3T3 fibroblast feeder layer31 33 49 50 and fibrin,37 should be evaluated to compare benefits and disadvantages between these substrates and AM.

In conclusion, in comparison with cell culture on plastic, culture on AM supports {Delta}Np63 protein expression of HLECs and maintains a higher resistance against phorbol ester. This indicates that characteristic features of limbal epithelial progenitor cells may be preserved during ex vivo expansion on AM, whereas they are suppressed on plastic surfaces. These data support the use of AM as substrate for tissue engineering and an alternative therapeutic strategy for corneal surface reconstruction in distinct ocular surface diseases.


   Acknowledgements
 
The authors thank Karl Meller (Department of Cytology, Institute of Anatomy, University of Bochum, Bochum, Germany) for helpful discussions of the manuscript.


   Footnotes
 
Presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2002.

Supported in part by DAAD-FUNDAYACUCHO (A/99/25975), Bonn, Germany, and IFORES, Essen, Germany.

Submitted for publication August 1, 2002; revised January 1 and January 29, 2003; accepted February 16, 2003.

Disclosure: E.E. Hernandez Galindo, None; C. Theiss, None; K.-P. Steuhl, None; D. Meller, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Daniel Meller, Department of Ophthalmology, University of Essen, 45122 Essen, Germany; daniel.meller{at}uni-essen.de.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Irwin, MS, Kaelin, WG. (2001) p53 family update: p73 and p63 develop their own identities Cell Growth Diff 12,337-349[Free Full Text]
  2. Strano, S, Rossi, M, Fontemaggi, G, et al (2001) From p63 to p53 across p73 FEBS Lett 490,163-170[CrossRef][Medline][Order article via Infotrieve]
  3. Flores, ER, Tsai, KY, Crowley, D, et al (2002) p63 and p73 are required for p53-dependent apoptosis in response to DNA damage Nature 416,560-564[CrossRef][Medline][Order article via Infotrieve]
  4. Liefer, KM, Koster, MI, Wang, XJ, et al (2000) Down-regulation of p63 is required for epidermal UV-B-induced apoptosis Cancer Res 60,4016-4020[Abstract/Free Full Text]
  5. Yang, A, Kaghad, M, Wang, Y, et al (1998) p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities Mol Cell 2,305-316[CrossRef][Medline][Order article via Infotrieve]
  6. Yang, A, Schweitzer, R, Sun, D, et al (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development Nature 398,714-718[CrossRef][Medline][Order article via Infotrieve]
  7. Parsa, R, Yang, A, McKeon, F, Green, H. (1999) Association of p63 with proliferative potential in normal and neoplastic human keratinocytes J Invest Dermatol 113,1099-1105[CrossRef][Medline][Order article via Infotrieve]
  8. Bamberger, C, Pollet, D, Schmale, H. (2002) Retinoic acid inhibits downregulation of DeltaNp63alpha expression during terminal differentiation of human primary keratinocytes J Invest Dermatol 118,133-138[CrossRef][Medline][Order article via Infotrieve]
  9. Noszczyk, BH, Majewski, ST. (2001) p63 expression during normal cutaneous wound healing in humans Plast Reconstr Surg 108,1242-1247[CrossRef][Medline][Order article via Infotrieve]
  10. Di Como, CJ, Urist, MJ, Babayan, I, et al (2002) p63 expression profiles in human normal and tumor tissues Clin Cancer Res 8,494-501[Abstract/Free Full Text]
  11. Pellegrini, G, Dellambra, E, Golisano, O, et al (2001) p63 identifies keratinocyte stem cells Proc Natl Acad Sci USA 98,3156-3161[Abstract/Free Full Text]
  12. Lehrer, MS, Sun, TT, Lavker, RM. (1998) Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation J Cell Sci 111,2867-2875[Abstract]
  13. Potten, CS, Loeffler, M. (1990) Stem cells: attributes, cycles, spirals, pitfalls and uncertainties: lessons for and from the crypt Development 110,1001-1020[Abstract/Free Full Text]
  14. Lavker, RM, Sun, TT. (1983) Epidermal stem cells J Invest Dermatol 81,121S-127S[CrossRef][Medline][Order article via Infotrieve]
  15. Schermer, A, Galvin, S, Sun, T-T. (1986) 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 103,49-62[Abstract/Free Full Text]
  16. Cotsarelis, G, Cheng, SZ, Dong, G, et al (1989) Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells Cell 57,201-209[CrossRef][Medline][Order article via Infotrieve]
  17. Tseng, SCG. (1996) Regulation and clinical implications of corneal epithelial stem cells Mol Biol Rep 23,47-58[CrossRef][Medline][Order article via Infotrieve]
