Plasticity of the urothelial phenotype: effects of gastro-intestinal mesenchyme/stroma and implications for urinary tract reconstruction

The present study tests the hypothesis that heterotypic stromal-epithelial interactions cause phenotypic changes in urothelium. The rational for the experimental design is to simulate heterotypic stromal-epithelial interactions that are created at the anastomotic site of intestinal-bladder augmentations and internal urinary diversions where the urothelium is in direct contact with the gastro-intestinal tract tissues. Tissue recombination experiments were performed by combining 14-day embryonic rat and mouse rectal mesenchyme with urothelium from embryonic, newborn, and adult mice or rats. All tissue recombinants were grown beneath the renal capsule of athymic mouse hosts for 6-16 weeks. Analyses were performed to detect expression of uroplakins, cytokeratin 7, 14, 19 and mucin secreting epithelial cells via Periodic Acid-Schiff (PAS). The phenotype of both mouse and rat urothelium was changed to a glandular morphology under the influence of rectal mesenchyme. Immunohistochemical staining revealed a loss of the urothelial specific uroplakins and cytokeratins 7, 14, and 19 (characteristic of urothelium). Histologic analysis revealed the presence of mucin secreting glandular structures which stained positive for PAS. The urothelial transdifferentiation into glandular epithelium was not a function of epithelial age and occurred in the embryonic, newborn and adult urothelium. Likewise, rectal mesenchyme from embryonic, neonatal, and adult animals was able to induce glandular differentiation in bladder epithelium. Urothelium exhibits the plasticity to change into an intestinal like epithelium as a result of mesenchymal/stromal stimulation from the gastro-intestinal tract. This experimental result is germane to heterotypic stromal-epithelial interactions that are created in patients with urinary tract reconstructions (intestinal augmentations, de-mucosalized urothelial lined bladder patches, and internal urinary diversion such as ureterosigmoidostomies). We propose that heterotypic stromal-epithelial interactions may play a role in determining histodifferentiation of urothelial cells at the anastomotic site between bowel and bladder tissue in patients with gastro-intestinal urothelial reconstructions.     

Freeze-fracture replica immunolabelling reveals urothelial plaques in cultured urothelial cells

The primary function of the urothelium is to provide the tightest and most impermeable barrier in the body, i.e. the blood-urine barrier. Urothelial plaques are formed and inserted into the apical plasma membrane during advanced stages of urothelial cell differentiation. Currently, it is supposed that differentiation with the final formation of urothelial plaques is hindered in cultured urothelial cells. With the aid of the high-resolution imaging technique of freeze-fracture replica immunolabelling, we here provide evidence that urothelial cells in vitro form uroplakin-positive urothelial plaques, localized in fusiform-shaped vesicles and apical plasma membranes. With the establishment of such an in vitro model of urothelial cells with fully developed urothelial plaques and functional properties equivalent to normal bladder urothelium, new perspectives have emerged which challenge prevailing concepts of apical plasma membrane biogenesis and blood-urine barrier development. This may hopefully provide a timely impulse for many ongoing studies and open up new questions for future research.     

Impaired Expression of Prostaglandin E2 (PGE2) Synthesis and Degradation Enzymes during Differentiation of Immortalized Urothelial Cells from Patients with Interstitial Cystitis/Painful Bladder Syndrome

Purpose

The differentiated superficial cells of the urothelium restrict urine flow into the bladder wall. We have demonstrated that urothelial cells isolated from bladders of patients with interstitial cystitis/painful bladder syndrome (IC/PBS) fail to release PGE₂ in response to tryptase. This study examines the expression of PGE₂ synthesis and degradation enzymes in urothelial cells during differentiation.

