Stratified Urothelium Research Articles

MP60-14 A CELLULAR ATLAS OF THE NORMAL ADULT HUMAN FEMALE URETHRA

You have accessJournal of UrologyCME1 Apr 2023MP60-14 A CELLULAR ATLAS OF THE NORMAL ADULT HUMAN FEMALE URETHRA John Lafin, Alicia Malewska, Philippe Zimmern, Maude Carmel, Gary Lemack, Douglas Strand, and Ramy Goueli John LafinJohn Lafin More articles by this author , Alicia MalewskaAlicia Malewska More articles by this author , Philippe ZimmernPhilippe Zimmern More articles by this author , Maude CarmelMaude Carmel More articles by this author , Gary LemackGary Lemack More articles by this author , Douglas StrandDouglas Strand More articles by this author , and Ramy GoueliRamy Goueli More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003318.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Urinary voiding dysfunction is highly prevalent in aging women. A deeper understanding of the cellular composition of female urethra in the non-diseased state could provide insights into the etiology of dysfunction. Single cell RNA sequencing was performed on female urethra to generate a cellular atlas. METHODS: Urethra from female organ donors aged 18-45 (N=4) was collected fresh from surgery and enzymatically digested into a single cell suspension. Single cell RNA sequencing was performed using 10x Genomics V3 chemistry. Data were analyzed in Seurat for cell type annotation. Immunohistochemical validation of cell types was performed on FFPE sections using differentially expressed genes from clustered data. RESULTS: The female proximal urethral lumen is lined with a stratified urothelium composed of KRT5+/KRT14- basal cells with KRT13+ hillock epithelia and KRT4+ luminal epithelia. Ducts emerging from dorsal urethra (Skene's glands) contain basal and club cells similar to prostatic urethra. The urethral lumen is surrounded by two discrete layers of fibroblasts (peri-urethral and interstitial) and circumferential smooth muscle and skeletal muscle. CONCLUSIONS: The identification of 1) specialized fibroblasts surrounding the urethra, and 2) club cells in dorsal ducts could yield new insight into the regulation of infection, inflammation and obstruction, which are the putative etiologies of most urethral dysfunction. Source of Funding: DK115477, DK104310, DK129994 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e848 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information John Lafin More articles by this author Alicia Malewska More articles by this author Philippe Zimmern More articles by this author Maude Carmel More articles by this author Gary Lemack More articles by this author Douglas Strand More articles by this author Ramy Goueli More articles by this author Expand All Advertisement PDF downloadLoading ...

Transplantation of hBMSCs Loaded on Collagen Scaffolds for the Regeneration of Canine Tissue-Engineered Urethras

Objective: To estimate the function of human bone marrow derived mesenchymal stem cells (hBMSCs) loaded on collagen scaffolds in the regeneration of canine tissue-engineered urethras. Methods: In this study, hBMSCs (1×106 hBMSCs per 1cm2 collagen scaffold) were loaded on tubular collagen scaffolds (5cm in length). A 5cm long urethra segment near the penis bone was excised surgically. A collagen scaffold seeded with hBMSCs was sutured to two ends of the host urethra for the urethral repair in the experimental group, whereas the unseeded scaffold was used in the control group. Retrograde urethrography was performed before the operation and at 6 months after the surgery. At 6 months postsurgery, the animals were sacrificed, and the reconstructed urethras were collected for histological observation. An anti-human nuclei monoclonal antibody was used for immunofluorescent staining to detect the human cells in the reconstructed urethras. The growth of the urothelium, smooth muscles and blood vessels were evaluated by immunofluorescent staining. Results: The macroscopic observation and retrograde urethrography demonstrated that varying degree of urethra strictures were observed in all the animals of two groups. But the severer urethrostenosis usually appeared in the control group. Histologically, the scaffolds seeded with the hBMSCs formed a stratified urothelium layer that was similar to the appearance of the normal urothelium; however, in most areas, the unseeded scaffolds formed a discontinuous mucosa with a monolayer of urothelial cells. There were more smooth muscles and blood vessels growing than those in controls, although they were far fewer than normal. Positive cells were found in the experimental group with anti-human nuclei immunofluorescence staining. Conclusion: In this study, hBMSCs loaded on collagen scaffolds were able to promote the regeneration of canine tissue-engineered urethras, predominantly for the urothelium.

