Modeling cerebral development in vitro with L-MYC-immortalized human neural stem cell-derived organoids

A promising advance for ex vivo studies of human brain development and formulation of therapeutic strategies has been the adoption of brain organoids that, to a greater extent than monolayer or spheroid cultures, recapitulate to varying extents the patterns of tissue development and cell differentiation of human brain. Previously, such studies been hampered by limited access to relevant human tissue, inadequate human in vitro models, and the necessity of using rodent models that imperfectly reproduce human brain physiology. Here we present a novel organoid-based research platform utilizing L-MYC-immortalized human fetal neural stem cells (LMNSC01) grown in a physiological 4% oxygen environment. We visualized developmental processes in LMNSC01 brain organoids for over 120 days in vitro by immunofluorescence and NanoString gene expression profiling. Gene expression patterns revealed by NanoString profiling were quantitatively compared to those occurring during normal brain development (BrainSpan database) using the Singscore method. We observe similar developmental patterns in LMNSC01 organoids and developing cortex for genes characterizing neurons, astrocytes, and oligodendrocytes, and multiple pathways including those involved in apoptosis, neuronal cytoskeleton, neurotransmission, and metabolism. Notable properties of this LMNSC01 platform are its initiation with immortalized authentic human neural stem cells, growth in a physiological oxygen environment, the consistency of the organoids produced, and favorable comparison of their gene expression patterns with those reported for normal cortical development. SUMMARYEx vivo studies of human brain development has been advanced by adoption of organoids recapitulating to varying extents in utero patterns of tissue development and cell differentiation. We here present an organoid-based human cortical development platform employing immortalized fetal neural stem cells (LMNSC01) grown in a physiological (4% oxygen) environment. Characterizing LMNSC01 organoids for over 120 days in vitro by immunofluorescence and expression profiling (using NanoString), and then comparing these profiles to those of normal cortical development (BrainSpan database), revealed similar developmental patterns for neurons, astrocytes and oligodendrocytes. Notable properties of this platform are its initiation with immortalized authentic human NSCs, growth at physiological oxygen concentration, and subsequent favorable comparison of their gene expression patterns with those observed during cortical development.