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. Accepted: 16 September 1999     

Detection of a glycosphingolipid antigen in bladder cancer cells with monoclonal antibody MRG-1

Summary The monoclonal antibody MRG-1 has been evaluated for the immunohistochemical detection of the type 3 chain of blood group A in human normal bladder epithelium and bladder tumours. Light microscope examination of paraffin sections demonstrated that this antigen was present in normal epithelium and superficial bladder tumour in patients with blood group A or AB, but was absent in the invasive type of bladder tumour. In normal epithelium, the plasma membrane was positive for this antigen, and the cytoplasm was diffusely stained. In superficial transitional cell carcinoma, the plasma membrane was negative, whereas the cytoplasm was intensely stained in the perinuclear region. This pattern was different from that observed for type 1 and 2 group A antigen, which was recognized mainly at the plasma membrane. However, in superficial transitional cell carcinoma, the staining was also seen on the plasma membrane. The pattern of the localization of this antigen in this carcinoma was influenced by the treatment of organic solvents. Electron microscopical observations confirmed that this antigen was localized on the plasma membrane and also in the Golgi apparatus of the superficial tumour.These results proved that the type 3 chain of blood group A is present in human bladder epithelium and low grade tumours in correspondence with the blood type, but disappears in tumours with high malignant potential. However, its expression is independent of the expressions of the other subtypes which have been studied. Furthermore, the changes in the staining pattern caused by pretreatment with organic solvents suggested possible differences in the microenvironment of the glycolipids containing this type of sugar chain.     

Uroplakins and cytokeratins in the regenerating rat urothelium after sodium saccharin treatment

A sodium saccharin (NaSac) diet was used to induce cell damage and regeneration in the urothelium of the male rat urinary bladder. Foci of terminally differentiated superficial cell exfoliation were detected after 5 weeks and their number increased after 10 and 15 weeks of the diet. At the sites of superficial cell loss, regenerative simple hyperplasia developed. Within 5 weeks of NaSac removal, regeneration re-established normal differentiated urothelium. In order to follow urothelial differentiation during regeneration we studied the expression of uroplakins and cytokeratins by means of immunocytochemistry and immunohistochemistry, respectively. Normal urothelium was characterised by terminally differentiated superficial cells which expressed uroplakins in their luminal plasma membrane and cytokeratin 20 (CK20) in the cytoplasm. Basal and intermediate cells were CK20 negative and cytokeratin 17 (CK17) positive. In hyperplastic urothelium all cells synthesised CK17, but not CK20. Differentiation of the superficial layer was reflected in three successive cell types: cells with microvilli, cells with rounded microridges and those with a rigid-looking plasma membrane on the luminal surface. The cells with microvilli did not stain with anti-uroplakin antibody. When the synthesis of uroplakins was detected rounded microridges were formed. With the elevated expression of uroplakins the luminal plasma membrane becomes rigid-looking which is characteristic of asymmetric unit membrane of terminally differentiated cells. During differentiation, syn-thesis of CK17 ceased in superficial cells while the synthesis of CK20 started. These results indicate that during urothelial regeneration after NaSac treatment, specific superficial cell types develop in which the switch to uroplakin synthesis and transition from CK17 to CK20 synthesis are crucial events for terminal differentiation. Accepted: 19 August 1997     

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.     

Autophagic activity in the mouse urinary bladder urothelium as a response to starvation

