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Lab Invest. 1994 Oct;71(4):583-94.

Normal human urothelial cells in vitro: proliferation and induction of stratification.

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1
Imperial Cancer Research Fund Cancer Medicine Research Unit, University of Leeds, U.K.

Abstract

BACKGROUND:

The purpose of the work was to establish urothelium as an in vitro model for the study of proliferation, stratification, and differentiation in "complex" epithelia.

EXPERIMENTAL DESIGN:

Normal human urothelial cells were cultured in a serum-free medium. The effects of epidermal growth factor (EGF), cholera toxin (CT), extracellular calcium and 13-cis-retinoic acid on cell growth, morphology, phenotype, and cytodifferentiation were studied using phase-contrast microscopy and indirect immunofluorescence. Stratification-related changes were additionally analyzed by transmission electron microscopy.

RESULTS:

Under optimized conditions, long-term cultures were successful in 44 (74.5%) out of 59 specimens. Bacterial infection was the most common cause of failure (9 cases). Primary urothelial cells required an initial plating density of > or = 10(4) cells/cm2 for survival; passaged cells survived much lower plating densities (> or = 2.5 x 10(2) cells/cm2). CT significantly improved cell attachment, but neither CT nor EGF were essential for growth. By contrast, cells failed to proliferate without bovine pituitary extract. In media containing bovine pituitary extract, CT, and EGF, cultures had a mean population doubling time of 14.7 +/- 1.8 hours, maintained a nonstratified phenotype, and expressed the cytokeratin (CK) profile of basal/intermediate urothelium: CK7, CK8, CK17, CK18 and CK19, with variable expression of CK13. CK20 was not expressed in vitro. CK14 and CK16 were also expressed, suggestive of squamous metaplasia in culture, which could be inhibited with 13-cis-retinoic acid. Increasing extracellular calcium from 0.09 to 0.9-4.0 mM slowed cell proliferation, induced stratification and desmosome formation, and increased expression of E-cadherin. High calcium, EGF, CT, and retinoic acid did not induce markers of late/terminal urothelial cytodifferentiation.

CONCLUSIONS:

We describe a simplified technique for the isolation and long-term culture of human urothelial cells. Urothelial cells in vitro are capable of rapid proliferation and can be induced to form integrated stratifying cell layers in high calcium medium. Stratification-related changes are not necessarily accompanied by urothelial cell maturation and differentiation.

PMID:
7967513
[Indexed for MEDLINE]
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