Establishment and functional characterization of the reversibly immortalized mouse glomerular podocytes (imPODs)

Abstract

Glomerular podocytes are highly specialized epithelial cells and play an essential role in establishing the selective permeability of the glomerular filtration barrier of kidney. Maintaining the viability and structural integrity of podocytes is critical to the clinical management of glomerular diseases, which requires a thorough understanding of podocyte cell biology. As mature podocytes lose proliferative capacity, a conditionally SV40 mutant tsA58-immortalized mouse podocyte line (designated as tsPC) was established from the Immortomouse over 20 years ago. However, the utility of the tsPC cells is hampered by the practical inconvenience of culturing these cells. In this study, we establish a user-friendly and reversibly-immortalized mouse podocyte line (designated as imPOD), on the basis of the tsPC cells by stably expressing the wildtype SV40 T-antigen, which is flanked with FRT sites. We show the imPOD cells exhibit long-term high proliferative activity, which can be effectively reversed by FLP recombinase. The imPOD cells express most podocyte-related markers, including WT-1, Nephrin, Tubulin and Vinculin, but not differentiation marker Synaptopodin. The imPOD cells do not form tumor-like masses in vivo. We further demonstrate that TGFβ1 induces a podocyte injury-like response in the FLP-reverted imPOD cells by suppressing the expression of slit diaphragm-associated proteins P-Cadherin and ZO-1 and upregulating the expression of mesenchymal markers, α-SMA, Vimentin and Nestin, as well as fibrogenic factors CTGF and Col1a1. Collectively, our results strongly demonstrate that the newly engineered imPOD cells should be a valuable tool to study podocyte biology both under normal and under pathological conditions.

BMP9‐initiated osteogenic/odontogenic differentiation of mouse tooth germ mesenchymal cells (TGMCS) requires Wnt/β‐catenin signalling activity

“…The use of the retroviral vector SSR #41 to express SV40 T antigen flanked with the FRT sites have been described previously. [15][16][17][18] Briefly, the SSR #41 vector and pCL-Ampho packaging vector were co-transfected into293 Phoenix Ampho (293PA) cells to produce the packaged retrovirus. Exponentially growing TGMCs were infected with the SSR #41 retrovirus and subjected to hygromycin B selection (0.4mg/mL) for 3-5 days twice in complete DMEM at 37°C, yielding the stably immortalized tooth germ mesenchymal cells, designated as the iTGMC line.…”

Bone morphogenetic protein 4 (BMP4) alleviates hepatic steatosis by increasing hepatic lipid turnover and inhibiting the mTORC1 signaling axis in hepatocytes

“…To assess the duration of adenovirus-mediated gene expression in liver, we injected the CsCl gradient purified Ad-FLuc into mouse liver (4-week-old male, 10 10 pfu/injection/mouse). Whole body optical imaging was performed at day 5 after injection by using D-Luciferin Potassium (Gold Biotechnology Inc.) as luciferase substrate and assessed with the Xenogen IVIS 200 Imaging System as described [67–70].…”

FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian Cells

“… 50 , 51 The above cells were cultured in DMEM containing 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, CA, USA), supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in 5% CO 2 as described. 19 , 26 , 41 , 52 , 53 , 54 Restriction endonucleases, NEBuilder HiFi DNA assembly master mix, Phusion high-fidelity DNA polymerase, and the electrocompetent DH10B cells were purchased from New England Biolabs (NEB, Ipswich, MA, USA). Blasticidin S was ordered from InvivoGen (San Diego, CA, USA).…”

“…The retroviral vector-mediated transduction and establishment of stable cell lines were carried out as previously described. 19 , 22 , 53 , 54 , 57 , 58 , 59 , 60 Briefly, the retroviral transfer vectors pSiEB-simSmad4, pSiEB-simSmad1458, and pSiEB-simSmad4158 were co-transfected with pCL-Ampho into subconfluent 293 Phoenix Ampho (293PA) cells to package retroviruses. The retrovirus-containing supernatants were harvested at 36–72 h after transfection and pooled to infect subconfluent BMSCs.…”