The urinary bladder urothelium is subjected to mechanical forces during cycles of distension and contraction, and its superficial cells are constantly flushed by toxic urine. Yet, the urothelium shows a very slow turnover of cells and superficial cells are extremely long lived. Autophagy has a well-known role in tissue homeostasis and serves as a protective mechanism against cellular stress. Therefore, the presence of autophagy as one of possible processes of survival in an unpleasant environment and during long lifetime of superficial cells was examined in mouse urothelium. We detected and evaluated autophagic activity of superficial urothelial cells under normal and stress conditions, caused by short-term starvation of newborn and 24-h-starved adult mice. Immunolabeling and Western blotting of essential effectors of autophagy, LC3 and Beclin 1, showed a weak signal in superficial urothelial cells. On the other hand, ultrastructural analysis, which proved to be the most reliable method in our study, revealed the presence of autophagic vacuoles, some of them containing specific urothelial structures, fusiform vesicles. Quantitative analysis showed increased autophagy in newborn and starved mice in comparison to a low basic level of autophagy in the urothelium of normal mice. Interestingly, some superficial cells of adults and neonates exhibit intense immunoreactions against LC3 and Beclin 1 and the typical ultrastructural characteristics of autophagy-dependent cell death. We conclude that autophagy, despite low basic activity under physiological conditions, plays an important role in urothelial homeostasis and stability under stress.     

Proliferating cell nuclear antigen in normal urothelium and urothelial lesions of the urinary bladder: a quantitative assessment using a true color image analysis system

To evaluate proliferating cell nuclear antigen (PCNA) staining for assessing proliferative activity in routine pathology specimens of urinary bladder, the bladder carcinoma cell line J82 and a total of 122 specimens of normal bladder and urothelial lesions were stained with the antibody clone PC10 against proliferating cell nuclear antigen. In in vitro plateau cultures the proportion of PCNA-positive cells exceeded that of Ki-67-positive cells, and only very few cells were negative. In formalin-fixed tissues, the PCNA staining pattern, which should be confined to replicon units in the nucleus, was optimized by 1 h postfixation in an organic solvent (methacarn). Sections showed positive nuclear staining confined to basal and some suprabasal cells in normal urothelium and grade 1 dysplasias, but more generalized nuclear staining in all other neoplastic lesions. In addition, stromal cells adjacent to invasive tumors showed nuclear positivity in some instances. Using quantitative true color image analysis of sections counterstained with hemalum, the degree of brown staining of the PCNA reaction product is contrasted with the blue staining of the nuclear area. With this method low contrast specific staining not appreciated optically can be reliably detected. Image analysis data confirmed observations made on noncounterstained sections and showed significant differences between grade 1 and 2 dysplasias as well as between grade 1 dysplasia and all grades of papillary tumor. Furthermore, a significant difference in PCNA staining indices was found between grade 1 and 3 bladder carcinomas. The results indicate that PCNA staining using the PC10 antibody is not confined to the proliferative fraction of neoplastic urothelium. In contrast with data from normal tissue and malignant hematological neoplasms, the amount of PCNA is regulated differently in urothelial neoplasms, emphasizing the biological differences between the following two sets: mild dysplasia and moderate dysplasia; mild dysplasia and papillary carcinomas. The use of image analysis to standardize the detection process after controlled staining conditions is advisable in order to provide reliable data. Supported by the DFG project: Knuechel/Urothelcarcinom 263     

Options for histological study of the structure and ultrastructure of human urinary bladder epithelium

The urothelium lines all urinary passages, with exception of the distal portions of the urethra. For the first time the structure of the human bladder was described by Leonardo Da Vinci in 15^th century, however, the exact ultrastructure and function of the bladder’s epithelium have not been fully understood. The aim of our study was to investigate the structure of normal human urinary bladder epithelium with methods of classical histology, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). We obtained biopsies from non-tumor areas from the human urinary bladder of tumor-bearing patients during transurethral resections of these tumours in general or spinal anaesthesia. Totally we investigated biopsies from 20 patients, 16 males and 4 females. The mean age of this group of patients was averaged 66.5 years. The urothelium is comprised of three cell types including polyhedral basal cells, piriform intermediate cells, and superficial umbrella cells. In human urinary bladder epithelium we found a direct connection between intermediate cells and the basement membrane. These thin cytoplasmic projections are detectable not only on slides for light microscopy (semi-thin sections), but also in transmission electron-micrographs. In semi-thin sections we found also direct connections between superficial umbrella cells and basement membrane. These connections we were not able to verify via transmission electron-microscopy. Nevertheless our results show that the human urinary bladder urothelium is a special type of pseudostratified epithelium and each cell has a thin cytoplasmic projection with a direct contact with basement membrane.     

