Urothelial progenitor cells: regional differences in the rat bladder

OBJECTIVES: Because the trigone is a unique region in the caudal bladder with a higher risk of neoplasia, we hypothesized that this area would have a high proportion of progenitor cells. As yet there is no marker nor methodology to specifically isolate urothelial stem cells, and thus demonstrate multi-potential differentiation and self-renewal. Here, our goal was to evaluate the distribution of progenitor cells that carry two general major attributes of stem cells: clonogenicity and proliferative capacity. MATERIALS AND METHODS: The bladders of Fisher rats were divided into caudal and cephalic segments and primary cultures were established from the harvested urothelial cells. RESULTS: We found that colony-forming efficiency was almost 2-fold higher for cells from the caudal bladder compared to the cephalic bladder. Doubling time was significantly faster for cells harvested from the caudal bladder at initial plating. This suggested that the caudal bladder harbours a higher density of urothelial progenitor cells. With passage to p4, the differences between the upper and lower bladder were lost, suggesting selection of proliferative cells with serial passage. Based on Ki-67 staining, there was no geographical difference in cell proliferation under normal homeostatic in vivo conditions. CONCLUSIONS: These results demonstrate geographical sequestration of urothelial progenitor cells to the area of the bladder that encompasses the bladder neck and trigone, which may be a factor in pathological disparities between the trigone and remaining bladder.     

I. A subpopulation of human urothelial cells is stimulated to proliferate by treatment in vitro with lipoteichoic acid,a cell wall component of Streptococcus faecalis

Urinary tract infection with gram-positive bacteria is common. Avenues for ingress of bacteria into the bladder include luminal and suburothelial infection. Terminally differentiated superficial urothelial cells lining the lumen of the bladder are often shed in response to infection. In contrast, infection-induced altered function of progenitors of urothelial cells residing in the basal layer of the urothelium is likely to have long lasting effects on the structure and function of the urothelium. The main objective of the present studies was to investigate in vitro the possibility that exposure to lipoteichoic acid, a cell wall component of the gram-positive Streptococcus faecalis (LT-2), stimulates basal urothelial cells to proliferate. To simulate conditions that restrict proliferation and inhibit terminal differentiation of urothelial cells in the basal layer, secondary cultures of urothelial cells (UT) were grown on collagen or fibronectin-coated substrate in medium containing low levels of Ca²⁺ (0.2 mM) and growth factors (0.005% bovine pituitary extract [BPE]). Under these conditions, UT cultures displayed a highly reproducible colony size distribution, possibly due to the fact that colonies were progeny of basal cells with various proliferative potentials, retained in vitro. In cultures grown under growth-restricting conditions, the majority of progenitors appeared to be quiescent, just like stem cells in the basal layer of the urothelium. Thus, the population of large colonies (more than six cells/colony), was small when a steady state of growth was achieved, 3–7 days after seeding. Growth factors (0.005–0.5% BPE) caused a dose-dependent increase in this population of large colonies. Moreover, treatment of UT grown under growth-restricting conditions (0.005% BPE) with LT-2 increased steady-state levels of the population of large colonies to levels obtained in cultures growing under optimal conditions with respect to growth factors. These results indicated that the subpopulation of progenitors, quiescent under normal conditions, could be stimulated to proliferate. Two lines of evidence were consistent with the possibility that treatment with LT-2 stimulated proliferation of the subpopulation of progenitors and that large colonies were the progeny of this subpopulation of single cells: (1) treatment with LT-2 increased the percentage of single cells that incorporated bromodeoxyuridine (i.e., proliferated) in a time-dependent manner; (2) An increase in the percentage of large colonies was found following LT-2-triggered proliferation of single cells. We propose that, under normal conditions, cells produced in response to LT-2-triggered proliferation of stem cells are removed from the system due to an increased rate of differentiation followed by apoptosis. Recurrent infection and inflammation may not allow these processes to proceed effectively, resulting in chronic injury to the bladder. Moreover, under conditions in which stem cells accumulate mutations that incapacitate their progeny to undergo apoptosis, LT-triggered proliferation could be a contributing factor to tumorigenesis. © 1996 Wiley-Liss, Inc.     