Materials and Methods

We measured immunoprotein expression of cyclooxygenase-2 (COX-2), prostaglandin E₂ synthase (PGES) and 15-hydroxyprostaglandin dehydrogenase (PGDH) in human urothelial cells and in immortalized urothelial cells isolated from the bladders of IC/PBS patients or normal subjects during stratification and differentiation produced by increased calcium and fetal bovine serum (Ca/FBS) in the culture medium for 1, 3 and 7 days.

Results

PGES immunoprotein expression increased during differentiation in normal and IC/PBS urothelial cells. COX-2 expression also increased in cells from normal patients following differentiation. Remarkably, no COX-2 expression was detectable in urothelial cells isolated from 3 out of 4 IC/PBS patients. PGDH immunoprotein expression decreased in normal cells after 1 and 3 days of Ca/FBS addition, but returned to normal after 7 days. PGDH expression was unchanged during differentiation at 1 and 3 days, but was more than 2-fold higher at 7 days compared to day 0 in the IC/PBS cells. Urothelial cells isolated from IC/PBS patients demonstrated no PGE₂ release in response to tryptase under any of the experimental conditions studied.

Conclusions

Taken together, our results indicate that PGE₂ release is compromised during stratification and differentiation in IC/PBS urothelium and may contribute to impaired barrier function.     

Urinary cytology of tuberculosis of the bladder

The cytologic findings in the urinary sediment of 13 patients with tuberculosis (TB) of the bladder were compared with the histologic findings. TB was cytologically recognized in 11 patients because of the presence of epithelioid and/or Langhans' giant cells. The morphologic deviations of the urothelial cells and the urothelium were classified according to the criteria of Koss. The type I atypical urothelial cells found in ten patients were correlated to the urothelial hyperplasia found at histology; the follow-up data documented the benign reversible nature of the urothelial hyperplasia as the focal expression of the reactive potential of urothelium to various forms of inflammatory processes. The presence of type II atypical urothelial cells reflected moderate and severe atypia of the urothelium at biopsy. In order to assess the reactive, reversible or precancerous nature of these forms of atypia associated with TB, an accurate follow-up is necessary. Since TB of the bladder may cause the presence of atypical cells in the urine, this lesion is of special interest in the differential diagnosis between inflammatory processes and carcinoma.     

Prevention of Anti-microbial Peptide LL-37-Induced Apoptosis and ATP Release in the Urinary Bladder by a Modified Glycosaminoglycan

Interstitial cystitis (IC), often referred to in combination with painful bladder syndrome, is a chronic inflammatory disease of the bladder. Current therapies primarily focus on replenishing urothelial glycosaminoglycan (GAG) layer using GAG analogs and managing pain with supportive therapies. However, the elusive etiology of IC and the lack of animal models to study the disease have been major hurdles developing more effective therapeutics. Previously, we showed an increased urinary concentration of antimicrobial peptide LL-37 in spina bifida patients and used LL-37 to develop a mouse model of cystitis that mimics important clinical findings of IC. Here we investigate (1) the molecular mechanism of LL-37 induced cystitis in cultured human urothelial cells and in mice, (2) the protective effects of GM-0111, a modified GAG, within the context of this mechanism, (3) the physiological and molecular markers that correlate with the severity of the inflammation, and (4) the protective effects of several GAGs using these biomarkers in our LL-37 induced cystitis model. We find that LL-37 quickly induces release of ATP and apoptosis in the urothelium. These changes can be inhibited by a chemically-modified GAG, GM-0111. Furthermore, we also find that GAG analogs provide varying degrees of protection against LL-37 challenge in mice. These findings suggest that GM-0111 and possibly GAG molecules prevent the development of cystitis by blocking the apoptosis and the concurrent release of ATP from the urothelium.     