  18. Lavker, RM, Sun, TT. (2000) Epidermal stem cells: properties, markers, and location Proc Natl Acad Sci USA 97,13473-13475[Free Full Text]
  19. Dua, HS, Azuara-Blanco, A. (2000) Limbal stem cells of the corneal epithelium Surv Ophthalmol 44,415-425[CrossRef][Medline][Order article via Infotrieve]
  20. Daniels, JT, Dart, JK, Tuft, SJ, Khaw, PT. (2001) Corneal stem cells in review Wound Repair Regen 9,483-494[CrossRef][Medline][Order article via Infotrieve]
  21. Barrandon, Y, Green, H. (1987) Three clonal types of keratinocytes with different capacities for multiplication Proc Natl Acad Sci USA 84,2302-2306[Abstract/Free Full Text]
  22. Pellegrini, G, Golisano, O, Paterna, P, et al (1999) Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface J Cell Biol 145,769-782[Abstract/Free Full Text]
  23. Yuspa, SH, Ben, T, Hennings, H. (1983) The induction of epidermal transglutaminase and terminal differentiation by tumor promoters in cultured epidermal cells Carcinogenesis 4,1413-1418[Abstract/Free Full Text]
  24. Yuspa, SH, Ben, T, Hennings, H, Lichti, U. (1982) Divergent responses in epidermal basal cells exposed to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate Cancer Res 42,2344-2349[Abstract/Free Full Text]
  25. Kruse, FE, Tseng, SCG. (1993) A tumor promoter-resistant subpopulation of progenitor cells is present in limbal epithelium more than corneal epithelium Invest Ophthalmol Vis Sci 34,2501-2511[Abstract/Free Full Text]
  26. Lavker, RM, Wei, ZG, Sun, TT. (1998) Phorbol ester preferentially stimulates mouse fornical conjunctival and limbal epithelial cells to proliferate in vivo Invest Ophthalmol Vis Sci 39,301-307[Abstract/Free Full Text]
  27. Tseng, SCG, Sun, T-T. (1999) Stem cells: ocular surface maintenance Brightbill, FS eds. Corneal Surgery: Theory, Technique, and Tissue 3rd ed. Mosby St. Louis. chap. 2
  28. Tseng, SCG. (1994) Conjunctival grafting for corneal diseases Tasman, W Jaeger, EA eds. Duane’s Clinical Ophthalmology 6 JB Lippincott Philadelphia. chap. 34
  29. Holland, EJ, Schwartz, GS. (1996) The evolution of epithelial transplantation for severe ocular surface disease and a proposed classification system Cornea 15,549-556[Medline][Order article via Infotrieve]
  30. Holland, EJ. (1996) Epithelial transplantation for the management of severe ocular surface disease Trans Am Ophthalmol Soc 94,677-743[Medline][Order article via Infotrieve]
  31. Pellegrini, G, Traverso, CE, Franzi, AT, et al (1997) Long-term restoration of damaged corneal surface with autologous cultivated corneal epithelium Lancet 349,990-993[CrossRef][Medline][Order article via Infotrieve]
  32. Tsai, RJF, Li, L-M, Chen, J-K. (2000) Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells N Engl J Med 343,86-93[Abstract/Free Full Text]
  33. Schwab, IR, Reyes, M, Isseroff, RR. (2000) Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease Cornea 19,421-426[CrossRef][Medline][Order article via Infotrieve]
  34. Koizumi, N, Fullwood, NJ, Bairaktaris, G, et al (2000) Cultivation of corneal epithelial cells on intact and denuded human amniotic membrane Invest Ophthalmol Vis Sci 41,2506-2513[Abstract/Free Full Text]
  35. Koizumi, N, Inatomi, T, Suzuki, T, et al (2001) Cultivated corneal epithelial transplantation for ocular surface reconstruction in acute phase of Stevens-Johnson syndrome Arch Ophthalmol 119,298-300[Free Full Text]
  36. Koizumi, N, Inatomi, T, Suzuki, T, et al (2001) Cultivated corneal epithelial stem cell transplantation in ocular surface disorders Ophthalmology 108,1569-1574[CrossRef][Medline][Order article via Infotrieve]
  37. Rama, P, Bonini, S, Lambiase, A, et al (2001) Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency Transplantation 72,1478-1485[CrossRef][Medline][Order article via Infotrieve]
  38. Meller, D, Pires, RT, Tseng, SC. (2002) Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures Br J Ophthalmol 86,463-471[Abstract/Free Full Text]
  39. Grueterich, M, Espana, E, Tseng, SCG. (2002) Connexin 43 expression and proliferation of human limbal epithelium on intact and denuded amniotic membrane Invest Ophthalmol Vis Sci 43,63-71[Abstract/Free Full Text]
  40. Han, B, Schwab, IR, Madsen, TK, Isseroff, RR. (2002) A fibrin-based bioengineered ocular surface with human corneal epithelial stem cells Cornea 21,505-510[CrossRef][Medline][Order article via Infotrieve]
  41. Koizumi, N, Inatomi, T, Quantock, AJ, et al (2000) Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits Cornea 19,65-71[CrossRef][Medline][Order article via Infotrieve]
