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Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.

Background: Pig‐derived tissues could overcome the shortage of human donor organs in transplantation. However, the glycans with terminal α‐Gal and Neu5Gc, which are synthesized by enzymes, encoded by the genes GGTA1 and CMAH, are known to play a major role in immunogenicity of porcine tissue, ultimately leading to xenograft rejection. Methods: The N‐glycome and glycosphingolipidome of native and decellularized porcine pericardia from wildtype (WT), GGTA1‐KO and GGTA1/CMAH‐KO pigs were analyzed by multiplexed capillary gel electrophoresis coupled to laser‐induced fluorescence detection. Results: We identified biantennary and core‐fucosylated N‐glycans terminating with immunogenic α‐Gal‐ and α‐Gal‐/Neu5Gc‐epitopes on pericardium of WT pigs that were absent in GGTA1 and GGTA1/CMAH‐KO pigs, respectively. Levels of N‐glycans terminating with galactose bound in β(1‐4)‐linkage to N‐acetylglucosamine and their derivatives elongated by Neu5Ac were increased in both KO groups. N‐glycans capped with Neu5Gc were increased in GGTA1‐KO pigs compared to WT, but were not detected in GGTA1/CMAH‐KO pigs. Similarly, the ganglioside Neu5Gc‐GM3 was found in WT and GGTA1‐KO but not in GGTA1/CMAH‐KO pigs. The applied detergent based decellularization efficiently removed GSL glycans. Conclusion: Genetic deletion of GGTA1 or GGTA1/CMAH removes specific epitopes providing a more human‐like glycosylation pattern, but at the same time changes distribution and levels of other porcine glycans that are potentially immunogenic. [ABSTRACT FROM AUTHOR]