One-Stage Tissue Engineering of Bladder Wall Patches for an Easy-To-Use Approach at the Surgical Table

We present a method for producing a cell-scaffold hybrid construct at the bedside. The construct is composed of plastic-compressed collagen together with a poly(ɛ-caprolactone) (PCL)-knitted mesh that yields an integrated, natural-synthetic scaffold. This construct was evaluated by seeding of minced bladder mucosa, followed by proliferation in vitro. High mechanical strength in combination with a biological environment suitable for tissue growth was achieved through the creation of a hybrid construct that showed an increased tensile strength (17.9 ± 2.6 MPa) when compared to plastic-compressed collagen (0.6 ± 0.12 MPa). Intimate contact between the collagen and the PCL fabric was required to ensure integrity without delamination of the construct. This contact was achieved by surface alkaline hydrolysis of the PCL, followed by adsorption of poly(vinyl) alcohol. The improvement in hydrophilicity of the PCL-knitted mesh was confirmed through water contact angle measurements, and penetration of the collagen into the mesh was evaluated by scanning electron microscopy (SEM). Particles of minced bladder mucosa tissue were seeded onto this scaffold, and the proliferation was followed for 6 weeks in vitro. Results obtained from phase contrast microscopy, SEM, and histological staining indicated that cells migrated from the minced tissue particles and reorganized on the scaffold. Cells were viable and proliferative, with morphological features characteristic of urothelial cells. Proliferation reached the point at which a multilayer with a resemblance to stratified urothelium was achieved. This successful method could potentially be used for in vivo applications in reconstructive urology as an engineered autologous tissue transplant without the requirement for in vitro culture before transplantation.

Differentiation of adipose-derived stem cells promotes regeneration of smooth muscle for ureteral tissue engineering

BackgroundThe purpose of the present study was to assess the differentiation potential of adipose-derived stem cells (ASCs) into smooth muscle cells (SMCs) and their potential for promoting regeneration of smooth muscle for ureteral tissue engineering. MethodsASCs were isolated, proliferated, and identified in vitro. SMC differentiation was induced using SMC induction medium. Gene expression was evaluated by quantitative polymerase chain reaction, immunofluorescence, and Western blotting. Vessel extracellular matrix was obtained by a decellularization process. The induced cells were seeded onto vessel extracellular matrix for ureter reconstitution. Grafts were obtained for evolutionary histologic studies. Renal function and ureteral patency was evaluated by intravenous urography at 16 wk. ResultsFlow cytometry demonstrated that the ASCs expressed CD90, but did not express CD45 or CD34. After 6 wk of induction, upregulation of α-smooth muscle actin expression was determined by quantitative polymerase chain reaction, and smooth muscle myosin heavy chain expression was confirmed by immunofluorescence and Western blotting in the induced cells. Vessel extracellular matrix exhibited a nontoxic and bioactive effect on the induced cells. Histologically, stratified urothelium and organized muscle bundles were observed in the grafts at 16 wk. Intravenous urography demonstrated no ureteral stricture or hydroureteronephrosis. ConclusionsThese results have demonstrated that ASCs can be differentiated into SMCs and this potential promoted smooth muscle regeneration for ureteral tissue engineering.

