Using Synthetic Organizers to Drive Multi-Axis Patterning of Brain Organoids - Project Summary Current methods for generating brain organoids are limited in their ability to mimic the spatial and structural complexity of the brain. Most organoid models lack reproducibility in cell-type patterning and tissue architecture, and they fail to capture the full cellular diversity seen in the brain. Brain organoids are typically generated by protocols that isotropically applying exogenous morphogens in the media, but they often produce oversimplified, poorly organized architectures and lack multi-regional interactions. A major limitation of these protocols is the lack of spatial control over morphogen signaling, which is a key driver of morphogenesis during natural development. Thus, there remains a critical need for scalable strategies that enable fine spatial and temporal control of morphogen signals to drive coordinated multi-axis and multi-regional patterning in brain organoids. We recently developed platform of engineered synthetic organizer cells – spatially self-assembling cells that produce morphogens from spatially defined positions. Here, we propose to use synthetic organizers to induce multi-axis patterning in 3D brain organoids. Our long-term goal of this exploratory project is to build platforms that produce reproducible, spatially organized brain tissues with greater complexity and functional relevance. Our specific aims are: Aim1: Construct synthetic organizers to control brain organoid axis formation. Engineer synthetic organizer cells (from cell lines) that produce morphogens involved in brain A–P and D–V differentiation (WNT3A, DKK1, FGF, CER, NOGGIN, BMP4 and SHH), providing a core toolkit. Aim2: Create synthetic gradients to shape A–P axis of brain organoids. Using tunable organizers at opposing poles that produce A–P morphogens (e.g. DKK1-Wnt), we can produce embryoids that cover selective ranges of the A–P axis body plan, including embryoids that primarily encompass the brain. We will optimize these organizer-driven brain organoids and use IHC, RNA scope and scRNA-seq to analyze the structures for neural subtypes and formation of key subregions (hind, mid and forebrain, as well as cortical layers). Aim3: Integrative multi-axis patterning in brain organoids. We will combine multiple synthetic organizers, arranged orthogonally to each other, to establish intersecting A–P and D–V axes. We will systematically and independently tune the following morphogen production organizers: WNT3A (posterior), DKK1/CER1/FGF8 (anterior), BMP4 (dorsal) and SHH (ventral). We will assess whether the resulting brain domains exhibit spatially organized A–P and D–V subregions and connections between subregions. Our central hypothesis is that synthetic organizers (programmable cellular morphogen sources) can be used to precisely and reproducible drive symmetry breaking and yield more complex and functional neural tissues. This work may provide a path toward more structured, reproducible, and physiologically relevant brain organoids, with applications in developmental biology, neuroscience, disease modeling, and neural engineering
