Abstract
Organ explant cultures are well-established in vitro models that are used to study normal cell biological and regeneration processes as well as carcinogenesis. Primary urothelial cultures from bladder mucosa explants are highly differentiated and are thus broadly used as in vitro experimental equivalents of native urothelial tissue. Since experiments on differentiated urothelial cultures from bladder mucosa explants currently allow only a single use of explants, establishment of sufficient quantities of cultures requires large numbers of sacrificed animals. There is thus a great need for a cheaper approach with less ethical dilemmas. Herein, we demonstrate that mouse bladder mucosa explants can be reused. Reused explants produce outgrowths with highly differentiated urothelia, just like primary explants. Even after being recycled ten times, urothelial outgrowths have the supramolecular and ultrastructural features that are comparable to the native urothelium. Ten times reused explants produce superficial urothelial cells that express uroplakins in the apical plasma membrane, claudin-8 in the tight junctions, and have a subapical network of cytokeratin 20. Basal urothelial cells in urothelial outgrowths of ten times reused explants express p63 which indicates that these urothelial outgrowths have a persistent proliferative capacity. Using our approach, one can perform experiments that were previously not feasible due to low quantities of donor tissue. The method also offers opportunity for effective use of scarce healthy human urothelial tissue.
References
Acharya P, Beckel J, Ruiz WG, Wang E, Rojas R, Birder L, Apodaca G (2004) Distribution of the tight junction proteins ZO-1, occludin, and claudin-4, -8, and -12 in bladder epithelium. Am J Physiol Renal Physiol 287(2):F305–F318. https://doi.org/10.1152/ajprenal.00341.2003
Barbieri CE, Pietenpol JA (2006) p63 and epithelial biology. Exp Cell Res 312(6):695–706. https://doi.org/10.1016/j.yexcr.2005.11.028
Bindels EM, Vermey M, Rebel JM, Zwarthoff EC, Van Der Kwast TH (1997) In vitro modulation of implantation and intraepithelial expansion of bladder tumor cells by epidermal growth factor. Exp Cell Res 235(2):395–402. https://doi.org/10.1006/excr.1997.3705
Bindels EM, Vermey M, van den Beemd R, Dinjens WN, Van Der Kwast TH (2000) E-cadherin promotes intraepithelial expansion of bladder carcinoma cells in an in vitro model of carcinoma in situ. Cancer Res 60(1):177–183
de Boer WI, Rebel JM, Vermey M, de Jong AA, van der Kwast TH (1994) Characterization of distinct functions for growth factors in murine transitional epithelial cells in primary organotypic culture. Exp Cell Res 214(2):510–518. https://doi.org/10.1006/excr.1994.1288
Hicks RM (1966) The function of the golgi complex in transitional epithelium. Synthesis of the thick cell membrane. J Cell Biol 30(3):623–643
Hicks RM (1975) The mammalian urinary bladder: an accommodating organ. Biol Rev Camb Philos Soc 50(2):215–246
Hudoklin S, Zupancic D, Romih R (2009) Maturation of the Golgi apparatus in urothelial cells. Cell Tissue Res 336(3):453–463. https://doi.org/10.1007/s00441-009-0779-9
Kachar B, Liang F, Lins U, Ding M, Wu XR, Stoffler D, Aebi U, Sun TT (1999) Three-dimensional analysis of the 16 nm urothelial plaque particle: luminal surface exposure, preferential head-to-head interaction, and hinge formation. J Mol Biol 285(2):595–608. https://doi.org/10.1006/jmbi.1998.2304
Karni-Schmidt O, Castillo-Martin M, Shen TH, HuaiShen T, Gladoun N, Domingo-Domenech J, Sanchez-Carbayo M, Li Y, Lowe S, Prives C, Cordon-Cardo C (2011) Distinct expression profiles of p63 variants during urothelial development and bladder cancer progression. Am J Pathol 178(3):1350–1360. https://doi.org/10.1016/j.ajpath.2010.11.061
Kreft ME, Robenek H (2012) Freeze-fracture replica immunolabelling reveals urothelial plaques in cultured urothelial cells. PLoS One 7(6):e38509. https://doi.org/10.1371/journal.pone.0038509
Kreft ME, Romih R, Sterle M (2002) Antigenic and ultrastructural markers associated with urothelial cytodifferentiation in primary explant outgrowths of mouse bladder. Cell Biol Int 26(1):63–74. https://doi.org/10.1006/cbir.2001.0829