Re‐epithelialization of demucosalized stomach patch with tissue‐engineered urothelial mucosa combined with Botox A in bladder augmentation

Re-epithelialization demucosa stomach patch is important to prevent the patch being exposed to urine that might cause patch shrinkage and fibrosis formation due to urine-derived chemical irritation. Additionally, Botox A acts by blocking the transmission of nerve impulses to smooth muscles and so paralysing the muscles, which is commonly used to relax muscle for treatment of oesophageal achalasia due to overactive smooth muscle and sphincters of gastrointestinal tract. We fabricated in vitro tissue engineered urothelial mucosa with multi-layers of urothelium and smooth muscle layers seeded on SIS scaffold and then used this cell-scaffold construct to cover nuke gastro patch combining with Botox A for gastrocystoplasty in a canine model. To evaluate the demucosalized stomach patch covered with tissue-engineered urothelium for gastrocystoplasty and to determine whether or not injections of Botox A into the re-epithelialized stomach patch can protect the graft from contraction in a canine bladder reconstruction model. Gastrocystoplasty was performed in 10 adult beagles after hemi-cystectomy using five types of stomach patch (n = 2 per group): entire stomach patches (group I); demucosalized patches (group II); demucosalized patches covered with cell-free small intestinal submucosa (SIS) (group III); demucosalized patches with urothelial and smooth muscle cell-seeded SIS (group IV); and demucosalized patches with the cell-seeded SIS combined with injections of Botox A (group V). The bladder volume/pressure and the graft sizes were measured before surgery and again 10 weeks after bladder augmentation. The graft tissues were examined both histologically and using immunohistochemistry. All dogs survived and their gastric grafts were all vital with a good blood supply. Gastric metaplasia of urothelium appeared on the top of stomach mucosa patches in two animals in group I. There was calcification formation at the centre of the graft in one animal in group II. As compared with urothelium that was partially covered over with stomach patches in groups II and III, stratified urothelium completely covered the demucosalized gastric patches in groups IV and V. There was less shrinkage of the stomach grafts in groups I and V, which shrank to half of their original size, than of the stomach grafts in groups II, III, and IV, which shrank significantly to one-quarter of their original sizes. Botox A injections appear to protect the graft contraction in the re-epithelialized stomach flaps. The gastrocystoplasty using demucosalized patches covered with tissue-engineered urothelial mucosa combined with an injection of Botox A could have clinical potential for use in bladder reconstruction.

Mechanical Stimuli-induced Urothelial Differentiation in a Human Tissue-engineered Tubular Genitourinary Graft

BackgroundA challenge in urologic tissue engineering is to obtain well-differentiated urothelium to overcome the complications related to other sources of tissues used in ureteral and urethral substitution. ObjectiveWe investigated the effects of in vitro mechanical stimuli on functional and morphologic properties of a human tissue-engineered tubular genitourinary graft (TTGG). Design, setting, and participantsUsing the self-assembly technique, we developed a TTGG composed of human dermal fibroblasts and human urothelial cells without exogenous scaffolding. Eight substitutes were subjected to dynamic flow and hydrostatic pressure for up to 2 wk compared to static conditions (n=8). MeasurementsStratification and cell differentiation were assessed by histology, electron microscopy, immunostaining, and uroplakin gene expression. Barrier function was determined by permeation studies with carbon 14–urea. Results and limitationsDynamic conditions showed well-established stratified urothelium and basement membrane formation, whereas no stratification was observed in static culture. The first signs of cell differentiation were perceived after 7 d of perfusion and were fully expressed at day 14. Superficial cells under perfusion displayed discoidal and fusiform vesicles and positive staining for uroplakin 2, cytokeratine 20, and tight junction protein ZO-1, similar to native urothelium. Mechanical stimuli induced expression of the major uroplakin transcripts, whereas expression was low or undetectable in static culture. Permeation studies showed that mechanical constraints significantly improved the barrier function compared to static conditions (p<0.01 at 14 d, p<0.05 at 7 d) and were comparable to native urothelium. ConclusionsMechanical stimuli induced in vitro terminal urothelium differentiation in a human genitourinary substitute displaying morphologic and functional properties equivalent to a native urologic conduit.

Immunoreactivity of p63 in Monolayered and In Vitro Stratified Human Urothelial Cell Cultures Compared with Native Urothelial Tissue

ObjectiveTo 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. Methodsp63 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. ResultsIn 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. Conclusionsp63 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.

