MP09-14 ESTABLISHMENT OF IMMORTALIZED HUMAN BLADDER SMOOTH MUSCLE CELLS

Abstract

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2018MP09-14 ESTABLISHMENT OF IMMORTALIZED HUMAN BLADDER SMOOTH MUSCLE CELLS Masaki Yoshida, Naohiro Hashimoto, Hisae Nishii, and Masanori Nomiya Masaki YoshidaMasaki Yoshida More articles by this author , Naohiro HashimotoNaohiro Hashimoto More articles by this author , Hisae NishiiHisae Nishii More articles by this author , and Masanori NomiyaMasanori Nomiya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.340AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Smooth muscle cell (SMC) cultures are an excellent tool for the study of cellular events and molecular mechanisms by which the phenotypic plasticity is controlled. Although many SMC systems have been previously analyzed, human bladder cell biology has not been fully evaluated. Therefore we attempted to establish the immortalized human bladder smooth muscle cells. METHODS In immortalization of human smooth muscle cells, primary cultured human bladder smooth muscle cells derived from a 68 year old man were transduced with recombinant lentiviruses and retroviruses encoding human cyclin D1, human mutant CDK4, and human telomerase. Immunoblotting analysis were performed using primary antibodies included mouse monoclonal antibodies for calponin, α-smooth muscle actin (α-SMA), ?-smooth muscle actin (γ-SMA) and rabbit monoclonal antibodies for muscarinic acetylcholine receptor M2 and M3 and rabbit polyclonal antibodies for smooth muscle myosin heavy chain 11 (MYH11). In addition, immunofluorescence analyses for connexin 43 were also performed. Live imaging of intracellular calcium evaluated with Calcium Kit-Fluo 4 after stimulation with high potassium medium and carbachol. Smooth muscle contraction were also evaluated live imaging of F-actin using SiR-A. RESULTS We have established an immortalized human bladder SMC line designated as hBS11. hBS11 cells exhibited continuous cell proliferation for more than 50 populations under optimized culture conditions. The cell cycle interval was 18.0±3.2h. A chromosome analysis of the cells revealed a normal 46XY diploid karyotype. hBS11 cells expressed smooth muscle differentiation marker proteins, including MYH11, α-SMA, γ-SMA, and calponin. The connexin 43 signals were also detected as spotty signals on the surface of hBS11 cells. M2 and M3 receptors expressed in differentiated hBS11 cells. Both the cholinergic receptor agonist calbachol and a high concentration of extracellular potassium increased intracellular calcium in differentiated hBS11 cells. Differentiated hBS11 cells stimulated by calcium ionophore (A23187) exhibited contractility. CONCLUSIONS The present study demonstrates that the hBS11 cell maintains plasticity of the differentiation that a smooth muscle cell originally has. Immortalized hBS11 cell provides useful analytical system for elucidation of the pathophysiological mechanism of lower urinary tract dysfunction and the development of the new treatment options, in addition to the elucidation of molecular differentiation mechanism, reproduction mechanism, and the function control mechanism of the smooth muscle cell. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e111-e112 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Masaki Yoshida More articles by this author Naohiro Hashimoto More articles by this author Hisae Nishii More articles by this author Masanori Nomiya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...