Cortical neurons derived from equine induced pluripotent stem cells are susceptible to neurotropic flavivirus infection and replication: an in vitro model for equine neuropathic diseases

Horses are susceptible to a number of neurotropic viruses, including West Nile virus (WNV), which is a pathogen of global significance in both horses and humans. However, there are no in vitro models with which to study infectious neuropathic diseases in the horse. In an effort to redress this we have generated neurons from equine induced pluripotent stem cells (equiPSCs) that express a range of cortical neuron-specific markers, in addition to the membrane-bound ligand Ephrin B3, which plays an important role in axon guidance as well as functioning as the receptor through which henipaviruses, such as Hendra virus, enter mammalian neurons. EquiPSC-derived neurons spontaneously depolarise with waves of depolarisation conducted unidirectionally to adjacent neurons. We sought to confirm that equiPSC-derived neurons are a possible in vitro model for viral neuropathic diseases in the horse by examining their susceptibility to infection with flaviviruses that are known to be neurotropic in horses, including WNV and Murray Valley encephalitis virus (MVEV), and to compare these to non-pathogenic flaviviruses such as Fitzroy River virus (FRV) and Bamaga virus (BgV). All three strains of WNV tested in this study grew to high titres in the equiPSC-derived neurons, inducing a strong cytopathic effect, as did MVEV. In contrast, FRV showed restricted replication, and no cytopathic effect, which is consistent with the observation that FRV infects, but does not cause disease, in horses. Bamaga virus, which is thought to infect only marsupials, did not replicate in the equiPSC-derived neurons. Hence, our equiPSC-derived neurons display virus-specific differences in terms of viral titre and cytopathic effect that are similar to observations made in vivo, thus supporting their use as an in vitro model for neurotropic viral infection in horses

Platypus Induced Pluripotent Stem Cells: The Unique Pluripotency Signature of a Monotreme

The platypus (Ornithorhynchus anatinus) is an egg-laying monotreme mammal whose ancestors diverged ∼166 million years ago from the evolutionary pathway that eventually gave rise to both marsupial and eutherian mammals. Consequently, its genome is an extraordinary amalgam of both ancestral reptilian and derived mammalian features. To gain insight into the evolution of mammalian pluripotency, we have generated induced pluripotent stem cells from the platypus (piPSCs). Deep sequencing of the piPSC transcriptome revealed that piPSCs robustly express the core eutherian pluripotency factors POU5F1/OCT4, SOX2, and NANOG. Given the more extensive role of SOX3 over SOX2 in avian pluripotency, our data indicate that between 315 and 166 million years ago, primitive mammals replaced the role of SOX3 in the vertebrate pluripotency network with SOX2. DAX1/NR0B1 is not expressed in piPSCs and an analysis of the platypus DAX1 promoter revealed the absence of a proximal SOX2-binding DNA motif known to be critical for DAX1 expression in eutherian pluripotent stem cells, suggesting that the acquisition of SOX2 responsiveness by DAX1 has facilitated its recruitment into the pluripotency network of eutherians. Using the RNAseq data, we were also able to demonstrate that in both fibroblasts and piPSCs, the expression ratio of X chromosomes to autosomes (X X:AA) is approximately equal to 1, indicating that there is no upregulation of X-linked genes. Finally, the RNAseq data also allowed us to explore the process of X-linked gene inactivation in the platypus, where we determined that for any given gene, there is no preference for silencing of the maternal or paternal allele; that is, within a population of cells, the silencing of X-linked genes is not imprinted