CREATION OF A LUMINAL TISSUE COVERED WITH A DIFFERENTIATED UROTHELIUM FROM AUTOLOGOUS UROTHELIAL CELLS CULTURED ON FIBRIN GEL/ATELOCOLLAGEN MATRIX AFTER IMPLANTATION INTO MESENTERIUM IN RATS

AIM: The aim of this study was to create a urothelial lining tissue made by implantation of cultured autologous urothelial cells onto mesenterium. METHODS: Cultured rat urothelial cells were seeded onto fibrin gel/atelocollagen sponge matrix and that was folded into two with the urothelial cells inside and implanted onto the mesenterium. Then, it was serially evaluated histologically. RESULTS: The implanted matrix showed cystic appearance on day 7. Stratified urothelium was observed on the luminal surface and infiltration of stromal cells and microvessels was identified in the matrix. Cystic appearance and urothelial lining remained on the day 14 and the regenerated urothelium showed the same lectinohistochemical staining as normal bladder. The infiltrated stromal cells showed positive staining to both alpha smooth muscle actin and desmin. On day 28 cystic appearance was observed in 2 of 6. On the luminal surface of the matrices with cystic appearance, urothelial lining was identified and two layers were noted within the matrices; the inner layer with a large amount of stromal cells and the outer layer with fewer stromal cells. In the matrices which lost the cystic appearance, on the contrary, urothelial lining was not identified. CONCLUSIONS: We created a mimetic urological organ covered with differentiated urothelium from cultured urothelial cells and fibrin gel/atelocollagen sponge matrix. This technique will contribute to the reconstructive surgery of the urologic organ.

Induction of urothelial cell proliferation by fibroblast growth factor-7 in RAG1-deficient mice.

Fibroblast growth factor-7 (FGF-7, keratinocyte growth factor, KGF) is a 163 amino acid glycoprotein synthesized and secreted by mesenchymal cells (e.g. fibroblasts/fibrocytes) in epithelial organs, thereby functioning as a paracrine mediator of epithelial cell proliferation. In the urinary bladder, FGF-7 is transported from the lamina propria across the urothelial basement membrane to where it ultimately binds to splice variants of the FGFR2 receptor present on the basolateral surface of transitional epithelial cells. We administered 100 micrograms/ml (i.p.) recombinant FGF-7 (rFGF-7) to RAG1-deficient mice (n = 3) for 7 days and observed a striking expansion of the urinary bladder urothelium. This expansion was characterized by a layer of stratified urothelium > 20 cells thick and by positive immunostaining for the proliferation marker Ki-67. In contrast, RAG1-deficient mice (n = 3) that received only buffer injection did not exhibit detectable urothelial expansion. rFGF-7 was detected by immunoblot analyses in the serum, but not in the urine, from RAG1-deficient mice that received the recombinant protein. Mice that have a targeted disruption in the gene encoding the V(D)J recombination activation gene RAG1 have small lymphoid organs with no mature B and T lymphocytes, due to the inability of cell progenitors to perform V(D)J recombination. The biological activity of FGF-7 in RAG-1 mice indicates that immuno-dependent mechanisms are not required for the induction of urothelial cell proliferation by this epithelial cell-specific growth factor.

Specific heterodimer formation is a prerequisite for uroplakins to exit from the endoplasmic reticulum.

