Bioengineering Morphogenesis and Differentiation in Human Brain Organoids

Abstract

The developmental processes that give rise to the distinctive human cerebral cortex are ex- perimentally inaccessible, and only partially recapitulated in animal models. Brain organoids derived from human stem cells offer a unique window into these events at the cellular level. However, organoids deviate from the single-lumen morphology and polarized tissue archi- tecture of the embryonic cortex, which orchestrate cell-cell interactions critical for normal development in vivo. Here, I develop techniques for culturing 3D in vitro human organoid mod- els of the cerebral cortex that maintain a single ventricle-like structure and display biomimetic polarization of the cortical neuroepithelium. I apply these models to investigate the role of tissue-level mechanical forces in balancing between proliferation and differentiation of neural stem cells. I find that manually inflating organoids by injection of biocompatible fluids to increase intraluminal pressure promotes the maintenance and expansion of endogenous-like tissue architecture. This injection protocol prolongs the survival of apicobasally polarized architecture from the previously reported maximum of 22 days in culture to at least 49 days, maintains this architecture as organoids grow to over a millimeter in diameter, and can preserve the overall single-lumen structure for up to three months. Leveraging the ability of this model to experimentally vary mechanical strain on human neuroepithelium-like tissue, I demonstrate that inflation of ventricular structures in organoids delays neurogenesis, favoring proliferative divisions of neural progenitors at the expense of differentiation. Conversely, pressure release is rapidly followed by an increase in neuronal differentiation. This finding suggests that mechani- cal forces naturally present in the early brain could play a key role in regulating the timing and extent of stem cell proliferation relative to differentiation, serving as a previously unrecognized driver of final cortex size and cellular composition. As a step towards future models that could enable experimental exposure of biomimetic human neuroepithelia to realistic polarized biochemical signals, I also completed the design and pilot experiments for a microfluidically perfused neural tube model. Together, my results show that organoid-derived neural stem cells retain the ability to form large-scale tissues with high fidelity to in vivo histology when provided with appropriate mechanical cues, and illustrate the potential for bioengineered brain models to investigate fundamental questions in developmental neurobiology.