  42. Hernandez Galindo, EE, Theiss, C, Steuhl, K-P, Meller, D. (2003) Gap junctional communication in microinjected human limbal and peripheral corneal epithelial cells cultured on preserved and intact human amniotic membrane Exp Cell Res 76,303-314
  43. Meller, D, Dabul, V, Tseng, SCG. (2002) Expansion of conjunctival epithelial progenitor cells on amniotic membrane Exp Eye Res 74,537-545[CrossRef][Medline][Order article via Infotrieve]
  44. Tseng, SCG, Prabhasawat, P, Lee, S-H. (1997) Amniotic membrane transplantation for conjunctival surface reconstruction Am J Ophthalmol 124,765-774[Medline][Order article via Infotrieve]
  45. Meller, D, Tseng, SCG. (1999) Conjunctival epithelial cell differentiation on amniotic membrane Invest Ophthalmol Vis Sci 40,878-886[Abstract/Free Full Text]
  46. Yang, A, McKeon, F. (2000) P63 and P73: P53 mimics, menaces and more Nature Review Molecular Cell Biology 1,199-207
  47. Matic, M, Petrov, IN, Chen, S, et al (1997) Stem cells of the corneal epithelium lack connexins and metabolite transfer capacity Differentiation 61,251-260[CrossRef][Medline][Order article via Infotrieve]
  48. Wolosin, JM, Xiong, X, Schutte, M, et al (2000) Stem cells and differentiation stages in the limbo-corneal epithelium Prog Retinal Eye Res 19,223-255[CrossRef][Medline][Order article via Infotrieve]
  49. Gallico, GG, III, O’Connor, NE, Compton, CC, et al (1984) Permanent coverage of large burn wounds with autologous cultured human epithelium N Engl J Med 311,448-451[Medline][Order article via Infotrieve]
  50. Lindberg, K, Brown, ME, Chaves, HV, et al (1993) In vitro preparation of human ocular surface epithelial cells for transplantation Invest Ophthalmol Vis Sci 34,2672-2679[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 IOVSHome page
N. Dong, W. Li, H. Lin, H. Wu, C. Li, W. Chen, W. Qin, L. Quyang, H. Wang, and Z. Liu
Abnormal Epithelial Differentiation and Tear Film Alteration in Pinguecula
Invest. Ophthalmol. Vis. Sci., June 1, 2009; 50(6): 2710 - 2715.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
S. Raeder, T. P. Utheim, O. A. Utheim, B. Nicolaissen, B. Roald, Y. Cai, K. Haug, A. Kvalheim, E. B. Messelt, L. Drolsum, et al.
Effects of Organ Culture and Optisol-GS Storage on Structural Integrity, Phenotypes, and Apoptosis in Cultured Corneal Epithelium
Invest. Ophthalmol. Vis. Sci., December 1, 2007; 48(12): 5484 - 5493.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
W. Li, Y. Hayashida, H. He, C.-L. Kuo, and S. C. G. Tseng
The Fate of Limbal Epithelial Progenitor Cells during Explant Culture on Intact Amniotic Membrane
Invest. Ophthalmol. Vis. Sci., February 1, 2007; 48(2): 605 - 613.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
K.-S. Park, C. H. Lim, B.-M. Min, J. L. Lee, H.-Y. Chung, C.-K. Joo, C.-W. Park, and Y. Son
The side population cells in the rabbit limbus sensitively increased in response to the central cornea wounding.
Invest. Ophthalmol. Vis. Sci., March 1, 2006; 47(3): 892 - 900.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
H. He, H.-T. Cho, W. Li, T. Kawakita, L. Jong, and S. C. G. Tseng
Signaling-Transduction Pathways Required for Ex Vivo Expansion of Human Limbal Explants on Intact Amniotic Membrane
Invest. Ophthalmol. Vis. Sci., January 1, 2006; 47(1): 151 - 157.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
P. Arpitha, N. V. Prajna, M. Srinivasan, and V. Muthukkaruppan
High Expression of p63 Combined with a Large N/C Ratio Defines a Subset of Human Limbal Epithelial Cells: Implications on Epithelial Stem Cells
Invest. Ophthalmol. Vis. Sci., October 1, 2005; 46(10): 3631 - 3636.
[Abstract] [Full Text] [PDF]

Home page
 Br. J. Ophthalmol.Home page
D G Harkin, Z Barnard, P Gillies, S L Ainscough, and A J G Apel
Analysis of p63 and cytokeratin expression in a cultivated limbal autograft used in the treatment of limbal stem cell deficiency
Br. J. Ophthalmol., September 1, 2004; 88(9): 1154 - 1158.
[Abstract] [Full Text] [PDF]

Home page
 Br. J. Ophthalmol.Home page
N Koizumi and S Kinoshita
Ocular surface reconstruction, amniotic membrane, and cultivated epithelial cells from the limbus
Br. J. Ophthalmol., December 1, 2003; 87(12): 1437 - 1439.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Galindo, E. E. H.
Right arrow Articles by Meller, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Galindo, E. E. H.
Right arrow Articles by Meller, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS