Differentiation Of Urothelial Cells Research Articles

Supraphysiologic Vaginal Estrogen Therapy in Aged Mice Mitigates Age-Associated Bladder Inflammatory Response to Urinary Tract Infections.

Bladder diseases characterized by chronic inflammation are highly prevalent in older women, as are recurrent urinary tract infections (rUTIs). Recurrent urinary tract infections lead to chronic inflammation of the bladder mucosa and cause lower urinary tract symptoms that persist even after the infection is cleared. Vaginal estrogen therapy (VET) has long been used for the treatment of rUTIs; however, its mechanism of action remains unclear. The objective of this study was to examine the mechanism(s) by which VET affects bladder inflammation and response to rUTIs. Here, we induced surgical menopause in aged (18 months old) mice followed by VET. Mice were then infected with uropathogenic Escherichia coli , and course of infection was investigated. Inflammatory cytokine response was assessed before and during infection using enzyme-linked immunosorbent assay. RNA sequencing analysis was used to compare the inflammatory status of the young versus aged bladder and principal changes confirmed via quantitative reverse transcriptase-polymerase chain reaction to determine the effects of VET on bladder inflammation. Impact on age-associated bladder tertiary lymphoid tissue formation was evaluated histologically. In the ovariectomized aged model, VET not only mitigated uterine atrophy but was also associated with reduced rUTIs, number of bacterial reservoirs, dampened immune response, and promotion of terminal differentiation of urothelial cells. Bladder tertiary lymphoid tissue lesions were also reduced with VET, with an associated decrease in signals important for bladder tertiary lymphoid tissue formation. Finally, we determined that VET reverses age-associated upregulation of inflammatory genes and pathways. Our data suggest that VET is effective by reducing age-associated hyperinflammatory conditions in bladder mucosa and in enhancing the host response to infection.

The Golgi complex: An organelle that determines urothelial cell biology in health and disease

The Golgi complex undergoes considerable structural remodeling during differentiation of urothelial cells in vivo and in vitro. It is known that in a healthy bladder the differentiation from the basal to the superficial cell layer leads to the formation of the tightest barrier in our body, i.e., the blood–urine barrier. In this process, urothelial cells start expressing tight junctional proteins, apical membrane lipids, surface glycans, and integral membrane proteins, the uroplakins (UPs). The latter are the most abundant membrane proteins in the apical plasma membrane of differentiated superficial urothelial cells (UCs) and, in addition to well-developed tight junctions, contribute to the permeability barrier by their structural organization and by hindering endocytosis from the apical plasma membrane. By studying the transport of UPs, we were able to demonstrate their differentiation-dependent effect on the Golgi architecture. Although fragmentation of the Golgi complex is known to be associated with mitosis and apoptosis, we found that the process of Golgi fragmentation is required for delivery of certain specific urothelial differentiation cargoes to the plasma membrane as well as for cell–cell communication. In this review, we will discuss the currently known contribution of the Golgi complex to the formation of the blood–urine barrier in normal UCs and how it may be involved in the loss of the blood–urine barrier in cancer. Some open questions related to the Golgi complex in the urothelium will be highlighted.

Surface Shave: Revealing the Apical-Restricted Uroglycome.

This study aimed to investigate the highly differentiated urothelial apical surface glycome. The functions of the mammalian urothelium, lining the majority of the urinary tract and providing a barrier against toxins in urine, are dependent on the correct differentiation of urothelial cells, relying on protein expression, modification, and complex assembly to regulate the formation of multiple differentiated cell layers. Protein glycosylation, a poorly studied aspect of urothelial differentiation, contributes to the apical glycome and is implicated in the development of urothelial diseases. To enable surface glycome characterization, we developed a method to collect tissue apical surface N- and O-glycans. A simple, novel device using basic laboratory supplies was developed for enzymatic shaving of the luminal bladder urothelial surface, with subsequent release and mass spectrometric analysis of apical surface O- and N-glycans, the first normal mammalian urothelial N-glycome to be defined. Trypsinization of superficial glycoproteins was tracked using immunolabeling of the apically expressed uroplakin 3a protein to optimize enzymatic release, without compromising the integrity of the superficial urothelial layer. The approach developed for releasing apical tissue surface glycans allowed for comparison with the N-glycome of the total porcine bladder urothelial cells and thus identification of apical surface glycans as candidates implicated in the urothelial barrier function. Data are available in MassIve: MSV000087851.

