Development of pluripotent stem cells from domestic and wild suidae for the study of host pathogen interactions in vitro
Watson, Thomas Milne
African Swine Fever (ASF) disease is caused by infection of susceptible Suidae with African Swine Fever Virus (ASFV). In Eurasian domestic pigs, and closely related wild boar, ASF is characterised by a haemorrhagic fever and a high mortality rate of 95-100%. There are currently no effective treatments for ASF, and as such, ASF is a major threat to pig production worldwide. An outbreak of ASF across Eurasia lead to an estimated global loss of around ¼ of all domestic pigs between 2018 and 2019. By contrast, although native African pig species such as the warthog, red river hog (RRH) and bush pigs are infected by ASFV, they carry a lower viral load and do not show clinical signs of infection. The current gold standard model for the study of ASFV in vitro are primary macrophages harvested from domestic pigs. Primary macrophages have limited proliferative potential in vitro and their use requires a constant supply from sacrificed animals. This is at odds with the 3Rs principal to reduce animal use in research. Moreover, primary macrophages are not readily amenable to genetic modification, making it difficult to interrogate gene function in this cell culture model. There is limited access to primary macrophages from wild suid species, leaving the genetic variation responsible for ASFV tolerance in wild African species largely unexplored. This project aims to produce better in vitro models to study ASFV using induced pluripotent stem cells (iPSCs). IPSCs can be expanded indefinitely in culture and can differentiate into any cell type of the three germ layers. This provides a genetically stable platform for in vitro gene editing and allows for the functional analysis of the genetic variation seen in wild Suidae, including ASFV tolerant RRHs and ASFV susceptible wild boar. This project has used tetracycline regulatable reprogramming factors to produce iPSCs from the domestic pig, RRH, and wild boar. The iPSCs were dependant on exogenous reprograming factor expression for maintenance of the undifferentiated state, but were shown to self-renew and produce derivatives of all three germ layers, including macrophages, after directed differentiation in vitro. Moreover, they were amenable to genetic modification using CRISPR/Cas9 as shown through the generation of REX1-GFP stem cell reporter lines. IPSC-derived macrophages (iPSCdM) exhibited molecular signatures of macrophage identity as determined by RT-qPCR, surface marker expression and RNA-Seq. In addition, they were able to phagocytose foreign particles and could be infected by macrophage specific pathogens including porcine reproductive and respiratory syndrome virus (PRRSV). Domestic pig iPSCdMs supported replication of ASFV to the same level as pig embryonic stem cell derived macrophages and pig ex vivo derived macrophages, making them an appropriate model for the study of ASFV in vitro. Comparison of ASFV infection between domestic pig and RRH iPSCdMs matched the in vivo infection kinetics, with lower levels of viral replication in the ASFV tolerant RRH. RNA-Seq analysis, and confirmatory RT-qPCR, indicated that infection and immunity related genes, including the pattern recognition receptors TLR2, TLR4 and inflammasome regulator CASP1, were downregulated in RRH macrophages. Corresponding differences were also noted in the iPSCdMs in response to immune stimulation. As a proof of principle, TLR2 knock-out domestic pig iPSCs were generated as a model to study the role of this pattern recognition receptor during ASFV infection in future experiments. Taken together, the results in this thesis show that iPSCdMs provide a useful new model for the study of ASFV tolerance in vitro. This also shows the potential of iPSC technology to effectively capture and interrogate biologically interesting and important genetic variation of wild animal species.