Detection of chromogranin A in human gastric adenocarcinomas using a sensitive immunohistochemical technique

Neuroendocrine cells are often disclosed in human gastric adenocarcinomas and may be recognised by their immunoreactivity towards chromogranin A. However, in dedifferentiated neuroendocrine tumour cells, the chromogranin A content may be reduced making it difficult to detect with conventional immunohistochemical methods. We therefore used a sensitive signal amplification technique in order to evaluate chromogranin A immunoreactivity and thus neuroendocrine differentiation in 40 gastric adenocarcinomas.Neuroendocrine cells were visualised by means of a monoclonal chromogranin A antibody and the avidin–biotin peroxidase complex technique, without and with addition of tyramide signal amplification. Double immunohistochemistry towards chromogranin A and Ki-67 were used to disclose proliferation in the neoplastic cells.A marked increase in the number of carcinomas containing chromogranin A-immunoreactive neoplastic cells was noted when applying the tyramide signal amplification technique. In addition, the number of immunoreactive cells within each tumour increased, and in some cases almost all the neoplastic cells became immunoreactive. Chromogranin A-immunoreactive tumour cells showing signs of proliferation were found in the majority of these carcinomas.In conclusion, we have disclosed widespread immunoreactivity towards chromogranin A in a proportion of gastric adenocarcinomas when enhancing the signal with tyramide signal amplification. Neuroendocrine differentiation is thus a common finding in gastric carcinomas when using sensitive methods.     

Tumor-associated neoexpression of the pS2 peptide and MUC5AC mucin in primary adenocarcinomas and signet ring cell carcinomas of the urinary bladder

To gain more detailed insight into the histogenesis of primary nonurachal adenocarcinomas and signet ring cell carcinomas of the urinary bladder, we analyzed by immunohistochemistry the expression of a broad panel of proteins, associated with cell differentiation (pS2 peptide, MUC5AC, MUC6, spasmolytic polypeptide, cyclooxygenases-1 and -2, caveolin-1), and of various novel known or candidate tumor suppressors (14-3-3 sigma, SYK, PTEN, maspin). Included were 12 adenocarcinomas admixed to urothelial carcinomas, 10 pure adenocarcinomas and 5 signet ring cell carcinomas. As the most important finding, the majority of signet ring cell carcinomas and three quarters of the adenocarcinomas (72.7%) expressed the pS2 peptide, and nearly half of the adenocarcinomas (45.5%) as well as most of the signet ring cell carcinomas were observed to secrete the MUC5AC apomucin. Since expression of both proteins was absent in the normal nonneoplastic urothelium, their tumor-associated appearance is regarded as a neoexpression or reexpression, respectively, of normally cryptic antigenic determinants, and is assumed to be involved in the phenotypical formation of vesical adenocarcinomas, including signet ring cell carcinomas. The expression of both pS2 and MUC5AC in variants of urothelial carcinomas with a glandular differentiation and an extracellular mucus production support the concept that adenocarcinomas may histogenetically develop from preexistent TCC. Adenocarcinomas which secrete the pS2 peptide and the MUC5AC glycoprotein are proposed to be subclassified as adenocarcinomas of the intestinal type, as distinguished from those of the common type lacking an expression. The tumor suppressor genes showed a loss of protein expression in adenocarcinomas, ranging from 54.5% (14-3-3 sigma), to 31.8 (PTEN), 27.3% (SYK) and 18.2% (maspin). Similar expression profiles in the coexistent urothelial carcinomas argue against a specific involvement of these genes during the morphogenesis of adenocarcinomas.     

Plenary Symposia

Studies of the urothelium, the specialized epithelial lining of the urinary bladder, are critical for understanding diseases affecting the lower urinary tract, including interstitial cystitis, urinary tract infections and cancer. However, our understanding of urothelial pathophysiology has been hampered by a lack of appropriate model systems. Here, we describe the isolation and characterization of a non-transformed urothelial cell line (TRT-HU1), originally explanted from normal tissue and immortalized with hTERT, the catalytic subunit of telomerase. We demonstrate responsiveness of the cells to anti-proliferative factor (APF), a glycopeptide implicated in the pathogenesis of interstitial cystitis. TRT-HU1 carries a deletion on the short arm of chromosome 9, an early genetic lesion in development of bladder cancer. TRT-HU1 urothelial cells displayed growth and migration characteristics similar to the low-grade papilloma cell line RT4. In contrast, we observed marked differences in both phenotype and gene expression profiles between TRT-HU1 and the highly malignant T24 cell line. Together, these findings provide the first demonstration of a non-transformed, continuous urothelial cell line that responds to APF. This cell line will be valuable for studies of both benign and malignant urothelial cell biology.     