The potential for designing urothelial carcinomas using pluripotent stem cell-based systems

Despite a variety of therapeutic options for urothelial carcinoma of the bladder (UCB) in the early stages, the efficacy of treatment options in metastatic disease remains modest. Investigating the influence of different mutational patterns during the complex process of carcinogenesis may enable the development of innovative therapeutic agents. The differentiation of human pluripotent stem cells (hPSCs) into mature bladder urothelial cells offers a distinct platform to study embryonic development and disease modelling in the human urothelium. Given the precisely defined genetic background of those cells, it becomes evident that hPSCs-derived urothelium provides a valuable tool to study the processes during early carcinogenesis upon defined oncogenic stimuli. Differentiation of hPSCs into urothelial cells was performed by mimicking different stages of embryonic development with the induction of definitive endoderm, a hindgut differentiation step and finally, the induction of mature urothelium. To study the influence of different genes on the carcinogenesis of UCB, knockouts of TP53, BRCA2 and ATM were generated by CRISPR-Cas9 mediated gene editing. A protocol for the differentiation of urothelial cells from hPSCs was established by reaching different milestones of embryonic development (1. definitive endoderm, 2. hindgut, 3. urothelium). The induction of urothelium was confirmed by upregulation of uroplakins (UPK2, UPK3a) in the differentiated cells. Functional relevance of the knockouts was confirmed by reduced gene and protein expression (BRCA2, TP53), the presence of truncated proteins (ATM and BRCA2) and enhanced sensitivity to genotoxic stress. An efficient generation of urothelial cells from hPSCs is feasible. The functional relevance of different gene knockouts underlines the validity of the gene-editing strategy. In future studies, the significance of exogenous and endogenous carcinogenic stimuli in the context of distinct mutational signatures during early carcinogenesis of UCB can be investigated in this defined genetic background.

GABPA is a master regulator of luminal identity and restrains aggressive diseases in bladder cancer

TERT promoter mutations occur in the majority of glioblastoma, bladder cancer (BC), and other malignancies while the ETS family transcription factors GABPA and its partner GABPB1 activate the mutant TERT promoter and telomerase in these tumors. GABPA depletion or the disruption of the GABPA/GABPB1 complex by knocking down GABPB1 was shown to inhibit telomerase, thereby eliminating the tumorigenic potential of glioblastoma cells. GABPA/B1 is thus suggested as a cancer therapeutic target. However, it is unclear about its role in BC. Here we unexpectedly observed that GABPA ablation inhibited TERT expression, but robustly increased proliferation, stem, and invasive phenotypes and cisplatin resistance in BC cells, while its overexpression exhibited opposite effects, and inhibited in vivo metastasizing in a xenograft transplant model. Mechanistically, GABPA directly activates the transcription of FoxA1 and GATA3, key transcription factors driving luminal differentiation of urothelial cells. Consistently, TCGA/GEO dataset analyses show that GABPA expression is correlated positively with luminal while negatively with basal signatures. Luminal tumors express higher GABPA than do basal ones. Lower GABPA expression is associated with the GABPA gene methylation or deletion (especially in basal subtype of BC tumors), and predicted significantly shorter patient survival based on TCGA and our cohort of BC patient analyses. Taken together, GABPA dictates luminal identity of BC cells and inhibits aggressive diseases in BC by promoting cellular differentiation despite its stimulatory effect on telomerase/TERT activation. Given these biological functions and its frequent methylation and/or deletion, GABPA serves as a tumor suppressor rather than oncogenic factor in BC. The GABPA effect on oncogenesis is context-dependent and its targeting for telomerase inhibition in BC may promote disease metastasizing.