Much of the lower urinary tract, including the bladder, is lined by a stratified urothelium forming a highly differentiated, superficial umbrella cell layer. The apical plasma membrane as well as abundant cytoplasmic fusiform vesicles of the umbrella cells is covered by two-dimensional crystals that are formed by four membrane proteins named uroplakins (UPs) Ia, Ib, II, and III. UPs are synthesized on membrane-bound polysomes, and after several co- and posttranslational modifications they assemble into planar crystals in a post-Golgi vesicular compartment. Distension of the bladder may cause fusiform vesicles to fuse with the apical plasma membrane. We have investigated the early stages of uroplakin assembly by expressing the four uroplakins in 293T cells. Transfection experiments showed that, when expressed individually, only UPIb can exit from the endoplasmic reticulum (ER) and move to the plasma membrane, whereas UPII and UPIII reach the plasma membrane only when they form heterodimeric complexes with UPIa and UPIb, respectively. Heterodimer formation in the ER was confirmed by pulse-chase experiment followed by coimmunoprecipitation. Our results indicate that the initial building blocks for the assembly of crystalline uroplakin plaques are heterodimeric uroplakin complexes that form in the ER.

A study of cytokeratin 20 immunostaining in the urothelium of neuropathic bladder of patients with spinal cord injury.

BackgroundNormal urothelium is characterised by terminally differentiated superficial cells, which express cytokeratin 20 in the cytoplasm. In contrast, cultured human stratified urothelium, which does not undergo complete terminal differentiation of its superficial cells, does not express cytokeratin 20. If spinal cord injury (SCI) affects urothelial differentiation or induces squamous or other metaplastic change undetected by histological analysis, the superficial urothelial cells of the neuropathic bladder might be expected to show absence of immunostaining for cytokeratin 20.Patients and MethodsWe studied immunostaining for cytokeratin 20 in bladder biopsies taken from 63 consecutive SCI patients. Immunostaining was performed on paraffin-embedded tissue using a mouse monoclonal antibody (clone: Ks20.8).ResultsOf 63 biopsies, the epithelium was scarce in two. Eight biopsies showed squamous metaplasia and immunostaining for cytokeratin 20 was absent in all the eight biopsies. Of the remaining 53 cases, in which the umbrella cell layer of the urothelium was intact, immunostaining for cytokeratin 20 was seen only in ten biopsies.ConclusionSuperficial cells in the transitional epithelium showed immunostaining for cytokeratin 20 in 10 of 53 bladder biopsies taken from SCI patients. The reasons for this could be either that there is an underlying metaplasia or that changes in the neuropathic bladder affect urothelial differentiation. Taken with evidence from other systems, such as loss of cytokeratin 20 expression from static organ cultures of urothelial tissue, this might suggest that other factors, such as impairment of voluntary voiding in SCI patients, could affect expression of markers such as cytokeratin 20.

Expansion and long-term culture of differentiated normal rat urothelial cells in vitro.

The objective of this study is to establish a reliable cell culture system for the long-term culture of rat urothelial cells (RUC), in which the cells multiply in vitro and form stratified polarized urothelium. Urothelial cells were harvested by the enzymatic digestion of the urothelium exposed by the eversion of resected rat bladders. Primary cultures were initiated in keratinocyte serum-free medium (KSFM) for selective proliferation of urothelial cells. Subsequently, the cells were propagated in a mixture of conditioned medium (CM) derived from Swiss 3T3 cell culture supernatant and KSFM (CM-KSFM). Mean population doubling time was 13.8 +/- 0.9 h. RUC were successfully maintained for 18 passages over a period of 4-5 mo. Detailed investigations of culture conditions showed that CM-KSFM yielded a differentiated multilayer structure. The stratified urothelial sheets measuring 4 x 6 cm2 could be formed and then detached using dispase. Cytokeratin pattern in both the cultured urothelial monolayer and engineered stratified layers was similar to those seen in vivo, as assessed with monoclonal antibody against cytokeratin 17. Ultrastructural morphology showed microvilli, basal cell layer, and desmosomes between adjacent cells in the stratified urothelium.

In vitro engineering of human stratified urothelium: analysis of its morphology and function.

Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

IN VITRO ENGINEERING OF HUMAN STRATIFIED UROTHELIUM: ANALYSIS OF ITS MORPHOLOGY AND FUNCTION

Purpose: Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium.Materials and Methods: Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium.Results: As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression o...

