Abstract 4147265: Title: Immortalization of human cardiac fibroblasts using a combination of SV40 large T antigen and hTERT.

Background: Fibroblasts are critical regulators of health and disease. Unfortunately, primary isolated fibroblasts retain limited proliferation potential. To enable robust biochemical experimentation in cardiac fibroblasts we immortalized human cardiac fibroblasts using lentiviral delivered SV40 large T antigen (SV40), human telomerase reverse transcriptase (hTERT), or both tools in combination. Methods: Following virus treatment with immortalization genes and hygromycin selection, we characterized subsequent immortalized cell lines for proliferative capacity and ability to become activated in response to TGF-β1 treatment. Expression of Periostin (POSTN), alpha smooth muscle actin (αSMA) and Sertad4 was measured via qPCR after 48hrs of vehicle or TGF-β1 (10ng/ml) treatment. Western Blot analysis was performed to detect the phosphorylation of SMAD2, AKT and P38 in response to TGF-β1 and cell doubling time was calculated at each passage through passage 20. Results: Fibroblasts immortalized with SV40 did not proliferate past passage 18 while those immortalized with hTERT quiesced at passage 19. Only the combination treatment resulted in fibroblasts that proliferated through passage 20 (last measured doubling time of 3.25 days – shortest doubling time of 1.23 days at passage 11). Early passage fibroblasts (passage 7) from each immortalization strategy showed increased mRNA expression of Sertad4, POSTN and αSMA following TFG-β1 treatment. However, only the combination strategy resulted increased Sertad4 and POSTN expression in higher passage cells upon stimulation. All immortalization strategies resulted in decreased αSMA expression upon TGF-β1 treatment in fibroblasts beyond passage 10. All cell lines tested retained the ability to increase AKT and SMAD2 phosphorylation in response TGF-β1 treatment. Conclusions: These results indicate that fibroblast immortalization through a combined SV40 large T antigen and hTERT expression approach is superior to individual manipulation. The combination strategy resulted in increased proliferative capacity and more robust TGF-β responsiveness. These cells may be a useful tool for investigating molecular mechanisms of fibroblast activation when the inability to expand primary cultures would be limiting