Mesenchymal reprogramming of adult human epithelial differentiation

The objective of this study was to determine whether neonatal rat seminal vesicle mesenchyme (rSVM) can reprogram epithelial differentiation in a fully differentiated adult human bladder epithelium. For this purpose neonatal rSVM was isolated from newborn (0-day) Sprague-Dawley rats, and normal adult human bladder epithelium (hBLE) was isolated from radical cystoprostatectomy specimens to prepare rSVM+hBLE tissue recombinants in vitro. After overnight culture the tissue recombinants were grafted beneath the renal capsule of male athymic rodent hosts and allowed to grow in vivo for 6 months. As controls, rSVM and hBLE were grafted separately and allowed to grow for the same period. Tissue recombinants and control tissue grafts were harvested, and secretions were collected for biochemical studies. Tissues were fixed both for histologic as well as immunohistochemical staining. Neonatal rSVM induced normal adult human bladder urothelium to form glandular structures resembling prostate. The induced prostatic acini were filled with secretions that expressed human prostate-specific secretory proteins. These findings demonstrate that adult human urothelial cells retain a responsiveness to neonatal prostatic mesenchymal inductors. Change in urothelial histodifferentiation was associated with change in functional activity. The ability of the neonatal rat mesenchymal tissues to induce morphologic as well as biochemical changes in normal adult human urothelium provides a basis for human tissue engineering and organ reconstruction.     

Epithelial-stromal interactions in the adult bladder: urothelial growth, differentiation, and maturation on culture facsimiles of bladder stroma

Analysis of the responsiveness of isolated adult urothelium to a series of different stromal cell-extracellular matrix combinations demonstrated the capacity of stromal cells to induce and maintain normal patterns of urothelial growth, differentiation, and maturation in vitro. By incorporating embryonic mesenchymal derived (Swiss 3T3) cells into type I collagen matrices, simplified three-dimensional tissue-like facsimiles of bladder stroma were derived. When recombined with sheets of isolated urothelium these facsimiles could approximately reproduce the capacity of natural stromal tissue to support the expression of normal urothelial tissue specific characteristics. In contrast cocultures between urothelia and monolayers of 3T3 cells, applied to the surface of planar collagen substrata could only permit urothelial cell attachment but not growth or differentiation whereas lethally irradiated 3T3 (feeder) cells, under similar experimental conditions, could support the maintenance of an immature or incompletely differentiated urothelium. Conditioned medium elaborated by cultured 3T3 cells could not stimulate further differentiation in urothelia cultured alone on planar collagen substrata. These studies indicate that a significant portion of the regulatory capacity of the stroma in stromal-urothelial interactions can be accounted for by the activities of a closely applied population of stromal cells, provided the cells are viable and presented to the urothelium in a three-dimensional context in combination with collagen. The capacity of embryonic mesenchymal cells to express properties appropriate to the development of a multilayered terminally differentiated urothelium suggests that normal interactions between adult urothelium and stroma are of limited specificity with the urothelium requiring an essential input of permissive signals only.     

Morphological and biochemical investigation of nitric oxide synthase and related enzymes in the rat and pig urothelium.