Kreft ME, Hudoklin S, Sterle M (2005a) Establishment and characterization of primary and subsequent subcultures of normal mouse urothelial cells. Folia Biol (Praha) 51(5):126–132
Kreft ME, Sterle M, Veranic P, Jezernik K (2005b) Urothelial injuries and the early wound healing response: tight junctions and urothelial cytodifferentiation. Histochem Cell Biol 123(4–5):529–539. https://doi.org/10.1007/s00418-005-0770-9
Kreft ME, Sterle M, Jezernik K (2006) Distribution of junction- and differentiation-related proteins in urothelial cells at the leading edge of primary explant outgrowths. Histochem Cell Biol 125(5):475–485. https://doi.org/10.1007/s00418-005-0104-y
Kreft ME, Di Giandomenico D, Beznoussenko GV, Resnik N, Mironov AA, Jezernik K (2010a) Golgi apparatus fragmentation as a mechanism responsible for uniform delivery of uroplakins to the apical plasma membrane of uroepithelial cells. Biol Cell 102(11):593–607. https://doi.org/10.1042/BC20100024
Kreft ME, Hudoklin S, Jezernik K, Romih R (2010b) Formation and maintenance of blood-urine barrier in urothelium. Protoplasma 246(1–4):3–14. https://doi.org/10.1007/s00709-010-0112-1
Lasič E, Višnjar T, Kreft ME (2015) Properties of the urothelium that establish the blood-urine barrier and their implications for drug delivery. Rev Physiol Biochem Pharmacol 168:1–29. https://doi.org/10.1007/112_2015_22
Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I, Bondanza S, Ponzin D, McKeon F, Luca MD (2001) p63 identifies keratinocyte stem cells. Proc Natl Acad Sci USA 98(6):3156–3161
Rebel JM, de Boer WI, Thijssen CD, Vermey M, Zwarthoff EC, van der Kwast TH (1993) An in vitro model of intra-epithelial expansion of transformed urothelial cells. Int J Cancer 54(5):846–850
Rebel JM, De Boer WI, Thijssen CD, Vermey M, Zwarthoff EC, Van der Kwast TH (1994a) An in vitro model of urothelial regeneration: effects of growth factors and extracellular matrix proteins. J Pathol 173(3):283–291. https://doi.org/10.1002/path.1711730312
Rebel JM, Thijssen CD, Vermey M, Delouvée A, Zwarthoff EC, Van der Kwast TH (1994b) E-cadherin expression determines the mode of replacement of normal urothelium by human bladder carcinoma cells. Cancer Res 54(20):5488–5492
Resau JH, Sakamoto K, Cottrell JR, Hudson EA, Meltzer SJ (1991) Explant organ culture: a review. Cytotechnology 7(3):137–149
Sterle M, Kreft ME, Batista U (1997) The effect of epidermal growth factor and transforming growth factor beta 1 on proliferation and differentiation of urothelial cells in urinary bladder explant culture. Biol Cell 89(4):263–271
van der Kwast TH, van Rooy H, Mulder AH (1989) Establishment and characterization of long-term primary mouse urothelial cell cultures. Urol Res 17(5):289–293
Veranic P, Jezernik K (2002) Trajectorial organisation of cytokeratins within the subapical region of umbrella cells. Cell Motil Cytoskeleton 53(4):317–325. https://doi.org/10.1002/cm.10077
Višnjar T, Chesi G, Iacobacci S, Polishchuk E, Resnik N, Robenek H, Kreft M, Romih R, Polishchuk R, Kreft ME (2017) Uroplakin traffic through the Golgi apparatus induces its fragmentation: new insights from novel in vitro models. Sci Rep 7(1):12842. https://doi.org/10.1038/s41598-017-13103-x
Zupančič D, Romih R, Robenek H, Žužek Rožman K, Samardžija Z, Kostanjšek R, Kreft ME (2014) Molecular ultrastructure of the urothelial surface: insights from a combination of various microscopic techniques. Microsc Res Tech 77(11):896–901. https://doi.org/10.1002/jemt.22412
Acknowledgements
The authors acknowledge the financial support from the Slovenian Research Agency (research core Funding No. P3-0108, Young-researcher funding, and Project No. J3-7494). Thanks also to Sanja Čabraja, Nada Pavlica Dubarič, Linda Štrus, and Sabina Železnik for technical assistance.
Funding
This study was funded by Slovenian Research Agency (research core Funding No. P3-0108, Young-researcher funding, and Project No. J3-7494).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.
Rights and permissions
About this article
Cite this article
Jerman, U.D., Kreft, M.E. Reuse of bladder mucosa explants provides a long lasting source of urothelial cells for the establishment of differentiated urothelia. Histochem Cell Biol 150, 567–574 (2018). https://doi.org/10.1007/s00418-018-1704-7
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00418-018-1704-7