Growth hormone receptor expression in the nucleus and cytoplasm of normal and neoplastic cells

 Growth hormone (GH) exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific receptors which trigger a phosphorylation cascade, resulting in the modulation of numerous signalling pathways dictating gene expression. A panel of five monoclonal antibodies was used in mapping the presence and somatic distribution of the GH receptor by immunohistochemistry in normal and neoplastic tissues and cultured cells of human, rat and rabbit origin. A wide distribution of the receptor was observed in many cell types. Not all cells expressing cytoplasmic GH receptors displayed nuclear immunoreactivity. In general, the relative proportion of positive cells and intensity of staining was higher in neoplastic cells than in normal tissue cells. Immunoreactivity showed subcellular localisation of the GH receptor in cell membranes and was predominantly cytoplasmic, but strong nuclear immunoreaction was also apparent in many instances. Intense immunoreactivity was also observed in the cellular Golgi area of established cell lines and cultured tissue-derived cells in exponential growth phase, indicating cells are capable of GH receptor synthesis. The presence of intracellular GH receptor, previously documented in normal tissues of mostly animal origin, is the result of endoplasmic reticulum and Golgi localisation. Heterogeneity of immunoreactivity was found in normal and neoplastic tissue with a variable range of positive cells. The nuclear localisation of immunoreactivity is the result of nuclear GH receptor/binding protein, identically to the cytosolic and plasma GH-binding protein, using a panel of five monoclonal antibodies against the GH receptor extracellular region. The expression of GH receptors, not only on small proliferating tumour cells such as lymphocytes, but also on well differentiated cells including keratinocytes, suggests that GH is necessary not only for differentiation of progenitor cells, but also for their subsequent clonal expansion, differentiation and maintenance. Accepted: 4 July 1997     

Postnatal restoration of the mouse urinary bladder urothelium

Mouse urothelium is disrupted just before birth, followed by a postnatal restoration process which includes cell proliferation, death and differentiation. We assessed urothelial proliferation by the expression of proliferating cell nuclear antigen (PCNA), desquamation by electron microscopy, and apoptosis by TUNEL staining and urothelial differentiation by the expression of uroplakins and cytokeratin 20 (CK20) as well as the apical plasma membrane maturation. Our results indicated that urothelial proliferation was high from birth until about the 14th postnatal day. A majority of basal cells and even occasional superficial cells were PCNA positive during the first 5 postnatal days. Cell death occurred during the first 9 postnatal days. Between birth and day 5, single cells underwent apoptosis, whereas between days 6 and 9 cells mainly desquamated. CK20 and uroplakins were expressed in all superficial cells in postnatal urothelium. Their subcellular distribution characteristically changed in accordance with the progressive differentiation of superficial cells. During the urothelial postnatal development, proliferation activity slowly decreases to the proliferatively quiescent urothelium of the adult animal. Apoptosis is present in the first 9 postnatal days and within a few days of this period it appears simultaneously with desquamation. Superficial urothelial cells gradually differentiate, which is reflected in the changeable morphology of the apical plasma membrane.     

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.     

Overexpression of annexin a1 induced by terephthalic acid calculi in rat bladder cancer

