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European Urology
Volume 53, issue 5, pages 869-1100, May 2008From Lab to Clinic
Immunoreactivity of p63 in Monolayered and In Vitro Stratified Human Urothelial Cell Cultures Compared with Native Urothelial Tissue
Gerhard Feil 
1 
, Sabine Maurer 1, Udo Nagele, Jutta Krug, Conny Bock, Karl-Dietrich Sievert, Arnulf Stenzl, 1
Accepted 15 October 2007, Published online 23 October 2007, pages 1066 - 1073
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Abstract
Objective
To investigate the immunoreactivity of p63 in monolayered and stratified human urothelial cell cultures and in normal urothelial tissues to assess the differentiation status of in vitro stratified urothelial constructs.
Methods
p63 expression was detected immunohistochemically in native normal human bladder, ureter, and renal pelvis tissues and immunocytochemically in monolayered urothelial cell cultures and urothelial constructs stratified in vitro. Additionally, expression of pancytokeratin, cytokeratin 20 (CK20), uroplakin III, and fibroblast surface antigen was investigated.
Results
In native tissues, urothelial cell layers showed the most intensive p63 staining in the basal cells; the superficial umbrella cells were predominantly negative. Monolayered urothelial cell cultures revealed reduced p63 expression with ongoing culture passages. In vitro stratified urothelial constructs exhibited p63 expression similar to that of native urothelium. CK20-reactive cells were absent in the monolayered cultures but present in the stratified cell cultures and in the urothelial constructs. In native urothelium, only superficial cells stained positive for CK20. Uroplakin III was not present in either monolayered urothelial cell cultures or stratified urothelial constructs. Cultured cells were always positive for pancytokeratin and negative for fibroblast surface antigen.
Conclusions
p63 is a new biomarker for differentiation and stratification of urothelium created in vitro. For proposed clinical applications of in vitro stratified urothelium in reconstructive urology, urothelial constructs should exhibit expression of significant marker proteins similar to that of native urothelium. Our results show such similarity of expression for pancytokeratin, p63, and CK20, an encouraging possibility for confirming the functionality of tissue-engineered urothelia after clinical application.
Keywords: Cytokeratin 20, Epithelial differentiation, Fibroblast surface antigen, In vitro stratification, Pancytokeratin, p63, Tissue engineering, Uroplakin III, Urothelial monolayer, Urothelial multilayer.
Article Outline
1. Introduction
Urothelium consists of three cell types: basal cells, intermediate cells, and superficial umbrella cells [1], [2], [3], and [4]. The basal cells form a single cell layer that contacts the underlying connective tissue and serve as precursors for the other cell layers [4]. One to several layers of intermediate cells sit on top of the basal cells. The superficial umbrella cell layer consists of cells of very large size that are frequently multinucleated. An important function of the urothelium is to maintain the urine composition generated by the kidneys, which requires an efficient permeability barrier for electrolytes and nonelectrolytes [1], [2], [3], [4], and [5]. The barrier is located in the superficial umbrella cell layer [3], and [4] and consists of three components: the unique apical membrane, the high-resistance tight junctions, and an active membrane-transporting mechanism.
p63 is a member of the p53 tumour suppressor gene family and encodes at least six distinct protein isoforms. Alternative promoter usage results in two classes of p63 isoforms that contain (TA isoform) or lack (ΔN isoform) an amino terminal transactivation domain. Additionally, an alternative messenger RNA (mRNA) splicing involving the C-terminal domain results in the synthesis of three different TA and ΔN isoforms, α, β, and γ, respectively [6], [7], [8], [9], and [10]. p63 regulates genes with various roles in cellular function, including proliferation, cell-cycle control, and induction of apoptosis. The TAp63 isoform behaves more like p53 regarding tumour suppressor activity, whereas the ΔNp63 displays opposing functions against the TAp63 isoform and p53 [11], and [12]. The TAp63 isoform is expressed in the surface ectoderm prior to stratification and continues to be expressed during embryonic development [11]. Mills et al [13] reported absent development of stratified epidermis and a lack of differentiation markers, concluding that p63 is essential for the commitment of the developing ectoderm to an epidermal lane. In contrast, Yang et al [14] identified remnants of a stratified epidermis that stained positive for differentiation markers, suggesting that p63 is essential for the proliferative potential of stem cells in stratified epithelia. Stem cells lacking p63 undergo a premature proliferative rundown. Furthermore, p63 is dispensable for both the commitment and differentiation of these stem cells during tissue morphogenesis [15]. ΔNp63 isoforms are predominantly expressed during late embryonic and postnatal epidermal development. However, the function of these proteins remains elusive. Using an epidermal-specific inducible knockdown mouse model, Koster et al [16] demonstrated that ΔNp63 proteins are essential for maintaining basement membrane integrity and terminal differentiation of keratinocytes. Park et al [17], in their study of noncancerous bladder tissue specimens, revealed easily detectable expression of TAp63 mRNA, whereas ΔNp63 expression was extremely low or undetectable.