Structurally diverse peroxisome proliferator-activated receptor agonists induce apoptosis in human uro-epithelial cells by a receptor-independent mechanism involving store-operated calcium channels

Objectives:  Peroxisome proliferator-activated receptors (PPARs) are implicated in epithelial cell proliferation and differentiation, but investigation has been confounded by potential off-target effects of some synthetic PPAR ligands. Our aim was to determine mechanisms underlying the pro-apoptotic effect of synthetic PPAR agonists in normal human bladder uro-epithelial (urothelial) cells and to reconcile this with the role of PPARs in urothelial cytodifferentiation.
Materials and methods:  Normal human urothelial (NHU) cells were grown as non-immortal lines in vitro and exposed to structurally diverse agonists ciglitazone, troglitazone, rosiglitazone (PPARγ), ragaglitazar (PPARα/γ), fenofibrate (PPARα) and L165041 (PPARβ/δ).
Results:  NHU cells underwent apoptosis following acute exposure to ciglitazone, troglitazone or ragaglitazar, but not fenofibrate, L165041 or rosiglitazone, and this was independent of ERK or p38 MAP-kinase activation. Pro-apoptotic agonists induced sustained increases in intracellular calcium, whereas removal of extracellular calcium altered the kinetics of ciglitazone-mediated calcium release from sustained to transient. Cell death was accompanied by plasma-membrane disruption, loss of mitochondrial membrane-potential and caspase-9/caspase-3 activation. PPARγ-mediated apoptosis was unaffected following pre-treatment with PPARγ antagonist T0070907 and was strongly attenuated by store-operated calcium channel (SOC) inhibitors 2-APB and SKF-96365.
Conclusions:  Our results provide a mechanistic basis for the ability of some PPAR agonists to induce death in NHU cells and demonstrate that apoptosis is mediated via PPAR-independent mechanisms, involving intracellular calcium changes, activation of SOCs and induction of the mitochondrial apoptotic pathway.     

A correlation between ovarian follicular maturity and anchorage-independent growth of bovine granulosa cells

Previous studies have shown that a subset of bovine ovarian granulosa cells can proliferate to form clones of functional cells in suspension in a semisolid agar matrix, without the requirement for attachment to the substratum. These clonogenic anchorage-independent granulosa cells are responsive to epidermal growth factor and exhibit properties of a primitive progenitor cell population. We have used this assay system to analyze the proliferation of granulosa cells during ovarian follicular maturation. As the follicle increases in size, there is a progressive decline in the ability of granulosa cells to clone in agar, and the proliferative potential of these cells as measured by colony size also decreases. The ratio of large colonies with high proliferative potential (greater than 250 micron in diameter) to small colonies with limited capacity for growth falls from 1.92 in follicles less than 7 mm in diameter, to 0.32 in follicles larger than 10 mm in diameter. This occurs as the follicular content of granulosa cells with more limited capacity for clonal growth in agar undergoes expansion. Analysis of colony-forming cells in follicles harvested at early and late estrus suggests that these cells are regulated by complex intraovarian factors rather than circulating gonadotropin levels. Our data indicate that the granulosa cell lineage is an age-structured continuum of maturing and differentiating cells with a progressively restricted proliferative capacity.     

The role of food and colony size in sexual offspring production in a social insect: an experiment

Abstract.  1. Large colonies of ants are known to have a higher propensity for sexual offspring production, probably owing to their high capacity to exploit food resources.
2. The effects of food supplementation on the propensity for sexual offspring production, and whether it is linked with colony size, were investigated in an environment with poor resources (clear-cut areas).
3. Large colony size was associated with a higher propensity for sexual offspring production in food-supplemented colonies, whereas in non-supplemented control colonies an association with colony size was not found.
4. The results demonstrate that large colonies seem to have a higher capacity to exploit supplemented food. In addition, the production of sexual offspring was apparently limited by food availability in clear-cuts, especially for large colonies.     

