Human PSC-based cortical organoid and assembloid systems integrating pericyte and microglial lineages and signals - Project Summary Three-dimensional (3D) brain organoids from human pluripotent stem cells (hPSCs) provide a tremendous opportunity to model brain development and disease. Over the last few years, we have developed many hPSC-based protocols that now enable researchers to routinely generate > 50 distinct human cell types for modeling both Central Nervous System and Peripheral Nervous System disorders in a dish. More recently, our lab and others have translated those 2D approaches into 3D “guided” neural organoid protocols to generate specific regions of the human brain. Further complexity can be achieved by combining individual organoids into multi-region assembloids, or by spatially restricted patterning to induce distinct human brain regions within a single organoid. However, currently available organoid and assembloid models have major limitations, including a poor representation of several progenitor and neuronal cell populations such as outer radial glia (oRG) and several cortical interneuron lineages. We posit that the missing or underrepresented cell types may depend on signals provided by non-neural lineages (vascular and immune cells). In our preliminary work, we have developed an improved hPSC-based cortical organoid model which dramatically enhances the generation of oRG, cortical-derived interneurons, and cortical excitatory neurons with increased levels of maturity. This was achieved by treatment with leukemia inhibitor factor (LIF) resulting in the activation of STAT3 and mTOR signaling pathways. Data from human fetal cortex development identifies vascular pericytes are a major source of LIF. Remarkably, we demonstrate that integrating hPSC-derived brain pericytes can substitute for LIF treatment in cortical organoids. Here, in Aim 1 we will optimize and assess robustness of the LIF organoid protocol and the generation of oRG across multiple human PSC lines. We will further assess whether oRG give rise to intermediate progenitors, cortical interneurons and cortical excitatory neurons using a unique oRG-specific double reporter hPSC line and genetic fate mapping tools. Finally, we will determine how changes in mTOR signaling impact oRG behavior and whether phenotypes observed in models of Tuberous Sclerosis (TSC) are linked to aberrant oRG function. In Aim 2, we will build an organoid system that integrates key non-neural cell types. We will establish a modular platform that combines cortical organoids with 3D spheroids (“microtissues”) comprised of vascular brain pericytes and/or microglia. These novel microphysiological platform will be utilized to determine both LIF-dependent and LIF independent effects on cortical development. Finally, we will assess whether such a more complex microphysiological platform will enable improved modeling of the developmental defects related to mTOR signaling and TSC.
