Construction of CD163-knockout induced pluripotent stem cells using reprogramming technology

Abstract

As the key receptor protein mediating PRRSV infection, CD163 represents a critical target for blocking viral entry. To establish a robust in vitro cellular model for investigating PRRSV–host interactions, this study utilized CD163-knockout transgenic pigs previously generated in our laboratory and isolated porcine auricular marginal fibroblasts (PAMFs). Porcine-derived OSKM reprogramming factors (OCT4, SOX2, KLF4, c-MYC) were delivered via a Tet-On lentiviral system combined with an LBCSV reprogramming protocol, leading to the successful generation of a CD163-deficient porcine induced pluripotent stem cell line (CD163-KO iPSC). Effective integration of exogenous reprogramming factors was confirmed during induction, and alkaline phosphatase staining showed strong positivity in CD163-KO iPSC colonies, preliminarily demonstrating their pluripotent characteristics. Further analyses, including quantitative PCR, semi-quantitative PCR, immunofluorescence, and Western blot, confirmed successful reprogramming, as evidenced by the upregulated expression of the pluripotency markers NANOG and SALL4 at both the mRNA and protein levels, along with a considerable downregulation of the somatic cell marker THY1 at the mRNA level. RNA-seq analysis revealed that CD163-KO iPSC exhibited significant upregulation of pluripotency genes (e.g., OCT4, ESRRB) compared to porcine embryonic fibroblasts (PEFs), along with downregulation of developmental genes and enhanced basal metabolic activity. Embryoid body spontaneous differentiation and primordial germ cell-like cell induction experiments demonstrated that this cell line not only exhibited excellent gene editing compatibility but also possessed defined multilineage differentiation potential, thereby providing a reliable in vitro model platform for in-depth investigation of PRRSV infection mechanisms and host–pathogen interactions. This study not only offers a novel tool for elucidating viral entry mechanisms, but also lays a solid foundation for the development of antiviral strategies against PRRSV and the control of porcine viral diseases.