Showing papers by "Barbara D. Abbott published in 2001"

An in vitro model for murine ureteric epithelial cells.

Abstract: Dear Editor: Regulation of the proliferation and differentiation of ureteric epithelial cells is critical to formation and maintenance of ureteric lumen. Abnormal proliferation of this epithelial population during early morphogenesis can obstruct the lumen and produce hydronephrosis and damage to the kidney (Abbott and Birnbaum, 1990). Epithelial proliferation and differentiation is associated with specific patterns of expression of epidermal growth factor (EGF), transforming growth factor-alpha, and the EGF receptor (EGFR). Disrupted growth factor expression con'elates with ureterie epithelial hyperplasia and hydronephrosis. The culture models presented here allow in vitro manipulation of the proliferation and differentiation of fetal ureteric epithelial cells. Modifications to the culture medium were developed to facilitate detection of either stimulation or inhibition of proliferation and differentiation of these cells in euhure. This approach should be valuable in studies of biomoleeules, such as growth thetors, believed to regulate the cells endogenously, as well as for mechanistic studies of chemical agents that produce hydronephrosis. Female C57BL/6N mice (Charles River Laboratories, Raleigh, NC) were mated overnight and checked for vaginal plugs the next morning, gestation day (GD) 0. On GD 18, dams were sacrificed by CO2 inhalation, uteri exteriorized, and fetuses were removed. The urinary tracts were removed from the fetuses and ureters from 8-9 fetuses were pooled in phosphate-buffered saline. Ureters were minced and digested for 80 rain at 37 ~ C, in purified eollagenase blend, Liberase Blend # 1 (Roche Molecular Biochemieals, Indianapolis, IN), diluted to a final concentration of 1.35 Ixg/p~l. The digest was centrifuged to produce a cellular pellet and cells were resuspended in medium and titurated to give a single cell suspension. Digests from separate litters were pooled and diluted such that 250,000 cells in 100 Ixl of medium were plated per Millieell-CM insert (Millipore Corporation, Marlborough, MA). Prior to culture, the inserts were coated with mouse collagen IV and laminin and rinsed with medium. The basal medium with low calcium (keratinoeyte growth medium-2; Cloneties, San Diego, CA) was supplemented with trace elements, a complex mixture of lipids, a defined mixture of purified hormones and growth factors. The final concentration in medium and sources of the supplements were as follows: insulin-like growth factor II (IGF-II), 4 or 10 ng/ml (Beeton Diekinson Labware, Bedford, MA); free fatty acids mixture (Sigma Chemical Co., St. Louis, MO), 7.6 Ixeq/L; transferrin, 5 txg/mg (Becton Dickinson); insulin, 1 Ixg/ml (Beeton Dickinson); penicillin/streptomycin, 10 U/10 txg per ml (GIBCO/BRL Products, Grand Island, NY); calcium chloride, 0.3 mM (Sigma); fibroblast growth factor, 1 ng/ml (Beeton Dickinson); sodium selenite, 3 • 10 lo M (Sigma); zinc sulfate, 5 • 10 -11 M (Sigma); copper sulfate, 1 X 10 -1~ M (Sigma); bovine serum albumin (BSA), 1 mg/ml (Sigma) or human high-density lipoprotein (HDL), 10 Ixg/ml (isolated from fresh human plasma) (Chemicon International, Temecula, CA); EGF, 50 ng/ml (Becton Dickinson); and fetal bovine serum (FBS) (GIBCO/BRL) at 2% for the frst 48 h and 1% for the remaining culture period. The cultures were incubated at 37 ~ C in a humidified atmosphere containing 5% CO2 in air. Medium at plating contained 50 ng/ml EGF and 2% FBS. This was replaced after the initial 48 h of culture with medium containing only 1% FBS. Medium was replaced every 48 h until termination of the cuhure (Fig. 1A). In a modification of this regimen, the medium was replaced after the initial 24 h with medium which did not contain EGF, and at 48 h this was changed to medium with 1% FBS which was used every 48 h thereafter (Fig. 1B). Cultures initially included round, adherent cells (single cells or in clusters), and over time isolated epithelial sheets and fibroblasts were distinguishable, with later development 9 f confluent epithelial sheets and/or multilayered epithelium, w~th or without fibroblasts, depending on the culture conditions. Proliferation was assessed by the detection of 5-bromo-2'-deoxyufidine (BrDU, Boehfinger Mannheim Corporation, Indianapolis, IN) uptake into nuclei, using a purified, peroxidase-labeled, monoelonal antibody against mouse BrDU, diluted 1:100 (#347580; Beeton Dickinson, Two Oak Park, MA) and avidin-biotin peroxidase complex (ABC Kit; Vector Laboratories, Burlingame, CA). Differentiated epithelial and/or mesenehymal cells were identified by immunoassay for keratin or vimentin using affinity-purified antibodies (Sigma) localized with alkaline-phosphatase or avidin-biotin peroxidase (ABC Kit; Vector Laboratories). As shown in Fig. 1, two culture models are described. Model 1 promoted epithelial cell growth to confluence and supported longterm survival. In this protocol, cells were initially cultured with keratinoeyte growth medium-2 supplemented with 2% FBS, trace elements, a complex mixture of lipids, a defined mixture of purified hormones and growth factors, including HDL. This medium was replaced after 48 h with medium containing 1% FBS, and other supplements remained the same. Medium with 1% FBS was used every 48 h thereafter. There were 14 inserts from five different experiments which were observed and terminated at 23-30 d. These cultures displayed a confluent, multilayered epithelium. Reducing the time in culture to 13 or 8 d did not affect overall growth, and cultures reached confluence with few or no fibroblasts observed (seven inserts from three different experiments were evaluated after 13-14 d and 14 inserts from four different experiments after 8-10 d). This model could be used for evaluating agents with the potential to slow epithelial proliferation, induce apoptosis, or promote fibroblast growth or survival. This regimen could also have applications for testing agents that produce their deleterious effects on muhilayered epithelia. Model 2 (Fig. 1B) would permit the analysis of agents that stim-