Cell cycle of normal bladder urothelium in developing and adult mice

The present research has employed a novel, nonradioactive technique to quantitatively study normal urothelial proliferation in foetal, neonatal, juvenile and adult mouse bladder. Using whole mount histological preparations, the total number of urothelial nuclei per mouse bladder, and per given urothelial cell layer, have been assessed to provide data of the (unstimulated) kinetic behaviour of basal urothelial cells (the proliferative population), to analyse characteristics of the normal urothelial cell cycle. The urothelial cell cycle time increases from 30.6 h (foetal) to 40 weeks (adult), the duration of mitosis from 0.23 h (foetal) to 2.71 h (adult) and the duration of DNA synthesis from 2.52 h (neonatal) to 10.83 h (adult). These are average values for the urothelial cell cycle, which do not preclude the possible existence of proliferative units. The ratio of superficial nuclei to basal and intermediate nuclei, possibly indicative of a urothelial proliferative unit, declines to reach a plateau (1:40) in adult mice. These findings indicate that rapid urothelial proliferation during early murine development was likely to be a) biologically useful, since intrauterine foetal metabolic activity may require a functional bladder urothelium at an early stage, b) kinetically similar to acutely regenerating adult urothelial cells after cytotoxic insult. During murine life, the range of durations of mitosis and DNA synthesis is much less than the range of cell cycle times. Normal unstimulated urothelium of adult mice was confirmed to proliferate slowly.     

Nourishment affects colony demographics in the paper wasp Polistes metricus

Abstract  1. Colony survivorship and numbers of nest cells, pupae, and adult females were monitored throughout the nesting season for a cohort of 78 colonies of the paper wasp Polistes metricus Say. Thirty-nine colonies received a twice-weekly nourishment supplement of honey during pre-emergence and early emergence phases of the colony cycle; 39 colonies were unsupplemented controls.
2. Colony survivorship was unaffected by the supplemental nourishment. Loss of colonies to predation differed among three sites but was unaffected by supplementation.
3. Honey-supplemented colonies constructed more nest cells than did control colonies but this effect was not expressed until after supplementation had ceased.
4. Honey-supplemented colonies produced more pupae than did control colonies but the number of adult females at nests did not differ between supplemented and control colonies. Because honey-supplemented colonies had more offspring but fewer of them remained as workers at the nest, honey supplementation led to a lower frequency of workers and corresponding higher frequency of reproductives than in control colonies.
5. In a second year of study, colony survivorship and numbers of nest cells, pupae, and adult females were monitored from late pre-emergence until the end of the nesting season for a cohort of 32 colonies of Polistes metricus . In 16 colonies, trophallactic saliva was taken from final-instar larvae on nine dates in the late pre-emergence and early emergence periods; 16 colonies served as controls.
6. Saliva-diminished colonies had lower survivorship, fewer nest cells, fewer pupae, and fewer adult females at the nest than did control colonies.
7. These results show that variation in nourishment in the early to mid phases of the colony cycle can have significant effects on the subsequent colony demographics of Polistes metricus paper wasps.     

The characteristics of an anchorage-independent clonal agar assay for primary explanted bovine granulosa cells

Normal, primary explanted, bovine granulosa cells grow reproducibly in agar culture as anchorage-independent clones. Epidermal growth factor (EGF) and rat erythrocytes are effective stimulators of colony formation, and when both are added to the culture medium at optimal concentrations, there is an enhancement of colony numbers and colony size, indicative of an independent, and operationally additive, mode of action for the two factors. The ability of cells propagated from agar clones to secrete progesterone, and to augment progesterone secretion 4-fold in the presence of 1 mM dbcAMP is proof that colonies originate from and are composed of functional granulosa cells. Maximal colony numbers are present at day 10 of incubation, and colony forming cells undergo self-renewal as assessed by the ability of cells from primary colonies to reclone in agar. Absolute cloning efficiency, however, is dependent on a number of factors. Inherent variability exists in cloning efficiency of granulosa cells from individual follicles. Quantitative and qualitative clonal growth was improved at an osmolality of less than 300 mOsm when compared with higher osmolalities. Cl-1 medium and the alpha modification of Eagle's medium were equally effective in supporting agar clonogenic growth, whereas both Ham's F12 and NCTC 135 media exhibited poor clonogenic growth supporting properties. The substitution of agarose for agar did not affect colony numbers but colonies grown in the presence of agarose tended to be smaller and more uniform in size.     