Cytokeratins are the intermediate filament proteins expressed by cells of epithelial origin. Urothelial cytokeratin expression differs with the stratification and differentiation status of the tissue. CK20 is restricted to the superficial umbrella cells and very occasionally to intermediate cells [18].
Uroplakins are the major differentiation products of the urothelium and contribute to maintaining the permeability barrier [19], and [20]. The apical surface of mammalian urothelium is covered with membrane plaques containing four major proteins, uroplakins Ia, Ib, II, and III. Uroplakin III is present in all urothelial plaques. Thus, the expression of CK20 and the presence of uroplakin reveal the differentiation status and, indirectly, the function of urothelial tissue.
With respect to a projected clinical application of in vitro stratified urothelium in reconstructive urology, one basic requirement for urothelial constructs is the achievement of expression profiles of significant marker proteins that are similar to those of native tissue. The aim of this study was to investigate the immunoreactivity of p63 in normal urothelial tissues for comparison with monolayered and stratified urothelial cell cultures in the context of CK20 and uroplakin III differentiation status and the functionality of in vitro stratified urothelial constructs.
2. Materials and methods
2.1. Isolation and culture of human urothelial cells
Following informed consent, a total of 13 tissue samples from the lower urinary tract were obtained from adult patients undergoing open tumour surgery. Patients were aged 62 yr (range, 19–85). In those with a transitional cell carcinoma of the bladder in an area without any sign of tumour, verified by haematoxylin-eosin staining, intact tissue was collected. In those who underwent nephrectomy an entire section of the ureter or renal pelvis was harvested. Tissue specimens were immediately placed in ice-cold transport medium [2] consisting of Hank's balanced salt solution (Biochrom, Berlin, Germany), 10 mmol/l HEPES buffer (Invitrogen, Carlsbad, CA, USA), 100 KIU/ml aprotinin (Bayer, Leverkusen, Germany), and antibiotics (100 u/ml penicillin; 0.1 mg/ml streptomycin). The specimens were kept at 4 °C until further processing.
Urothelial cells were isolated as described previously [21]. Briefly, excess fat and connective tissue were removed from the tissue specimens. A small tissue section was fixed in 3.7% formalin for histology. Specimens were dissected into 1- to 2-cm2 pieces and transferred into 1% EDTA in Hank's balanced salt solution buffered with 10 mmol/l HEPES. After 3 h of incubation at 37 °C, the urothelium was removed from the underlying stroma by gentle scraping along the urothelial surface. To produce a single-cell suspension, the material obtained was digested with 100 units/ml collagenase type IV (Sigma-Aldrich, Saint Louis, MO, USA) for 20 min at 37 °C. The cells were washed and resuspended in complete keratinocyte serum-free medium, supplemented with 50 μg/ml bovine pituitary extract, 5 ng/ml human recombinant epidermal growth factor (Invitrogen), and 30 ng/ml cholera toxin (List Biological Laboratories, Inc, Campbell, CA, USA). Subsequently, cells were plated into plastic culture flasks coated with collagen A (Biochrom). The cultures were maintained at 37 °C in a humidified atmosphere with 5% CO2. The culture medium was replaced the following day and thereafter three times a week.
For further passages, subconfluent monolayers were incubated in phosphate-buffered saline (PBS) containing 0.1% EDTA (Biochrom) for 5–10 min at 37 °C, followed by exposure to TrypLE™ Express (Invitrogen) for another 1–2 min. The detached cells were resuspended in cell culture medium and seeded into Petri dishes 6 cm in diameter for histology and immunohistology of stratified cell sheets. For immunocytochemical investigations, cells were seeded onto 16-well glass chamber slides at 2 × 104 cells per well. Alternatively, 8-well Permanox™ chamber slides (Nunc, Roskilde, Denmark) were seeded at 4 × 104 cells per well. All culture dishes except the Permanox™ slides were coated with collagen A at 50 μg/cm2 prior to use.
2.2. Stratification of urothelial monolayers
Well-established monolayer cultures showing 100% confluence were used for the induction of stratification by addition of CaCl2 to the culture medium to a final concentration of 2.09 mmol/l. The stratification medium was changed every other day. Ten to 20 days after induction of stratification, urothelial sheets were detached from the culture dish by incubation with 2.5% dispase II (Invitrogen) at 37 °C for 7 min.
