(680c) Three-Dimensional Neural Microtissue Models Derived from Human Induced Pluripotent Stem Cells

Neural tissues derived from human induced pluripotent stem cells (hiPSCs) can provide invaluable models for the applications in drug discovery, neurological disease modeling, and cell therapy.  While the 3-D models were established using adult human neural stem cells, the availability of cell source limits the distribution of such models.  HiPSCs can generate allogeneic or patient-specific neural cells/tissues and even mini-brains that are physiologically relevant to model neural diseases.  Toward this goal, the objective of this study is to construct neural microtissues from hiPSCs through the scalable suspension culture which can generate cortical glutamatergic neurons and motor neurons by tuning the differentiation protocols.  The differentiation of human iPSK3 cells was induced using dual inhibition of SMAD signaling with LDN193189 and SB431542.  Then the neural tissue patterning was tuned through the treatment with cyclopamine (the antagonist of sonic hedgehog signaling) or purmorphamine (the agonist of sonic hedgehog signaling) along with other factors (fibroblast growth factor-2 and retinoic acid) and further maturation.  The neural microtissues were characterized at day 20, day 35, and day 55.  Abundant glutamatergic neurons (>60%) was observed with the cyclopamine treatment, while the cells were more enriched with motor neurons expressing Islet-1 (>40%) with the purmorphamine treatment.  The cells also expressed pre-synaptic marker synapsin I and post-synaptic marker PSD95 after maturation.  Characterization of electrophysiology with whole-cell patch clamp recordings showed normal Na+/K+ currents and action potentials in response to depolarizing current injections and spontaneous excitatory post-synaptic currents.  The influence of matrix metalloproteinases (MMPs) on the proliferation and neurite outgrowth of the replated neural microtissues was also investigated using MMP-2 and MMP-9 inhibitors, showing the important role of matrix remodeling.  The cells also responded to N-methyl-D-aspartate (NMDA) treatment, showing the NMDA-induced neurotoxicity.  This study should advance our understanding of hiPSC self-organization and development into neural microtissues and provide a transformative approach to establish 3-D models for disease modeling and drug discovery.