Spatial patterns, temporal variability, and the role of multi-nest colonies in a monogynous Spanish desert ant

Abstract 1. The colonies of the Spanish desert ant Cataglyphis iberica are polydomous. This study describes the temporal and spatial patterns of the polydomy in this species at two different sites, and presents analyses of its role in reducing the attacks of the queen over sexual brood, and in allowing better habitat exploitation.
2. The spatial distribution of nests was clumped while colonies were distributed randomly. Mean nearest neighbour distance ranged from 3.4 to 7.0 m for nests and from 12.3 to 14.1 m for colonies. Distance of foragers searching for food varied among nests: mean values were between 6.1 and 12.6 m.
3. At both sites, the maximum number of nests per colony occurred in summer, during the maximum activity period of the species. Colonies regrouped at the end of this period but overwintered in several nests.
4. Nest renewal in C. iberica colonies was high and showed great temporal variability: nests changed (open, close, re-open) continuously through the activity season and/or among years. The lifetime of up to 55% of nests was only 1–3 months.
5. Polydomy in C. iberica might decrease the interactions between the queen and the sexual brood. In all colonies excavated just before the mating period, the nest containing the queen did not contain any virgin female. Females were in the queenless nests of the colony.
6. The results also suggest that polydomous C. iberica colonies may enhance habitat exploitation because foraging activity per colony increases with nest number. The relationship between total prey input and foraging efficiency and number of nests per colony attains a plateau or even decreases after a certain colony size (four to six nests). This value agrees with the observed mean number of nests per colony in C. iberica .     

Extracting information on the evolution of living- and dead-cell fractions of Salmonella Typhimurium colonies in gelatin gels based on microscopic images and plate-count data

Aims:  The aim of this study was to extract information on cell number and colony volume dynamics of Salmonella Typhimurium colonies.
Methods and Results:  Both cell number and colony volume of Salmonella Typhimurium in gelatin were monitored during the exponential and the stationary phase with varying pH and water activity, by plate counts and microscopic image analysis respectively. The exponential growth rates of cell numbers and colony volumes were correlated. The exponential growth rate of cell numbers was estimated based on this correlation and a secondary model that describes the effect of pH and water activity on the growth rate of the colony volumes. During the stationary phase, the cell number was constant, while colony volume increased, thus indicating the formation of a dead fraction. Models were developed to describe the living and dead population.
Conclusions:  By comparing colony volumes and cell numbers, the formation of dead fraction can be noticed from the beginning of the stationary phase, which indicates that the stationary phase is a dynamic – including both cell death and cell growth – rather than a static phase.
Significance and Impact of the Study:  This study was the first to investigate the proportion of living and dead bacteria within a stationary colony quantitatively.     

Clonal variation in colony morphology and growth of CHO cells cultured on agar

Single Chinese hamster ovary (CHO) cells plated on agar form macroscopic colonies with high efficiency. Colonies produced by cells from the uncloned cell line increase in diameter continuously for 10–12 days after plating to form mounds of cells about 1 mm in diameter. With further incubation, some of these colonies do not increase in diameter (arrested dome), some form an expanding annular monolayer of cells around the central mound (fried egg), and some grow by enlarging the central mound into a low multilayered disc (saucer).These colony types on agar appear to be clonal characteristics of the CHO cell line. Cloning the line gives two kinds of isolates: one forms a mixture of arrested dome and fried egg colonies in an inheritable ratio, and the other forms saucer colonies. Cells from saucer colonies form saucer colonies when replated on agar. Cells from all colony types replate with similar efficiency on plastic or agar, and exhibit the same growth rate and cell size in liquid suspension culture. On plastic substrate, all these CHO cells form colonies which increase continuously in diameter for as long as 21 days, and little clonal difference in the morphology of colonies or of single cells is observed.These observations reveal a previously unsuspected heterogenieity in an established line of cultured mammalian cells and provide a method for studying new classes of In vitro growth control phenomena. These control phenomena may help in the building an in vitro model for tumor growth.     

