Effect of Scrapie Prion Infection in Ovine Bone Marrow-Derived Mesenchymal Stem Cells and Ovine Mesenchymal Stem Cell-Derived Neurons

Abstract

Scrapie is a prion disease affecting sheep and goats and it is considered a prototype of transmissible spongiform encephalopathies (TSEs). Mesenchymal stem cells (MSCs) have been proposed as candidates for developing in vitro models of prion diseases. Murine MSCs are able to propagate prions after previous mouse-adaptation of prion strains and, although ovine MSCs express the cellular prion protein (PrPC), their susceptibility to prion infection has never been investigated. Here, we analyze the potential of ovine bone marrow-derived MSCs (oBM-MSCs), in growth and neurogenic conditions, to be infected by natural scrapie and propagate prion particles (PrPSc) in vitro, as well as the effect of this infection on cell viability and proliferation. Cultures were kept for 48–72 h in contact with homogenates of central nervous system (CNS) samples from scrapie or control sheep. In growth conditions, oBM-MSCs initially maintained detectable levels of PrPSc post-inoculation, as determined by Western blotting and ELISA. However, the PrPSc signal weakened and was lost over time. oBM-MSCs infected with scrapie displayed lower cell doubling and higher doubling times than those infected with control inocula. On the other hand, in neurogenic conditions, oBM-MSCs not only maintained detectable levels of PrPSc post-inoculation, as determined by ELISA, but this PrPSc signal also increased progressively over time. Finally, inoculation with CNS extracts seems to induce the proliferation of oBM-MSCs in both growth and neurogenic conditions. Our results suggest that oBM-MSCs respond to prion infection by decreasing their proliferation capacity and thus might not be permissive to prion replication, whereas ovine MSC-derived neuron-like cells seem to maintain and replicate PrPSc.

Susceptibility of Ovine Bone Marrow-Derived Mesenchymal Stem Cell Spheroids to Scrapie Prion Infection

“…The presence/absence of PrP Sc in the CNS tissues was confirmed with two rapid diagnostic tests (Prionics-CheckWestern blotting; ThermoFisher Scientific and Idexx HerdChek; IDEXX, Westbrook, ME, USA) as previously descried [ 43 ]. CNS homogenates and microbe safety tests were prepared as described in previous works [ 35 ].…”

“…To test the ability to replicate PrP Sc of 2D oBM-MSCs and spheroids inoculated with scrapie-infected brain homogenate in growth and neurogenic conditions, the amount of pathogenic protein was quantified by ELISA at 2, 5, and 8 dpi using the EEB-Scrapie HerdCheck kit (IDEXX) and following the manufacturer’s recommendations. A previous study conducted by our group showed that this kit was suitable for the sensitive detection of PrP Sc in oBM-MSC cultures [ 35 ]. oBM-MSCs were seeded at 5000 cells/cm 2 in 6-well plates, and the retrieval of the cells was performed by means of trypsinization and subsequent centrifugation to obtain 3 technical replicates per condition and time (dpi).…”

“…A previous study conducted by our group evaluated the effect of scrapie prion infection in ovine bone marrow-derived mesenchymal stem cells (oBM-MSCs) cultured in growth conditions and subjected to neurogenic differentiation [ 35 ]. In this study, oBM-MSCs initially took up PrP Sc , but they were not able to maintain it over time.…”

“…In this study, oBM-MSCs initially took up PrP Sc , but they were not able to maintain it over time. MSC-derived neuron-like cells, on the contrary, absorbed and maintained stable PrP Sc levels throughout the duration of the culture [ 35 ]. These results were obtained using a 2D culture system approach that may not exactly mimic the physiological response of these cells to prion infection.…”

Stem Cells in Domestic Animals: Applications in Health and Production

“…The meta-analysis reported by the Authors showed that stem-cell therapy may improve heart function in large animal models and that the swine species is confirmed as a relevant animal model in the cardiovascular field. In this context, there is also the study of Garcia-Mendivil et al [ 3 ] regarding the development of in vitro cellular models using ovine MSCs for prion neurodegenerative disorders affecting both humans and animals, particularly ruminants. Indeed, the response of ovine bone marrow-derived MSCs and their neuron-like derivatives to prion infection allowed us to find that BM-MSC-derived neuron-like cells could be a good candidate for developing in vitro studies.…”