Understanding of many aspects of the human brain is currently limited due to the lack of appropriate model systems that recapitulate the heterogenous nature of the human brain and the ethical and practical limitations of working with human brain tissue from patients. To overcome these challenges, we employ three-dimensional brain organoids, derived from human pluripotent stem cells, that recapitulate key features of human cortical development. Current approaches to engineer higher levels of organization in human brain organoids, however, are still quite limited, with fusion of two organoids of disparate specification (assembloids) being the most common approach. While a basic level of self-organization has been achieved, these models do not mimic the signaling that occurs during human forebrain formation or the resulting cortical organization seen at similar fetal developmental states. Another missing component in current organoid systems are the meninges, known to be a fundamental driving factor in cortical development. Our goal is to increase the level of complexity and organization in human brain organoids and, additionally, to understand and mimic the contribution of the meningeal cells to cortical development.
In Aim 1, we will develop a platform using fluidic channels within a large hydrogel to expose embedded organoids to user-defined gradients of soluble morphogens. We will employ well-established hydrogel materials as well as a novel bioinstructive hydrogel modified with N-cadherin extracellular peptide epitope (this material can be patterned with channels, exhibits physiological stiffness, and early studies indicate is suitable for organoid embedding). We propose to use these platforms to mimic cues occurring during development and thereby direct an embedded human brain organoid to polarize and to exhibit higher-order cortical organization.
In Aim 2, we plan to elucidate and mimic the contribution of the meninges to cortical development. We will fuse murine meninges to brain organoids, and characterize the thickness and composition of the subventricular and intermediate progenitor zones as well as the cortical plate using immunohistochemistry, scRNAseq and mass cytometry. We will also develop new strategies to differentiate human neural crest stem cells into human meningeal-like cells. We will then expose assembloids and polarized organoids from Aim 1 to meningeal tissue and meningeal-derived soluble signaling factors and determine their effects on polarization and interneuron migration. Completion of these Aims will pave the way for the next generation of human brain organoid research, enabling higher degrees of complexity and more biomimetic organization to facilitate new insight into human brain development and pathology, as well as to rationalize treatments for neurological disorders.
As human brain organoid technology evolves and structures, hierarchies, and cell behavior observed in this system more closely replicate that observed in vivo, there is increasing potential to provide critical insight needed to understand and treat currently incurable neurological disorders. Current approaches, however, do not mimic the spatially-varying signaling cascades typical of natural development, and have not integrated supportive tissue crucial for achieving crucial polarization and higher-order organization of the neocortex. To address these critical limitations, we will combine our expertise in developmental cell biology (Gama ? PI) and tissue engineering/microfluidics (Bellan ? PI) with the goal of understanding and recapitulating the in vivo signaling environment required to induce higher order forebrain organization, and additionally elucidate and mimic the contribution of the meninges to the development of the cortex.
