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.
