Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Neurons as Models for Toxoplasma gondii Infection and Persistence

Toxoplasma gondii infection may be asymptomatic in healthy people but results in the lifelong persistence of cysts containing bradyzoites in brain and muscle. We used human induced pluripotent stem cell-derived cardiomyocytes (iCMs), cardiac fibroblasts (iCFs) and neurons (iNEs) from a single human donor to study the interactions of T. gondii with human cells in vitro. iCMs, iCFs and iNEs infected with the T. gondii TypeI/III EGS reporter strain expressing SAG1-mCherry (tachyzoite) and LDH2-sfGFP (bradyzoite) displayed higher rates of bradyzoite formation in iCMs and iNEs, compared to iCFs from the same donor at 72 hours post-infection by fluorescence microscopy of live cells and flow cytometry. Bradyzoite induction occurred spontaneously in normal media.

Using RNA-seq we compared uninfected iCMs, iCFs and iNEs to cells infected for 24 hours and 72 hours. Both iCFs and iCMs showed thousands of differentially expressed genes (DEGs) at both timepoints. In contrast, iNEs infected for 24 hours showed 190 upregulated and 321 downregulated DEGs whereas iNEs infected for 72 hours showed 244 upregulated and 91 downregulated DEGs. Enrichment analysis of gene sets (GSEA) regulated by infection was performed using the hallmark gene set collection from the Molecular Signatures Database. To correlate the higher rate of bradyzoite formation in iCMs and iNEs to the relevance of each GSEA hallmark pathway, we identified hallmark pathways having enriched DEGs in a similar manner between iCMs and iNEs but different from iCFs. At 24 hours post-infection, fatty acid metabolism and oxidative phosphorylation pathways were upregulated in iCFs but unchanged in iCMs or iNEs, whereas MYC targets pathway was downregulated in iCMs or iNEs but unchanged in iCFs. At 72 hours post-infection, glycolysis pathway was downregulated in iCMs or iNEs but unchanged in iCFs, whereas myogenesis and protein secretion pathways were upregulated in iCFs but were unchanged in iCMs or iNEs.

Our in vitro generated human iCMs, iCFs and iNEs provide a new model to understand the impact of host cell type and host-specific factors upon Toxoplasma infection. We anticipate this model can be used to develop cell-based assays for drug screening of potential bradyzoite therapeutic targets and to explore the role of host genetics in T. gondii pathogenesis and persistence.