Self-organizing, three-dimensional cerebral organoids differentiated from human induced pluripotent stem cells (hiPSC) have emerged as a new in vitro model to investigate pathogenesis of infectious diseases. Cerebral organoids are beneficial over animal models because they allow for the investigation of infectious disease pathogenesis in a human context. The cellular components and structural configuration of mature cerebral organoids resemble the developing human brain. Toxoplasma gondii displays a tropism for brain tissue after infecting humans and other warm-blooded animals. Within brain tissue, T. gondii tachyzoites transform into bradyzoites and encyst, leading to chronic, latent infection. Our goal is to develop a reliable model of chronic T. gondii infection in brain tissue using human-derived cerebral organoids to further our understanding of chronic infection and host-parasite interactions.

A commercially available kit (STEMdiff Cerebral Organoid kit, STEMCELL Technologies) was used to generate cerebral organoids using hiPSCs from a single human donor (ATCC-ACS-1026). Cerebral organoids matured for 4 months before infection with T. gondii. To investigate brain tissue tropism and bradyzoite transformation, we infected cerebral organoids with a T. gondii Type I/III EGS reporter strain that expresses SAG1-mCherry (tachyzoite stage) and LDH2-sfGFP (bradyzoite stage). Each cerebral organoid was exposed to 1,000 T. gondii tachyzoites for 4-hours then organoids were washed and re-incubated. Fluorescent microscopy was used to visualize tachyzoite proliferation and bradyzoite transformation in the cerebral organoids. After 17 days incubation cerebral organoids were fixed for cryosectioning. A cryotome was used to make 5 uM cryosections for fluorescent microscopy. Cryosections of cerebral organoids were stained using the following antibodies to verify cerebral organoid structural components: anti-SOX2 (radial glial cell, neuronal precursor marker) and anti-tubulin β III isoform (neuronal differentiation marker). DAPI DNA stain was used to visualize cell nuclei. Fluorescent microscopy verified T. gondii infection, tachyzoite to bradyzoite transformation, and the presence of cerebral organoid structural components. Additional fluorescent microscopy is underway to investigate T. gondii cyst formation and to verify presence of additional cerebral organoid structural components (astrocytes and oligodendrocytes).