Prolonged cell proliferation in response to irritation by bladder calculi can evoke malignant transformation of the urothelium. However, the molecular mechanisms responsible for calculi-associated bladder carcinogenesis are unknown. We compared the protein expression pattern of rat bladder transitional cell carcinomas (TCCs) induced by terephthalic acid with that of normal bladder tissues using 2-DE. Comparative analysis of the respective spot patterns on 2-DE showed 146 spots that were markedly changed in TCC samples. Subsequently, 56 of the variant protein spots were identified by MALDI-TOF MS. Among them, overexpression of annexin a1 (ANNA1) in rat TCCs was confirmed by Western blotting and real-time RT-PCR analysis. Immunohistochemical staining revealed that ANNA1, usually a cytoplasmic protein in normal urothelium, was translocated to the nucleus in rat bladder cancer cells. In contrast to the animal studies, examination of human clinical specimens showed that ANNA1 expression was reduced in TCC compared to normal urothelium. The expression of ANNA1 was inversely related to the level of differentiation of TCC. Our data suggest that overexpression of ANNA1 is involved in bladder carcinogenesis induced by bladder calculi and that translocation of the protein may be partly responsible for the effect. ANNA1 may serve as a new marker of differentiation for the histopathological grading of human TCC.     

Identification of potential bladder progenitor cells in the trigone

Urothelial cells are specialized epithelial cells in the bladder that serve as a barrier toward excreted urine. The urothelium consists of superficial cells (most differentiated cells), intermediate cells, and basal cells; the latter have been considered as urothelium progenitor cells. In this study, BrdU or EdU was administrated to pregnant mice during E8–E13 for 2 consecutive days when bladder development occurs. The presence of label retaining cells was investigated in bladders from offspring. In 6 months old mice ~1% of bladder cells retained labeling. Stem cell markers as defined for other tissues (e.g., p63, CD44, CD117, trop2) co-localized or were in close vicinity to label retaining cells, but they were not uniquely limited to these cells. Remarkably, label retaining cells were distributed in all three cell layers (p63+, CK7+, and CK20+) of the urothelium and concentrated in the bladder trigone. This study demonstrates that bladder progenitor cells are present in all cell layers and reside mostly in the trigone. Understanding the geographic location of slow cycling cells provides crucial information for tissue regenerative purposes in the future.     

Immunohistochemical demonstration and localization of Lewis a and Lewis b determinants in human urothelium

In order to obtain baseline information about Lewis antigen expression in human urothelium in order that changes during malignant transformation can be evaluated, urothelium from eight individuals of known erythrocyte Lewis types were stained by a Tween-modified indirect immunoperoxidase staining technique using goat antibodies directed toward the Lewis a and Lewis b determinants and mouse monoclonal antibodies directed toward the Lewis a determinant in serial dilutions. To evaluate the value of the method for tissue Lewis typing, eleven ureters from individuals of unknown erythrocyte Lewis types were stained using goat antibodies. The Lewis antigens were located on the cell membranes and in the cytoplasm of urothelial cells. Goat antibodies identified Lea-b-, Lea+b+, and Lea+b- urothelium. Monoclonal antibodies identified urothelium with both low and high Lewis a antigen expression as well as urothelium with no Lewis a antigen expression. Urothelial Lewis antigen expression correlated with erythrocyte Lewis types in Lea-b+ and Lea+b- individuals. In Lea-b- individuals Lewis determinants were either not detected or were expressed similarly to Lea-b+ individuals. Urothelial Lewis typing were doubtful in two out of the eleven ureters examined. The results imply that knowledge about erythrocyte Lewis type or normal tissue Lewis antigen expression is necessary for the immunohistochemical evaluation of changes in Lewis antigen expression in urothelial tumors.     

Correlative study of functional and structural regeneration of urothelium after chitosan-induced injury

High transepithelial electrical resistance (TEER) demonstrates a functional permeability barrier of the normal urothelium, which is maintained by a layer of highly differentiated superficial cells. When the barrier is challenged, a quick regeneration is induced. We used side-by-side diffusion chambers as an ex vivo system to determine the time course of functional and structural urothelial regeneration after chitosan-induced injury. The exposure of the urothelium to chitosan caused a 60 % decrease in TEER, the exposure of undifferentiated urothelial cells to the luminal surface and leaky tight junctions. During the regeneration period (350 min), TEER recovered to control values after approximately 200 min, while structural regeneration continued until 350 min after injury. The tight junctions are the earliest and predominant component of the barrier to appear, while complete barrier regeneration is achieved by delayed superficial cell terminal differentiation. The barrier function and the structure of untreated urothelium were unaffected in side-by-side diffusion chambers for at least 6 h. The urinary bladder tissue excised from an animal thus retains the ability to maintain and restore the transepithelial barrier and cellular ultrastructure for a sufficient period to allow for studies of regeneration in ex vivo conditions.     