2.3. Histology
Harvested cell sheets were washed twice in PBS, placed between two foam plastic sheets, fixed in 4% neutral buffered formalin for 24 h, and embedded in paraffin. Then 5-μm sections were deparaffinised in xylene, rehydrated in ethanol, and stained with haematoxylin-eosin.
2.4. Immunoassaying
2.4.1. Immunohistology
Samples of native tissues were fixed in 4% neutral buffered formalin, dehydrated through a graded series of ethanol to xylene, and embedded in paraffin. Sections 5 μm thick were deparaffinised in xylene and rehydrated.
2.4.2. Immunocytochemistry
Monolayer and multilayer chamber slide cultures were gently rinsed with PBS and subsequently fixed in 3.7% neutral buffered paraformaldehyde (PFA) for 10 min to label cell-surface antigens. For labelling of intracellular antigens, a permeabilisation step with PBS containing 0.1% saponin was performed after fixation with PFA. Additionally, primary isolated cells at passage 0 and enzymatically detached cells at passages 2, 3, 8, and 12 were fixed in 3.7% neutral buffered PFA for 10 min, rinsed with PBS, and centrifuged as cytospin spots on SuperFrost™ Plus glass slides (Langenbrinck, Teningen, Germany). Immediately after preparation, the cytospin slides were transferred into PBS and stored at 4 °C until further processing.
2.4.3. Antibodies
For detection of p63 expression, the mouse monoclonal anti-p63 antibody 4A4, recognising all p63 isoforms [22], was used. The mouse monoclonal anticytokeratin antibody cocktail AE1/AE3 was applied to identify cells of epithelial origin. To exclude the presence of mesenchymal cells, mouse monoclonal anti–smooth muscle α-actin antibody 1A4 and mouse monoclonal antifibroblast surface antigen–specific antibody TE-7 were used. CK20 expression was detected with the mouse monoclonal anti-CK20 antibody Ks20.8 and uroplakin III with the mouse monoclonal antiuroplakin III antibody AU1 (Table 1).
Table 1 Antibodies used for immunostaining
| Specificity | Clone | Dilution | Source |
|---|---|---|---|
| p63 | 4A4 | 1:300 | Lab Vision, Fremont, CA, USA |
| Pancytokeratin | AE1/AE3 | 1:200 | Chemicon, Temecula, CA, USA |
| Smooth muscle α-actin | 1A4 | 1:2000 | Sigma-Aldrich, Saint Louis, MO, USA |
| Fibroblast surface antigen | TE-7 | 1:200 | Chemicon, Temecula, CA, USA |
| Cytokeratin 20 | Ks 20.8 | 1:300 | Dako, Glostrup, Denmark |
| Uroplakin III | AU1 | 1:4 | Progen, Heidelberg, Germany |
2.4.4. Immunohistochemical and immunocytochemical staining
For immunohistology with AE1/AE3, TE-7, 4A4, and Ks 20.8, microwave epitope retrieval was performed in citrate buffer pH 6.0 (3 × 15 min). Subsequently, the samples were incubated with appropriately diluted primary antibody for 40 min at room temperature in a humidified chamber. Binding of the primary antibodies was assessed with the use of the DakoCytomation LSAB2 System detection kit (Dako, Glostrup, Denmark). As negative control, primary antibody was substituted with PBS. The nuclear counterstain with haematoxylin was applied except in cytospin slides used for quantification of p63-positive cells.
2.4.5. Quantification of p63 expression
The quantification of p63-positive cells was performed microscopically. In histological samples, p63 expression was calculated with a semiquantitative score from – for p63-negative cells to +++ for very high p63 expression. The percentage of p63-positive cells in the immunocytochemical assays was calculated in at least 10 visual fields, with a total of 1000–2000 cells.
3. Results
3.1. Immunohistochemistry in native tissues
A total of six bladder, three renal pelvis, and four ureter specimens were examined. All urothelial cells stained positive for pancytokeratin AE1/AE3, whereas CK20 was expressed only by superficial umbrella cells. p63 expression (Fig. 1) was found in all cell layers, with the highest expression in the basal cells. In contrast, p63 was usually absent in the superficial cells. In the semiquantification score from − to +++, the basal cells were quantified ++/+++, the intermediate cells ++, and the superficial cells −/+. Throughout the urothelium, no positive staining for anti–smooth muscle α-actin antibody or for antifibroblast surface antigen–specific antibody was observed.
