Project Summary A Fully Patterned Human Neural Tube Model Using Microfluidics The development of vertebrate central nervous system (CNS) begins with the formation of neural tube (NT) and its regional patterning to generate neuronal subtypes along the rostral (R)-caudal (C) and dorsal (D)- ventral (V) axes. Regional patterning of the human NT is a tightly regulated process, deviation from which can result in neurodevelopmental disorders and may lead to distinct neurological and psychiatric diseases later in life. Regional patterning of the human NT remains incompletely understood due to limited access to human embryonic tissues. Animal models have been instrumental in understanding the development of human CNS and associated disorders. However, they are limited in revealing some fundamental aspects of development, genetics, pathology, and disease mechanisms that are unique to humans. Stem cell-based in vitro models of human nervous system development, including neural organoids and bioengineered NT development models, are emerging as promising experimental tools. However, none of the current stem cell-based neural development models is capable of recapitulating neural patterning along two orthogonal axes in a 3D tubular geometry, the hallmark of NT patterning in vivo. Furthermore, the existing neural development models only recapitulate certain aspects of the development of either human brain or spinal cord regions but not both. In our preliminary study, we have successfully leveraged the developmental potential and self- organizing property of human pluripotent stem cells (hPSCs) in conjunction with microfluidics to develop the first of its kind, synthetic, fully patterned human NT model. Using this microfluidic platform, exogenous morphogen gradients along two orthogonal axes can be established to achieve regional patterning of the microfluidic human NT-like structure along both the R-C and D-V axes, in both brain-like and spinal cord-like regions. This microfluidic patterned human NT-like structure exhibits many hallmarks of NT development, including a tubular geometry, a single continuous central lumen enclosing by neuronal progenitor cells, patterned expression of canonical R-C and D-V regional markers including HOX genes, and the emergence of neural mesodermal progenitors and the isthmic organizer. Thus, the development of the microfluidic human NT-like structure closely mimics NT development, offering for the first time an in vivo-like tissue architecture with consistent spatiotemporal cell differentiation and organization. The goal of this R01 research is to develop this exciting microfluidic human NT-like model (Aim 1) and leverage its technical advantages to study the roles of different exogenous morphogen signals in neural patterning (Aim 2 & 3). Genetic perturbations and lineage tracing assays will be conducted to study human neural mesodermal progenitor development (Aim 2). In Aim 3 we further aim to achieve D-V patterned human NT-l...