Clonal analysis of oligodendrocyte development in culture: evidence for a developmental clock that counts cell divisions

The clonal development of oligodendrocytes was studied by culturing individual oligodendrocyte--type-2 astrocyte (O-2A) progenitor cells on monolayers of type-1 astrocytes, which stimulate O-2A progenitor cells to divide. Oligodendrocytes developed by a proliferative lineage in which clonal progeny differentiated together after a number of cell divisions. Most O-2A progenitor cells had similar cell cycle times (1-2 days), but their proliferative capacity varied greatly: some divided only once while others divided up to eight times before differentiating. sister cells behaved similarly when recultured separately on astrocyte monolayers. These findings are consistent with the cell-division-counting hypothesis previously proposed to explain the timing of oligodendrocyte differentiation. They also unambiguously establish the phenotype of O-2A progenitor cells in vitro and demonstrate that these cells respond directly to growth factors produced by type-1 astrocyte monolayers.     

Clonal nature of murine hemopoietic blast cell colonies

Murine hemopoietic blast cell colonies obtained from spleen cells of 5-fluorouracil (5-FU)-treated mice give rise to many multilineage colonies including granulocyte - erythrocyte - macrophage - megakaryocyte (GEMM) colonies in secondary cultures. Progenitor cells for blast cell colonies are considered to be more primitive than colony forming units (CFU)-GEMM. To determine whether they are clonal, we examined the phosphoglycerate kinase-1 (PGK-1) isozyme type of colonies originally grown from spleen cells of 5-FU-treated mice which had PGK-1 isozyme mosaicism. PGK assays of whole secondary colonies derived from one blast cell colony showed that they were either of type A or type B but not both. These results suggest that murine hemopoietic blast cell colonies are clonal.     

Queen lifespan and colony longevity in the ant Harpegnathos saltator

Abstract.  1. The longevity of field colonies was investigated in the ponerine ant Harpegnathos saltator (Jerdon) in which either reproductive workers (gamergates) or a single queen reproduce.
2. Data from 3 years were used to calculate the ratio between queen-right ( n  = 50) and gamergate ( n  = 12) colonies that can be used to derive the colony mortality of gamergate colonies. Using the survival rates of queens in the laboratory, extrinsic and intrinsic mortality rates of queen-right colonies were calculated.
3. No significant differences in the sizes of queen-right and gamergate colonies above 14 workers was found, suggesting that mortality of established colonies is not size related.
4. The mortality of gamergate colonies is 4.17 times higher than the intrinsic mortality of queen-right colonies.
5. In the laboratory, mean survival time of queens in colonies of more than 14 workers was 1.79 years.
6. Estimated mean survival time of queen-right and gamergate colonies in the field varies between 0.78 and 0.43 years respectively, when no costs of conflict during the replacement of queens occur; however, when the latter costs increase colony mortality to a level similar to extrinsic mortality, the calculated longevity of queen-right colonies would increase to 1.02 years.     

Analysis of pure and mixed murine mast cell colonies

Mast cells have been proposed to originate from diverse sources, including connective tissues, macrophages, T lymphocytes, and hemopoietic cells. Evidence for a hemopoietic origin of mast cells includes the presence of mast cell precursors in spleen colonies and the presence of mast cells in hemopoietic colonies in culture. Here we report a detailed analysis of mouse spleen mixed hemopoietic colonies containing mast cells. All of the colonies in cultures plated at low cell densities were individually removed for analysis by May-Grunwald-Giemsa staining on day 15 of culture. Examination of five dishes which contained a total of 82 colonies showed 16 pure mast cell colonies and 36 mixed mast cell colonies. Sixteen different combinations of cell types were seen and were not distinguishable from each other in situ. The most diverse type of mixed colony contained macrophages (m), neutrophils (n), eosinophils (e), mast cells (Mast), megakaryocytes (M), erythroid cells (E), and blast cells. The clonal origin of mixed mast cell colonies was established by the replating of single cells obtained from blast cell colonies. Individual cells were removed with a micromanipulator, replated, and allowed to grow for 15 days. Cytospin preparations of 10 such colonies showed diverse combinations of cell lineages which were seen in the different types of mixed mast cell colonies described above. Replating studies of mixed mast cell colonies were carried out and a high incidence of replating was seen. Approximately one half of these colonies formed only mast cell colonies upon replating. Further studies showed that pure mast cell colonies could be serially replated four to five times. The replating efficiency of cells in the primary mast cell colonies varied over a wide range (2.5–44%) with an average replating efficiency of 13%. The data also revealed that cells containing metachromatic granules possess significant proliferative capacity. From these studies of pure and mixed mast cell colonies, we concluded (1) that mast cells are in wide variety of types of mixed colonies and that the in situ identification of mixed colonies is unreliable, (2) that mast cells are derived from pluripotent hemopoietic stem cells, and (3) that mast cells with metachromatic granules can have a high proliferating ability.     