We investigated the enzymes involved in the NADPH-diaphorase (d) reaction in the rat and pig bladder urothelium. The urothelial cell layer displayed intense and uniform NADPH-d activity. Preincubation with the flavoprotein inhibitor diphenyleneiodionium chloride (DPI) and the alkaline phosphatase inhibitor levamisole concentration-dependently decreased the urothelial NADPH-d activity. Immunoreactivities to neuronal (n), endothelial (e), or inducible (i) nitric oxide synthase (NOS) were not detected in rat or pig urothelial cells. In rats, the urothelium was uniformly immunoreactive for NADPH cytochrome P450 reductase, whereas the pig urothelium displayed inconsistent labeling. In lipopolysaccharide (LPS)-treated rats, the bladder urothelium showed positive iNOS immunoreactivity. The iNOS labeling was found predominantly in cells located in the basal layer of the urothelium. In the pig bladder mucosa, a Ca2+-dependent NOS activity was evident in cytosolic and particulate fractions that was quantitatively comparable to the NOS activity found in the smooth muscle. In ultrastructural studies of urothelial cells, NADPH-d reaction products were found predominantly on membranes of the nuclear envelope, endoplasmatic reticulum and mitochondria. In conclusion, NADPH-d staining of the urothelium cannot be taken as an indicator for the presence of constitutively expressed NOS. Activity of alkaline phosphatase and cytochrome P450 reductase may account for part of the NADPH-d reaction in urothelial cells. However, LPS treatment of rats caused expression of iNOS in urothelial cells.     

Urothelial umbrella cells of human ureter are heterogeneous with respect to their uroplakin composition: different degrees of urothelial maturity in ureter and bladder?

Urothelial umbrella cells are characterized by apical, rigid membrane plaques, which contain four major uroplakin proteins (UP Ia, Ib, II and III) forming UPIa/UPII and UPIb/UPIII pairs. These integral membrane proteins are thought to play an important role in maintaining the physical integrity and the permeability barrier function of the urothelium. We asked whether the four uroplakins always coexpress in the entire human lower urinary tract. We stained immunohistochemically (ABC-peroxidase method) paraffin sections of normal human ureter (n = 18) and urinary bladder (n = 10) using rabbit antibodies against UPIa, UPIb, UPII and UPIII; a recently raised mouse monoclonal antibody (MAb), AU1, and two new MAbs, AU2 and AU3, all against UPIII; and mouse MAbs against umbrella cell-associated cytokeratins CK18 and CK20. Immunoblotting showed that AU1, AU2 and AU3 antibodies all recognized the N-terminal extracellular domain of bovine UPIII. By immunohistochemistry, we found that in 15/18 cases of human ureter, but in only 2/10 cases of bladder, groups of normal-looking, CK18-positive umbrella cells lacked both UPIII and UPIb immunostaining. The UPIb/UPIII-negative cells showed either normal or reduced amounts of UPIa and UPII staining. These data were confirmed by double immunofluorescence microscopy. The distribution of the UPIb/UPIII-negative umbrella cells was not correlated with localized urothelial proliferation (Ki-67 staining) or with the distribution pattern of CK20. Similar heterogeneities were observed in bovine but not in mouse ureter. We provide the first evidence that urothelial umbrella cells are heterogeneous as some normal-looking umbrella cells can possess only one, instead of two, uroplakin pairs. This heterogeneity seems more prominent in the urothelium of human ureter than that of bladder. This finding may indicate that ureter urothelium is intrinsically different from bladder urothelium. Alternatively, a single lineage of urothelium may exhibit different phenotypes resulting from extrinsic modulations due to distinct mesenchymal influence and different degrees of pressure and stretch in bladder versus ureter. Additional studies are needed to distinguish these two possibilities and to elucidate the physiological and pathological significance of the observed urothelial and uroplakin heterogeneity.     