The effect of a cyclic uniaxial strain on urinary bladder cells

PurposePre-conditioning of a cell seeded construct may improve the functional outcome of a tissue engineered construct for augmentation cystoplasty. The precise effects of mechanical stimulation on urinary bladder cells in vitro are not clear. In this study we investigate the effect of a cyclic uniaxial strain culture on urinary bladder cells which were seeded on a type I collagen scaffold.MethodsIsolated porcine smooth muscle cells or urothelial cells were seeded on a type I collagen scaffolds and cultured under static and dynamic conditions. A uniform cyclic uniaxial strain was applied to the seeded scaffold using a Bose Electroforce Bio-Dynamic bioreactor. Cell proliferation rate and phenotype were investigated, including SEM analysis, RT-PCR and immunohistochemistry for α-Smooth muscle actin, calponin-1, desmin and RCK103 expression to determine the effects of mechanical stimulation on both cell types.ResultsDynamic stimulation of smooth muscle cell seeded constructs resulted in cell alignment and enhanced proliferation rate. Additionally, expression of α-Smooth muscle actin and calponin-1 was increased suggesting differentiation of smooth muscle cells to a more mature phenotype.ConclusionsMechanical stimuli did not enhance the proliferation and differentiation of urothelial cells. Mechanical stimulation, i.e., preconditioning may improve the functional in vivo outcome of smooth muscle cell seeded constructs for flexible organs such as the bladder.

Combined cytotoxic effect of UV-irradiation and TiO2 microbeads in normal urothelial cells, low-grade and high-grade urothelial cancer cells.

The differentiation of urothelial cells results in normal terminally differentiated cells or by alternative pathways in low-grade or high-grade urothelial carcinomas. Treatments with traditional surgical and chemotherapeutical approaches are still inadequate and expensive, as bladder tumours are generally highly recurrent. In such situations, alternative approaches, using irradiation of the cells and nanoparticles, are promising. The ways in which urothelial cells, at different differentiation levels, respond to UV-irradiation (photolytic treatment) or to the combination of UV-irradiation and nanoparticles (photocatalytic treatment), are unknown. Here we tested cytotoxicity of UV-irradiation on (i) normal porcine urothelial cells (NPU), (ii) human low-grade urothelial cancer cells (RT4), and (iii) human high-grade urothelial cancer cells (T24). The results have shown that 1 minute of UV-irradiation is enough to kill 90% of the cells in NPU and RT4 cultures, as determined by the live/dead viability assay. On the other hand, the majority of T24 cells survived 1 minute of UV-irradiation. Moreover, even a prolonged UV-irradiation for 30 minutes killed <50% of T24 cells. When T24 cells were pre-supplemented with mesoporous TiO2 microbeads and then UV-irradiated, the viability of these high-grade urothelial cancer cells was reduced to <10%, which points to the highly efficient cytotoxic effects of TiO2 photocatalysis. Using electron microscopy, we confirmed that the mesoporous TiO2 microbeads were internalized into T24 cells, and that the cell's ultrastructure was heavily compromised after UV-irradiation. In conclusion, our results show major differences in the sensitivity to UV-irradiation among the urothelial cells with respect to cell differentiation. To achieve an increased cytotoxicity of urothelial cancer cells, the photocatalytic approach is recommended.

Brg1 Determines Urothelial Cell Fate during Ureter Development

Developing and adult ureters express the epigenetic regulator Brg1, but the role of Brg1 in ureter development is not well understood. We conditionally ablated Brg1 in the developing ureter using Hoxb7-Cre and found that Brg1 expression is upstream of p63, Pparγ, and sonic hedgehog (Shh) expression in the ureteral epithelium. In addition, epithelial stratification in the basal cells required Brg1-dependent p63 expression, whereas terminal differentiation of the umbrella cells required Brg1-dependent Pparγ expression. Furthermore, the loss of ureteric Brg1 resulted in failure of Shh expression, which correlated with reduced smooth muscle cell development and hydroureter. Taken together, we conclude that Brg1 expression unifies three aspects of ureter development: maintenance of the basal cell population, guidance for terminal differentiation of urothelial cells, and proper investment of ureteral smooth muscle cells.