Effects of resource availability and social parasite invasion on field colonies of Bombus terrestris

Abstract.  1. The survival, growth and fecundity of bumblebee colonies are affected by the availability of food resources and presence of natural enemies. Social parasites (cuckoo bumblebees and other bumblebees) can invade colonies and reduce or halt successful reproduction; however, little is known about the frequency of invasion or what environmental factors determine their success in the field.
2. We used 48 experimental colonies of the bumblebee Bombus terrestris , and manipulated both resource availability at the landscape scale and date of colony founding, to explore invasion rates of social parasites and their effect on the performance of host colonies.
3. Proximity to abundant forage resources (fields of flowering oilseed rape) and early colony founding significantly increased the probability of parasite invasion and thus offset the potential positive effects of these factors on bumblebee colony performance.
4. The study concludes that optimal colony location may be among intermediate levels of resources and supports schemes designed to increase the heterogeneity of forage resources for bumblebees across agricultural landscapes.     

Genetic analysis of the clonal origin of regenerating mouse spermatogenesis following transplantation

Spermatogonial transplantation provides a straightforward approach to quantify spermatogonial stem cells (SSCs). Because donor-derived spermatogenesis is regenerated in the form of distinct colonies, the number of functional SSCs can be obtained by simply counting the number of colonies established in recipient testes. However, this approach is legitimate only when one colony arises from one stem cell (one colony-one stem cell hypothesis). In this study, we evaluated the validity of this hypothesis. Two populations of donor cells were obtained from the testes of two transgenic mouse lines and mixed at a 1:1 ratio. Following transplantation of the cell mixture, donor-derived colonies were visualized and individually excised, and genomic DNA was extracted from each colony. Based on unique marker genes of the two transgenic lines, the genotype of the cells contained in a colony was examined by polymerase chain reaction. A colony was determined to be clonal when only one transgene was detected. The results showed that 100% and 90% of colonies were clonal when <5 and 19 colonies were formed per recipient testis, respectively. However, the clonality of colonies decreased as the colony number per recipient testis or the length of each colony increased. These results support the one colony-one stem cell hypothesis and demonstrate that spermatogonial transplantation provides a highly quantitative assay for SSCs; however, these conclusions are applicable under a defined transplantation condition.     

Nuclear heterogeneity and proliferative capacity of human adipose derived MSC-like cells

Adipose derived stem cells (ADSCs) are MSC-like cells which could be easily used for regenerative medicine. Here, the morphology and proliferative capacity of human ADSCs is discribed. ADSCs were analyzed after one month of cultivation at a density of 10 cells/cm2. 21 colonies were counted. Few atypical cells (huge nuclei and cytoplasm) were found in 9 out of 17 colonies analyzed. ANOVA demonstrated that colonies also differed (P = 0.0025) in nuclei dimensions and scatter in the dimensions in each colony. Nuclei dimensions and cell density logarithms correlated in reverse proportion (-0.7; P = 0.002). Thus, ADSCs were heterogeneous and represented two types of cells: small highly proliferative and large low proliferative cells. Cell heterogeneity observed in some colonies might be due to cells registered at different cell cycle phases. Stable and typical morphology, colony-formation capability and high proliferative capacity of cells indicate visceral adipose tissue as a rich source of ADSCs.