Differentiation-dependent localisation of tissue-type plasminogen activator in human bladder urothelium

Human bladder urothelium is able to secrete tissue-type plasminogen activator (tPA). The aim of our study was to analyse localisation of tPA antigen in comparison to differentiation state of cells in samples of histologically normal urothelium and non-invasive tumours of the human urinary bladder. Twenty-five samples of normal urothelium and 31 non-invasive papillary tumours from 36 patients were examined. The presence of tPA antigen was evaluated immunohistochemically. Differentiation of superficial cells was assessed by the presence of urothelial cell differentiation markers, uroplakins (UPs; immunohistochemistry) and cell's apical surface architecture (scanning electron microscopy). All tissue samples stained anti-tPA positive. In normal urothelium, the intensity of anti-tPA staining was the strongest in superficial cells, which were well-differentiated. In tumours, all cell layers stained anti-tPA positive. The intensity of anti-tPA positive reaction in the upper cell layer correlated with the percentage of anti-UP positive superficial cells. Superficial cells showed various differentiation states. The localisation of tPA antigen in human in vivo tissue is not confined to the well-differentiated superficial cells. Our results suggest a positive correlation between tPA secretion and cell differentiation.     

Bladder cancer cell in co-culture induces human stem cell differentiation to urothelial cells through paracrine FGF10 signaling

Fibroblast growth factor 10 (FGF10) is required for embryonic epidermal morphogenesis including brain development, lung morphogenesis, and initiation of limb bud formation. In this study, we investigated the role of FGF10 as a lead induction factor for stem cell differentiation toward urothelial cell. To this end, human multipotent stem cell in vitro system was employed. Human amniotic fluid stem cells were co-cultured with immortalized bladder cancer lines to induce directed differentiation into urothelial cells. Urothelial markers, uroplakin II, III, and cytokeratin 8, were monitored by RT-PCR, immunocytochemistry, and Western blot analysis. Co-cultured stem cells began to express uroplakin II, III, and cytokeratin 8. Targeted FGF10 gene knockdown from bladder cancer cells abolished the directed differentiation. In addition, when FGF10 downstream signaling was blocked with the Mek inhibitor, the co-culture system lost the capacity to induce urothelial differentiation. Exogenous addition of recombinant FGF10 protein promoted stem cell differentiation into urothelium cell lineage. Together, this report suggests that paracrine FGF10 signaling stimulates the differentiation of human stem cell into urothelial cells. Current study provides insight into the potential role of FGF10 as a lead growth factor for bladder regeneration and its therapeutic application for bladder transplantation.     

Urothelium-specific antibody and lectin surface mapping of bladder urothelium

Summary Coupled ligand-colloidal gold complexes were found to provide a convenient approach for the localization by scanning electron microscopy of cell surface membrane antigens and lectin-binding sites on bladder urothelium and for the immunocytochemical identification of urothelial cell populations at different stages of differentiation. The ligands used to probe the membrane were a urothelium-specific rabbit antibody raised to a urothelial membrane-associated antigen (UMA), and two lectins: Concanavalin A (Con A) and peanut agglutinin (PNA). A complex luminal surface distribution pattern was demonstrated by the UMA antigen related to the stage of urothelial cell maturation and differentiation. UMA could be detected on the surface of immature and early differentiating intermediate cells, but was absent from the late differentiation stage, becoming re-expressed as the cells matured and was found in greatest abundance on the terminally differentiated superficial cells. It was absent on cells in benign hyperplasia of the urothelium. Cellular and regional differences in lectin binding to the urothelial cell surface was suggested with Con A receptors localized uniformly over the superficial cells, and PNA receptors confined to linear arrays or occasional clusters over the apical surface but evenly dispersed over the lateral surface of these cells.     