Melatonin prevents the development of hyperplastic urothelium induced by repeated doses of cyclophosphamide

Repeated cyclophosphamide (CP) chemotherapy increases the risk of developing bladder cancer, which could be due to the extremely rapid proliferation of urothelial cells observed in hyperplastic urothelium induced by CP treatment. We investigated the effect of melatonin on the development of urothelial hyperplasia induced by repeated CP treatment. Male ICR mice were injected with CP (150 mg/kg) or melatonin (10 mg/kg) with CP once a week for 3, 4 and 5 weeks. Transmission and scanning electron microscopy, immunohistochemistry and Western blot analysis were used to study the ultrastructure, apoptosis, proliferation and differentiation of urothelial cells. Repeated doses of CP caused the development of hyperplastic urothelium with up to ten cell layers and increased proliferation and apoptotic indices regarding Ki-67 and active caspase-3 immunohistochemistry, respectively. Scanning electron microscopy observations, cytokeratin and asymmetrical unit membrane immunohistochemistry and Western blot analysis showed a lower differentiation state of superficial urothelial cells. Melatonin co-treatment prevented the development of hyperplastic urothelium, statistically significantly decreased proliferation and apoptotic indices after four and five doses of CP and caused higher differentiation state of superficial urothelial cells.

Maturation of the Golgi apparatus in urothelial cells

The differentiation of urothelial cells is characterized by the synthesis of uroplakins and their assembly into the asymmetric unit membrane. The Golgi apparatus (GA) has been proposed to play a central role in asymmetric unit membrane formation. We have studied the distribution and organization of the GA in normal mouse urothelial cells and in the superficial urothelial cells that undergo differentiation following cyclophosphamide-induced regeneration, in correlation with urothelial cell differentiation. In normal urothelium, immature basal cells have a simple GA, which is small and distributed close to the nucleus. In intermediate cells, the GA starts to expand into the cytoplasm, whereas the GA of terminally differentiated umbrella cells is complex, being large and spread over the whole basal half of the cytoplasm. During early stages of regeneration after cyclophosphamide treatment, the GA of superficial cells is simple and no markers of urothelial differentiation (uroplakins or asymmetric unit membranes, discoidal or fusiform vesicles, apical surface covered with microvilli) are expressed. At a later stage, the GA expands and, in the final stage of regeneration, when cells express all markers of terminal urothelial differentiation, the GA become complex once again. Our results show that: (1) GA distribution and organization in urothelial cells is differentiation-dependent; (2) the GA matures from a simple form in partially differentiated cells to a complex form in terminally differentiated superficial cells; (3) major rearrangements of GA distribution and organization correlate with the beginning of asymmetric unit membrane production. Thus, GA maturation seems to be crucial for asymmetric unit membrane formation.

Apical Plasma Membrane Traffic in Superficial Cells of Bladder Urothelium

Superficial urothelial cells that line the urinary bladder accommodate cyclical changes in organ volume while maintaining a permeability barrier between urine and tissue fluids. The specific apical plasma membrane traffic is necessary for their proper function. The composition of the apical plasma membrane is dramatically modified during differentiation of bladder urothelial cells, most notably by assembly of urothelial plaques containing uroplakins. However, the assembly of uroplakins into plaques, their insertion and removal from the apical surface, and the regulation of these processes are still poorly understood. This review examines the traffic (exocytosis/endocytosis) of the apical plasma membrane during differentiation of urothelial cells and focuses on the physiological and clinical significance of the apical plasma membrane traffic in bladder superficial urothelial cells.