Reconstruction of the Urinary Bladder Mucosa in Three-Dimensional Collagen Gel Culture: Fibroblast-Extracellular Matrix Interactions on the Differentiation of Transitional Epithelial Cells

The purpose of this study was to reconstruct a urinary bladder mucosa in three-dimensional collagen gel culture conditions that included fibroblasts. Transitional epithelial cells and fibroblasts, isolated respectively from the epithelial and lamina propria layers of porcine urinary bladder, were cultured in monolayer. These fibroblasts were embedded and cultured within a collagen gel matrix to reconstruct a lamina propria. The isolated transitional epithelial cells were then seeded in vitro on this reconstructed lamina propria on which the transitional epithelial cells formed a stratified urothelium composed of basal, intermediate and superficial layers. Urothelial differentiation was observed only on the fibroblast-containing collagen matrix. Differentiation did not occur on a cell-free collagen matrix through use of fibroblast conditioned medium. Thus, urothelial differentiation depended upon a fibroblast-extracellular matrix interaction. The differentiated transitional epithelial cell layer thus produced closely resembled a urothelium in vivo. This culture model may provide a useful system in which to study various diseases of the urinary bladder.

Growth and characterization of normal human urothelium in vitro.

A method for initiating rapidly growing cultures of normal human transitional cells from ureter and embryonic bladder specimens has been developed and quantified. A new microdissection technique was used to nonenzymatically separate the urothelium. The use of enriched medium containing 10 micrograms/ml insulin, 5 micrograms/ml transferrin, and 1 microgram/ml hydrocortisone resulted in improved growth. The use of thin collagen gel substrates (0.6 ml/60 mm petri dish) resulted in 97% attachment of explants compared to 77% attachment on plastic. Explants grown on thicker collagen (2 ml/60 mm petri dish) showed, in addition to better attachment, enhanced growth of cells as determined both by measurements of colony size and cell density. Cultures of transitional cells that were initiated using explants could be passed three to five times using 0.1% EDTA for dispersion. Autoradiography of [3H]thymidine-labeled cells showed an initial phase of rapid cell division in primary explant cultures and restimulation of cell division in passaged cultures. Transmission electron microscopy showed that the cells growing out from the explants were continuous with the stratified urothelium maintained in the original explant. Stratification of transitional cells occurred in cultures of both ureter and embryonic bladder cells. Surface cells were joined near their apices by junctional complexes. Desmosomes and Golgi vesicles were present in all cells. Passage in culture did not alter the morphological characteristics of cells.

Histology and histogenesis of two different types of inverted urothelial papillomas.

Inverted papillomas are rare tumors of the lower urinary tract. Among 1829 reclassified tumors of the urinary bladder, renal pelves, ureters, and urethra, there were 40 (2.2%) inverted papillomas. The great majority of these were localized in the bladder. Because of distinctive histologic features and patterns of growth it is possible to differentiate between two basic types of inverted papillomas which were termed "trabecular" and "glandular." The trabecular type consists of widely branched, anastomosing cords of urothelial cells originating directly from the overlying transitional epithelium. The trabeculae are arranged horizontally or perpendicularly to the surface epithelium and occasionally exhibit peripheral palisading of the cells. The glandular type is characterized by multiple round to oval islands of proliferated urothelial cells together with pseudoglandular and true glandular structures which are often still connected with the surface urothelium. The gland-like structures are lined by stratified urothelium, the true glands by mucus secreting columnar epithelium. Sometimes glandular metaplasia of an intestinal type with goblet cell formation could be observed. Inverted papillomas of the trabecular type arise histogenetically from a proliferation of the basal cells of the urothelium. The glandular type develops apparently from a proliferative cystitis cystica and glandularis which, therefore, should be considered a potentially preneoplastic lesion. The predominant view of the biological behavior of inverted papillomas is that of a benign neoplastic lesion. Morphologic findings supplied some arguments in favor of a low grade malignant potential of these tumors. However, their malignant transformation seemed to be much lower than that of exophytic papillomas.