Chromosomal abnormalities in macroscopically normal urothelium in patients with bladder pT1 and pT2a urothelial carcinoma: a fluorescence in situ hybridization study and correlation with histologic features

OBJECTIVE: To analyze chromosomal abnormalities in macroscopically normal urothelium in patients with bladder pT1 and pT2a urothelial carcinoma and correlate the changes with histologic features. STUDY DESIGN: Cytologic touch preparations of the tumors and of the adjacent and distant urothelium were obtained from 8 bladders with urothelial carcinoma. Fluorescence in situ hybridization (FISH) was used to detect abnormalities of chromosomes 3, 7, 9 and 17 and of the 9p21 locus. RESULTS: The macroscopically normal urothelium adjacent to and distantfrom neoplastic foci was either normal looking microscopically or showed histologic changes ranging from hyperplasia to dysplasia and carcinoma in situ. FISH analysis detected chromosome gains and 9p21 deletion similar to those present in the urothelial carcinoma even though the percentage of altered nuclei was lower, especially in hyperplasia. The microscopically normal urothelium showed minor abnormalities in terms of gain for all the chromosomes investigated. CONCLUSION: Even though urothelium looks normal from the macroscopic point of view, it frequently harbors histologic changes and chromosomal abnormalities. These findings are of clinical significance since they might represent genetic alterations involved in recurrence and/or progression of urothelial carcinoma.     

Host-pathogen checkpoints and population bottlenecks in persistent and intracellular uropathogenic Escherichia coli bladder infection

Bladder infections affect millions of people yearly, and recurrent symptomatic infections (cystitis) are very common. The rapid increase in infections caused by multidrug-resistant uropathogens threatens to make recurrent cystitis an increasingly troubling public health concern. Uropathogenic Escherichia coli (UPEC) cause the vast majority of bladder infections. Upon entry into the lower urinary tract, UPEC face obstacles to colonization that constitute population bottlenecks, reducing diversity, and selecting for fit clones. A critical mucosal barrier to bladder infection is the epithelium (urothelium). UPEC bypass this barrier when they invade urothelial cells and form intracellular bacterial communities (IBCs), a process which requires type 1 pili. IBCs are transient in nature, occurring primarily during acute infection. Chronic bladder infection is common and can be either latent, in the form of the quiescent intracellular reservoir (QIR), or active, in the form of asymptomatic bacteriuria (ASB/ABU) or chronic cystitis. In mice, the fate of bladder infection, QIR, ASB, or chronic cystitis, is determined within the first 24 h of infection and constitutes a putative host-pathogen mucosal checkpoint that contributes to susceptibility to recurrent cystitis. Knowledge of these checkpoints and bottlenecks is critical for our understanding of bladder infection and efforts to devise novel therapeutic strategies.     

Preparation and characteristics of growth and marker properties of urinary bladder mesenchymal stem cells

Mesenchymal stem cells (MSC) are able to transdifferentiate into cells with different functional phenotypes and considered as a promising resource for regenerative therapy. MSC derived from different tissues vary in their differentiation potential and in some cases express tissue specific markers indicating a kinship between mesenchymal and parenchymal phenotypes in the same tissue. It is possible that homorganic MSC can be more effectively induced to tissue specific differentiation and preferable for cell therapy of this organ as compared with bone marrow derived cells being commonly used for this purpose. Using bladder tissue explants, we prepared primary MSC cultures from the fetal (MSC-BF) and adult syngenic BALB/c mice and characterized their abilities during long-term passaging. In contrast to the cells from adult mice, the MSC-BF cells have the ability for a sustained growth in vitro, clonogenicity and differentiation into adipose and bone cells. Similar to the bone marrow MSC, MSC-BF express the mesenchymal markers CD29, CD44, CD49f, CD90, CD105 but not the leukocyte common antigen CD45. In normal conditions, MSC-BF produce such urothelial markers as CK14 and FOXA1 although their expression level is by far lower than in the bladder tissue. The hypomethylating agent, 5-azacytidine, induces in MSC-BF the expression of the urothelial differentiation activator PPARγ and the functional urothelium markers UP1a, UP1b, UP3a, UP3b. The data obtained suggest that MSC-BF can be epigenetically reprogrammed into urothelium by the 5-azacytidine treatment, and this may offer the novel strategy for cell therapy of bladder diseases.