Effect of melatonin on apoptosis, proliferation and differentiation of urothelial cells after cyclophosphamide treatment

Melatonin was recently shown to have protective effects against cyclophosphamide (CP)-induced hemorrhagic cystitis (HC) by diminishing bladder oxidative stress. HC is accompanied by destruction of the bladder urothelium and followed by apoptosis and rapid regeneration via proliferation and differentiation of urothelial cells, reaching complete restoration of normal urothelium in three weeks. Therefore, the effect of melatonin on apoptosis, proliferation and differentiation of urothelial cells, during destruction and regeneration of the urothelium three-weeks after a single dose CP treatment, was studied. F344 male rats were injected intraperitoneally with saline (control group) or melatonin (Mel group) or a single dose of CP (100 mg/kg; CP group) or melatonin (10 mg/kg) with CP (Mel + CP group). Melatonin co-treatment with CP significantly reduced apoptosis and increased proliferation of urothelial cells at day 1 and thus prevented extensive loss of cells from the urothelium. However, proliferation indices at days 4 and 7 after melatonin and CP co-treatment suddenly dropped and therefore the development of hyperplasia was prevented. Melatonin co-treatment with CP also resulted in earlier differentiation of superficial urothelial cells. Melatonin seems to have protective effect against CP-induced urothelial damage and a favorable impact on regeneration and restoration of normal urothelium, since it reduces the number of apoptotic and proliferating urothelial cells and results in their earlier differentiation.

A Comparative Ontogenic Study of Urinary Bladder: Impact of the Epithelial Differentiation in Embryonic and Newborn Rats

The present study aimed to show the cellular and subcellular distribution of glycogen content during the differentiation of urothelial cells from simple cuboidal to stratified transitional epithelium. Bladder samples were taken from rat embryos on the 15th to 19th days and newborn at 21st day. During the development of the bladder, the formation of fusiform vesicles, asymmetric unit membrane (AUM) and microridges were examined with staining with haematoxylin-eosin and periodic acid Schiff for light microscope and periodic acid-thiocharbohydrazide-silver proteinate for transmission electron microscope. The topographical changes of luminal differentiation were examined with the scanning electron microscope. The urothelium was simple cuboidal from 15th till the 17th days of gestation. Glycogen content was present in the cytoplasm till the 18th day of gestation. At the early stage (16th day) of gestation, the apical surface contains microvilli that points the undifferentiated cells. The density of microvilli decreased and ropy microridges appeared at the 17th day of gestation. The small discoid vesicles lined with AUM developed at the apical cytoplasm of the surface cells at the 17th day of gestation. After this stage, both the density of microridges and large and elongated fusiform vesicles increased. The differentiation of the urothelium begins with the formation of the round and small vesicles, continues with the formation of the AUM and at the final stage there is a decrease in both glycogen content and the appearance of the microridges at the luminal surface of the urothelial cells.

Activation of Peroxisome Proliferator-Activated Receptor-γ Reverses Squamous Metaplasia and Induces Transitional Differentiation in Normal Human Urothelial Cells

We observed that in urothelium, both cornifying and noncornifying forms of squamous metaplasia are accompanied by changes in the localization of the nuclear hormone receptors, peroxisome proliferator activated receptor gamma (PPAR-gamma) and retinoid X receptor (RXR-alpha). To obtain objective evidence for a role for PPAR-gamma-mediated signaling in urothelial differentiation, we examined expression of the cytokeratin isotypes CK13, CK20, and CK14 as indicators of transitional, terminal transitional, and squamous differentiation, respectively, in cultures of normal human urothelial cells. In control culture conditions, normal human urothelial cells showed evidence of squamous differentiation (CK14+, CK13-, CK20-). Treatment with the high-affinity PPAR-gamma agonist, troglitazone (TZ), resulted in gain of CK13 and loss of CK14 protein expression. The effect of TZ was significantly augmented when the autocrine-stimulated epidermal growth factor receptor pathway was inhibited and this resulted in induction of CK20 expression. The RXR-specific inhibitors PA452, HX531, and HX603 inhibited the TZ-induced CK13 expression, supporting a role for RXR in the induction of CK13 expression. Thus, signaling through PPAR-gamma can mediate transitional differentiation of urothelial cells and this is modulated by growth regulatory programs.

