Generation and characterization of an ovine cell line derived from peripheral blood and its potential use in the study of livestock and zoonotic viral infections

Abstract

[Abstract] Background: Mesenchymal stromal cells (MSCs) are a population of undifferentiated, non-hematopoietic fibroblast-like cells isolated from several tissues with multipotent differentiation capacity in vitro. This study focused on the establishment and characterization of a mesenchymal stromal cell line derived from ovine peripheral blood mononuclear cells (PBMCs).

Methods: MSCs were isolated from ovine blood and used to establish a continuous cell line. Several assays were performed to characterize this cell line and confirm its mesenchymal origin. First, cells were characterized by flow cytometry (FACS) using monoclonal antibodies specific for MSCs and hematopoietic non-stromal cell surface antigens. Second, real-time quantitative PCR (RT-qPCR) was performed using gene primer pairs specific for hematopoietic and mesenchymal cell surface markers. Third, we evaluated the potential of this cell line to differentiate in vitro into other lineages such as osteoblasts and neurons, which was confirmed by specific staining with Alizarin Red (AR), RT-qPCR and immunofluorescence (IFA) assays. Finally, we tested the susceptibility of this ovine cell line to diverse livestock, animal and human viruses.

Results: FACS and RT-qPCR analyses revealed that the ovine cell line expressed mesenchymal markers, and was negative for hematopoietic markers. In addition, our ovine cell line was able to differentiate into osteoblasts and neurons, as we observed via quantitative analyses of AR staining, RT-qPCR and IFA. Importantly, the ovine cell line was susceptible to infection by all the viruses tested, as confirmed by IFA and viral growth kinetics assays.

Conclusion: In this work, we isolated MSCs from ovine peripheral blood and established a continuous ovine cell line. The thorough characterization conducted as well as the osteogenic and neuronal differentiation capacity of these cells confirmed that the ovine cell line had a mesenchymal stromal origin. Interestingly, the newly established ovine cell line was permissive to all viruses studied in this work, suggesting its potential as a valuable tool for isolating and characterizing viruses and studying virus-host interactions.