01312-7/assets/gr1/gr1_584x349.jpg)
Fig. 1 Comparison of monolayered (a–c) and multilayered (d–f) urothelial cell culture and in vitro stratified urothelium (g–i) to native grown urothelium (j–l) for expression of the epithelial cell marker pancytokeratin (Pan-CK, top); p63 (centre), the marker for epithelium development, proliferation, differentiation, and maintenance; and cytokeratin 20 (CK20, bottom), the marker for terminal urothelial cell differentiation. Positive cells are marked by brown-coloured cytoplasm (Pan-CK, CK20) or nuclei (p63). Original magnification from ×10 (a–d, f, j, l), ×20 (g, i), and ×40 (e, h, k).
3.2. Immunocytochemistry in monolayered and stratified urothelial cell cultures
3.2.1. p63
Quantification of p63-positive cells was performed microscopically in monolayered cultures of urothelial cells as well as in cytospin slides. The chamber slide culture originated from a bladder tissue sample, and the cells used for the cytospin slides from a bladder and a renal pelvis tissue sample. Chamber slide cultures and cytospin spots were examined for positive staining of p63 and AE1/AE3 at culture passages 1–4, and 0, 2, 3, 8, and 12, respectively. In the assays performed, each passage showed a consistent expression of pancytokeratin AE1/AE3 in 100% of the cells, indicating the maintained epithelial phenotype. In the same assays, the cells always stained negative for the fibroblast surface antigen and for smooth muscle α-actin.
After reaching 100% confluence, urothelial monolayer cultures showed homogeneously distributed expression of p63. In confluent cultures maintained up to 14 d, p63 expression appeared spotwise in small cell groups. When stratification was induced in 100% confluent urothelial cell cultures, p63 was expressed only partially (Fig. 1). p63 expression of the cultured cells from bladder and renal pelvis continuously decreased with ongoing culture passages from 53–44% in passages 0, 1, and 2, respectively, to 2% positive cells in passages 8 and 12 (Fig. 2).
01312-7/assets/gr2/gr2_584x154.jpg)
Fig. 2 Percentages of p63 expression of human urothelial cells harvested from urinary bladder (left) and renal pelvis (right) in consecutive culture passages.
3.3. CK20 and uroplakin III
The urothelial monolayered cell cultures examined showed no expression of CK20, independent of culture passage and cultivation time within a passage. In contrast, stratified urothelial cell cultures revealed partially positive CK20 after induction of stratification. Fig. 1 shows CK20 expression of bladder urothelial cells at day 2 and day 14 in culture passage 3. The presence of uroplakin III was not detected in either the monolayered or stratified urothelial cell cultures.
3.4. Immunohistochemistry of detached multilayered urothelial sheets
Urothelial cells generated in vitro reached two to five cell layers. All cell layers of the investigated urothelial sheets stained 100% positive for the pancytokeratin antibody AE1/AE3, demonstrating the epithelial phenotype. Consistent with the immunocytochemical assays, the cells stained positive for CK20, although in a more inhomogeneous distribution compared with native urothelium. Investigation of p63 revealed partial distribution of p63-positive cells, as similarly observed in the stratified urothelial cultures (Fig. 1).
4. Discussion
Because in vitro stratified constructs of urothelial tissue might be useful for reconstructive purposes in the lower urinary tract, functional properties of native urothelium, as characterised by specific marker expression, should be present in tissue-engineered urothelial structures.
In fully developed mature tissues, the p63 protein is expressed at high levels in the basal or progenitor cell layers of many stratified epithelial structures, including human urothelium, skin, and oesophagus, as well as secretory epithelial structures like mammary and prostate glands [6], [23], and [24]. p63 plays a unique dual role in part of ectodermal development and morphogenesis: (1) the TAp63 isoforms are important for initiating epithelial stratification during embryogenesis; and (2) because TAp63 inhibits terminal differentiation, ΔNp63 keeps this isoform in check to maintain the proliferative potential of the basal keratinocytes in epidermis [11].
The intention of our in vitro experiments was to investigate p63 expression in both urothelial cell cultures and stratified urothelial cell sheets generated in vitro with regard to a clinical application of tissue-engineered urothelium for reconstructive purposes. The data found showed p63 expression in monolayered and multilayered cell cultures. As in native urothelia, basal and intermediate cell layers demonstrated the most intensive p63 reactivity. The superficial cells were predominantly negative for this epithelial marker but stained positive for the terminal differentiation marker CK20.
