title: Establishment of conditionally immortalized human glomerular mesangial cells in culture, with unique migratory properties.
abstract: The aim of this study was to establish an immortalized human mesangial cell line similar to mesangial cells in vivo for use as a tool for understanding glomerular cell function. Mesangial cells were isolated from glomerular outgrowths from a normal human kidney, then retrovirally transfected with a temperature-sensitive SV40T antigen+human telomerase (hTERT). Mesangial cells exhibited features of compact cells with small bodies in a confluent monolayer at 33°C, but the cell shape changed to flat and stellate after 5 days in growth-restrictive conditions (37°C). Western blot and immunofluorescence analysis showed that podocyte markers (nephrin, CD2AP, podocin, Wilms' tumor-1) and an endothelial-specific molecule (VE-cadherin) were not detectable in this cell line, whereas markers characteristic of mesangial cells (α-SMA, fibronectin, and PDGFβ-R) were strongly expressed. In migration assays, a significant reduction in wound surface was observed in podocyte and endothelial cells as soon as 12 h (75 and 62%, respectively) and complete wound closure after 24 h. In contrast, no significant change was observed in mesangial cells after 12 h, and even after 48 h the wounds were not completely closed. Until now, conditionally immortalized podocyte and endothelial cell lines derived from mice and humans have been described, and this has greatly boosted research on glomerular physiology and pathology. We have established the first conditionally immortalized human glomerular mesangial cell line, which will be an important adjunct in studies of representative glomerular cells, as well as in coculture studies. Unexpectedly, mesangial cells' ability to migrate seems to be slower than for other glomerular cells, suggesting this line will demonstrate functional properties distinct from previously available mesangial cell cultures. This conditionally immortalized human mesangial cell line represents a new tool for the study of human mesangial cell biology in vitro.
authors.affiliation: Children's Renal Unit and Academic Renal Unit, University of Bristol, Southmead Hospital, Bristol, United Kingdom.
authors.forename: Ramadan M
authors.initials: RM
authors.lastname: Sarrab
authors.forename: Rachel
authors.initials: R
authors.lastname: Lennon
authors.forename: Lan
authors.initials: L
authors.lastname: Ni
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Wherlock
authors.forename: Gavin I
authors.initials: GI
authors.lastname: Welsh
authors.forename: Moin A
authors.initials: MA
authors.lastname: Saleem
mesh_terms: D000199:Actins / Q000378:metabolism; D000963:Antimetabolites; D015415:Biomarkers; D015153:Blotting, Western; D001973:Bromodeoxyuridine; D002454:Cell Differentiation; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D003584:Cytological Techniques; D055724:Electrophysiological Phenomena; D004727:Endothelium / Q000502:physiology; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism; D050527:Mesangial Cells / Q000378:metabolism / Q000502:physiology*; D050199:Podocytes / Q000502:physiology; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000096:biosynthesis; D014945:Wound Healing / Q000502:physiology
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D000963:Antimetabolites; D015415:Biomarkers; D005353:Fibronectins; D008565:Membrane Proteins; C113267:nephrin; D020797:Receptor, Platelet-Derived Growth Factor beta; D001973:Bromodeoxyuridine