What determines differentiation of urothelial umbrella cells?

Cytokeratins, uroplakins and the asymmetric unit membrane are biochemical and morphological markers of urothelial differentiation. The aim of our study was to follow the synthesis, subcellular distribution and supramolecular organization of differentiation markers, cytokeratins and uroplakins, during differentiation of umbrella cells of mouse bladder urothelium. Regenerating urothelium after destruction with cyclophosphamide was used to simulate de-novo differentiation of cells, which was followed from day 1 to day 14 after cyclophosphamide injection. Cytokeratin 7 and uroplakins co-localized in the subapical cytoplasm of superficial cells from the early stage of differentiation on. At early stages of superficial cell differentiation cytokeratin 7 was filamentary organized, and rare uroplakins were found on the membranes of relatively small cytoplasmic vesicles, which were grouped in clusters under the apical membrane. Later, cytokeratin 7 gradually reorganized into a continuous trajectorial network, and uroplakins became organized into plaques of asymmetric unit membrane, which formed fusiform vesicles. After insertion of fusiform vesicles into the apical plasma membrane, the surface acquired microridged appearance of umbrella cells. Cytokeratin 20 appeared as the last differentiation marker of umbrella cells. Cytokeratin 20 was incorporated into the pre-existing trajectorial cytokeratin network. These results indicate that differentiation of urothelial cells starts with the synthesis of differentiation-related proteins i.e., cytokeratins and uroplakins, and later with their specific organization. We consider that the umbrella cell has reached its final stage of differentiation when uroplakins form plaques of asymmetric unit membrane that are inserted into the apical plasma membrane and when cytokeratin 20 becomes included in a trajectorial cytokeratin network in the subapical area of cytoplasm.

The effect of lamina propria on the growth and differentiation of urothelial cells in vitro.

The effect of lamina propria on the growth and differentiation of the mouse urinary bladder urothelial cells in vitro has been studied by light and electron microscopy using morphological and immunohistochemical methods. Three different types of urothelial cultures were maintained on a porous membrane in serum-free medium for 10 days. Our results showed that urothelial cells in culture types I and II were organised as the multilayer epithelium showing terminal differentiation, from the porous membrane toward the surface. On the surface of culture type I and II, superficial urothelial cells were highy differentiated. They formed tight junctions and had plaques of an asymmetric unit membrane, a hallmark of terminal differentiation in bladder superficial cells. These cultured superficial cells were also cytokeratin 20 positive. On the contrary, urothelial cells in culture type III have shown a limited capacity for survival and have grown as poorly differentiated monolayer. These results revealed that reciprocal intercellular signalling between the lamina propria and urothelial cells is essential to achieve terminal differentiation of urothelial cells in vitro.

Actin filaments during terminal differentiation of urothelial cells in the rat urinary bladder

The localisation of actin filaments was studied in rat urothelial cells during differentiation which accompanied regeneration after cell damage induced by cyclophosphamide (CP). By immunofluorescence it was established that actin filaments equally stained along the cell circumference in basal and intermediate cells, while basolateral cell membrane expression was found in terminally differentiated superficial cells. During regeneration, after CP treatment, simple urothelial hyperplasia developed with smaller cuboidal superficial cells, in which actin filaments were equally distributed under the apical and basolateral plasma membranes. As demonstrated by immunoelectron microscopy, the apical surface of these superficial cells was covered with microvilli containing bundles of actin filaments. Within 1 week, the urothelium reverted to its normal three-layer thickness. Superficial cells became larger and flattened and the unthickened apical plasma membrane matured into a thick asymmetric unit membrane. Concomitantly actin filaments disappeared from apical areas of superficial cells while remaining abundant at basolateral areas. Our results indicate that in the urothelium subcellular distribution of actin filaments can be considered as a marker of cell differentiation.