With a focus on urothelial development, Urist et al [25] found that p63-deficient mice develop a nontransitional cuboidal epithelium without the apical layer of umbrella cells. They concluded that p63 is thus not required for bladder epithelium formation but is essential for the specific terminal differentiation of the superficial cell layers. Consequently, all tissue-engineered urothelial constructs have to show p63 expression as a basis for the functional properties required, that is, epithelium development, epithelial proliferation, terminal differentiation regulation, and maintenance of stratified epithelia.
In case of urothelial injury, umbrella cells are rapidly regenerated. Cell division is induced and multinucleate umbrella cells arise from intermediate cell-cell fusion [4]. Lavelle et al [3] demonstrated that selective urothelial injury resulted in the loss of umbrella cells and in a rapid maturation of intermediate cells, indicated by increased expression of uroplakins and the formation of tight junctions.
According to other reported data [6], [22], [23], and [26], we confirmed p63 expression in the basal and intermediate cell layers of the urothelium in situ. In general, p63 was not expressed in the most superficial cells of the urothelium, the well-differentiated umbrella cells. The expression of CK20 and the presence of uroplakin give evidence of the differentiation status and, indirectly, of the function of urothelial tissue. The native tissue samples investigated showed expression of CK20 in the superficial cell layer. Uroplakin III was detected as part of the apical membrane. Because the functionality of native human bladder epithelium is associated with CK20 and uroplakin III expression, tissue-engineered urothelial constructs have to express these marker proteins. The data obtained demonstrate the expression of p63 and CK20 in vitro. Uroplakin might be induced in transplanted urothelial constructs by impulses given in vivo.
The investigated urothelial monolayer cultures originating from human bladder and human renal pelvis showed decreasing p63 expression with ongoing passages. Partial expression of p63 was observed in stratified urothelial cultures derived from monolayers of early cell culture passages. In correlation to the expression of CK20 in superficial cells of the stratified urothelial constructs, decreased p63 expression might rather reflect a switch to an increased expression of differentiation markers [11], and [15] than a tumourigenic process [27] (reviewed in [28]). Detached multilayered urothelial sheets also showed expression of p63, mainly in the basal and intermediate cell layers. As in native tissue, these cells contact the underlying connective tissue and serve as precursors for the other cell layers [4]. Therefore, the proliferative and differentiating potential of epithelial cells has to be assumed also for in vitro stratified urothelial constructs.
In concordance with Southgate et al [29], expression of CK20 in monolayer cultures and uroplakin also was not detected in the current work. In the stratified urothelial cell cultures and in detached urothelial sheets, CK20 was expressed only partially, not as continuously as in native tissue, indicating ongoing terminal differentiation. Uroplakin was not detected in either stratified urothelial cell cultures or detached multilayered urothelial cell sheets.
The current data provide the basis for further investigations to enhance terminal differentiation status of tissue-engineered multilayered urothelium with respect to its functional performance as new substitute for reconstructive urology. The insertion of urothelial constructs without complication-bearing matrices might be a treatment option for sustained urothelial reconstruction, especially in patients for whom other autologous grafts are not available. Additionally, cultured autologous urothelium might prevent immunological reactions followed by inflammation and fibrosis, development of functional problems like metabolic complications, mucus production, and urolithiasis [30], and [31]. Investigations on the effect of omental maturation of a seeded ureter-shaped construct revealed vascularised and terminally differentiated urothelium in a pig model [31]. First animal models on the outcome of tissue-engineered urothelial transplants in bladder augmentation demonstrated the rapid formation of a native-like epithelium. Hence, terminal differentiation could not be achieved [32], and [33]. The conclusion was that it is essential to generate urothelium with superior differentiated and functional properties for a proposed clinical application. We suppose that p63 is not only necessary for the development of primitive epithelia into stratified tissues [13], [14], and [16] and for the commitment and differentiation of epithelial stem cells during tissue morphogenesis [15], but is a key protein for the development of urothelial monolayers into layers of stratified and differentiated urothelia in vitro.
5. Conclusions
p63 is necessary for initial epithelium development and for epithelial proliferation, terminal differentiation regulation, and maintenance of stratified epithelia in vivo. In addition, p63 expression is a marker of proliferation, differentiation, and stratification of urothelium created in vitro. This new finding is encouraging for the potential functionality of tissue-engineered urothelia after an intended clinical application.
Conflicts of interest
The authors confirm that they have no disclosures to report.
Acknowledgements
We are grateful to Jörg Hennenlotter and Ursula Kuehs for preparing surgical tissues.
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Footnotes
Department of Urology, University of Tübingen, Hoppe-Seyler-Strasse 3, D-72076 Tübingen, Germany
Corresponding author. Tel. +49 7071 29 86657; Fax: +49 7071 29 5858.
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