Recent observations on the ultrastructure of human urothelium

Summary An electron microscopic study of normal bladder urothelium of elderly patients ranging in age from 61 to 82 years has shown the occurrence of unusually thin regions consisting of either one or two layers of undifferentiated cells interspersed between 3–4 cell layers thick regions. A morphometric study has confirmed the existence of a pattern of cytodifferentiation in cells of the thick region. The generally microvillous nature of the luminal surface is attributed to incompletely differentiated cells that have come to occupy the superficial layer. The lack of thickened and/or asymmetric membrane plaques in luminal plasma as well as the dearth of characteristic precursor vesicles in the cytoplasm are also explicable in terms of a failure of normal cell differentiation. It is suggested that the unusual features noted are consequences of tissue ageing rather than prognostic of cancer. There are indications that the aged urothelium may be prone to increased leakiness and the bladder tissues may therefore be at greater risk from urine-borne chemicals and carcinogens.     

Expression and localization of four uroplakins in urothelial preneoplastic lesions

In superficial umbrella cells of normal urothelium, uroplakins (UPs) are assembled into urothelial plaques, which form fusiform vesicles (FVs) and microridges of the apical cell surface. Altered urothelial differentiation causes changes in the cell surface structure. Here, we investigated ultrastructural localization of UPIa, UPIb, UPII and UPIIIa in normal and cyclophosphamide-induced preneoplastic mouse urothelium. In normal urothelium, terminally differentiated umbrella cells expressed all four UPs, which were localized to the large urothelial plaques covering mature FVs and the apical plasma membrane. The preneoplastic urothelium contained two types of superficial cells with altered differentiation: (1) poorly differentiated cells with microvilli and small, round vesicles that were uroplakin-negative; no urothelial plaques were observed in these cells; (2) partially differentiated cells with ropy ridges contained uroplakin-positive immature fusiform vesicles and the apical plasma membrane. Freeze-fracturing showed small urothelial plaques in these cells. We concluded that in normal urothelium, all four UPs colocalize in urothelial plaques. However, in preneoplastic urothelium, the growth of the uroplakin plaques was hindered in the partially differentiated cells, leading to the formation of immature FVs and ropy ridges instead of mature FVs and microridges. Our study demonstrates that despite a lower level of expression, UPIa, UPIb, UPII and UPIIIa maintain their plaque association in urothelial preneoplastic lesions.     

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.     

Cellular heterogeneity in human transitional cell carcinoma: an anlysis of optical properties and lectin binding

Summary Flow cytometry was used to measure the binding of a panel of ten fluorescein isothiocyanate(FITC)-conjugated lectins to fifteen samples of normal and neoplastic human urothelium. Concurrent measurement of light scattering and fluorescence permitted the quantification of lectin binding to cellular subpopulations defined by their light-scattering properties. In normal urothelium, we previously demonstrated levels of lectin binding to the cellular subpopulations derived from the superficial and intermediate cell layers which were higher than levels which bound to the subpopulation derived from the basal cell layer (Wardet al., 1987). This difference was most marked withMaclura pomifera agglutinin (MPA),Ricinus communis agglutinin (RCA) andUlex europus agglutinin (UEA). We now report a similar correlation between the degree of differentiation of a cellular subpopulation and the level of lectin binding in human transitional cell carcinomas (TCCs). Morphological differentiation in human TCCs is accompanied by alterations in cell-surface carbohydrates which are similar to those which accompany cellular differentiation in the corresponding normal tissue. No systematic difference in lectin binding was observed between the corresponding subpopulations of normal and neoplastic urothelial cells.     

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.