Fine Needle Aspiration Biopsy of the Penis: Transitional Cell Carcinoma of the Urinary Bladder with Mucinous Differentiation

We report a case of transitional cell carcinoma of the urinary bladder metastatic to the penis. The diagnosis was established by fine needle aspiration biopsy (FNAB). Malignant cells showed mucinous differentiation. Ancillary studies carried out in the FNA material as well as in the primary bladder carcinoma suggested transitional cell carcinoma with mucinous differentiation. FNAB proved to be effective in diagnosing a secondary neoplasm to the penis. Mucoid differentiation of urothelial cells can be seen in FNAB specimens and might pose a problem in differential diagnosis.

The effect of epidermal growth factor and transforming growth factor β1 on proliferation and differentiation of urothelial cells in urinary bladder explant culture

The effects of two growth factors, EGF and TGFβ1, on growth and differentiation of different populations of urothelial cells in explant cultures of mouse urinary bladder have been studied by electron microscopy and lectin analysis. In an explant culture 10 days after the implantation three different populations of urothelial cells can be distinguished. Original urothelial cells, which are integral part of the explant, new urothelial cells, which cover the baso‐lateral sides of the explant and are organized in a multilayer epithelium, and new urothelial cells, which are not any more in direct contact with the explant and grow over the membrane in epithelium‐like structure. Exogenously added EGF or TGFβ1 did not affect either the formation or the thickness of multilayered urothelium, so cells were proliferating on the free surfaces of stroma as well as on the epithelium expanding over the membrane. In the absence of growth factors in medium, the newly formed baso‐lateral multilayered epithelium bordering the stroma shows ultrastructural signs of terminal differentiation suggesting that for cell proliferation and differentiation the action of stroma is of crucial importance. On the other hand, the differentiation of the epithelium spreading over the membrane, but not its thickness, is affected by exogenously added TGFβ1. Solely in TGFβ1‐treated cultures a differentiation sirnilar to that in vivo takes place. The apoptosis of the urothelial cells was not increased by TGFβ1. The lectin analysis by WGA and ConA conjugated with ferritin revealed that ConA‐ferritin combines only with the surface cells which grow over the membrane in the absence of TGFβ1.

The effect of epidermal growth factor and transforming growth factor β1 on proliferation and differentiation of urothelial cells in urinary bladder explant culture

The effects of two growth factors, EGF and TGF β 1, on growth and differentiation of different populations of urothelial cells in explant cultures of mouse urinary bladder have been studied by electron microscopy and lectin analysis. In an explant culture 10 days after the implantation three different populations of urothelial cells can be distinguished. Original urothelial cells, which are integral part of the explant, new urothelial cells, which cover the baso-lateral sides of the explant and are organized in a multilayer epithelium, and new urothelial cells, which are not any more in direct contact with the explant and grow over the membrane in epithelium-like structure. Exogenously added EGF or TGF β 1 did not affect either the formation or the thickness of multilayered urothelium, so cells were proliferating on the free surfaces of stroma as well as on the epithelium expanding over the membrane. In the absence of growth factors in medium, the newly formed baso-lateral multilayered epithelium bordering the stroma shows ultrastructural signs of terminal differentiation suggesting that for cell proliferation and differentiation the action of stroma is of crucial importance. On the other hand, the differentiation of the epithelium spreading over the membrane, but not its thickness, is affected by exogenously added TGF β 1. Solely in TGFβ 1-treated cultures a differentiation sirnilar to that in vivo takes place. The apoptosis of the urothelial cells was not increased by TGF β 1. The lectin analysis by WGA and ConA conjugated with ferritin revealed that ConA-ferritin combines only with the surface cells which grow over the membrane in the absence of TGF β 1.