The human central nervous system (hCNS) is comprised of myriad neuronal and glial cell phenotypes that are morphogenetically patterned to discrete anatomical regions of highly structured tissues. Each of these cell phenotypes could be uniquely susceptible to neurodegenerative diseases or neurotoxins either autonomously or only within their tissue-specific context. Thus, hCNS models used in regulatory science should maximally recapitulate organotypic cell phenotype diversity and tissue structure to provide optimally predictive screening of therapeutic efficacy and neurotoxicity. Recent advances in harnessing the self-organizing properties of human pluripotent stem cell (hPSC)- derived neural stem cells (NSCs) to generate 3D stratified cortex and retinal tissues demonstrates the feasibility of creating organotypic hCNS models, a.k.a. organoids. However, the ability to generate analogous organoids for other hCNS regions remains elusive. In particular, derivation of the full spectrum of hindbrain and spinal cord tissues is hindered by the absence of a differentiation protocol that provides deterministic control of Hox gene expression, which regionalizes neural phenotypes to specific rostrocaudal positions along the posterior neuraxis. Additionally, methods have yet to be developed to recapitulate organotypic spatial patterns of cell phenotypes within hPSC-derived hindbrain and spinal cord tissues. We propose to overcome these limitations by combining novel neural differentiation protocols and emerging microfabrication methods to generate arrays of 3D hindbrain and spinal cord organoids within a microfluidic high-throughput screening (HTS) platform. A key strength of this application is the consistent use of chemically defined culture reagents and automated microfabrication techniques, which enable scalable and reproducible manufacture of the organoid arrays. Furthermore, we will use state-of-the-art genome editing tools to customize the organoid array platform for quantitative HTS of therapeutics and neurotoxins that affect hindbrain and spinal cord motor neuron fate.
2014 - presentpresent