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Engineering Conferences International ECI Digital Archives Cell Culture Engineering XV Proceedings Spring 5-12-2016 Neural patterning of human induced pluripotent stem cells for studying neurotoxicity Yan Li Florida State University, [email protected] Yuanwei Yan Florida State University Follow this and additional works at: http://dc.engconfintl.org/cellculture_xv Part of the Biomedical Engineering and Bioengineering Commons Recommended Citation Yan Li and Yuanwei Yan, "Neural patterning of human induced pluripotent stem cells for studying neurotoxicity" in "Cell Culture Engineering XV", Robert Kiss, Genentech Sarah Harcum, Clemson University Jeff Chalmers, Ohio State University Eds, ECI Symposium Series, (2016). http://dc.engconfintl.org/cellculture_xv/199 This Abstract is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in Cell Culture Engineering XV by an authorized administrator of ECI Digital Archives. For more information, please contact [email protected]. NEURAL PATTERNING OF HUMAN INDUCED PLURIPOTENT STEM CELLS FOR STUDYING NEUROTOXICITY Yuanwei Yan, Department of Chemical and Biomedical Engineering, Florida State University Julie Bejoy, Department of Chemical and Biomedical Engineering, Florida State University Junfei Xia, Department of Chemical and Biomedical Engineering, Florida State University Jingjiao Guan, Department of Chemical and Biomedical Engineering, Florida State University Yi Zhou, Department of Biomedical Sciences, Florida State University Yan Li, Department of Chemical and Biomedical Engineering, Florida State University [email protected] Key Words: pluripotent stem cell, neural patterning, neurotoxicity, three-dimensional Existing models using adult human neural stem cells have the restricted access. Human induced pluripotent stem cells (hiPSCs) can generate allogeneic or patient-specific neural cells/tissues and even mini-brains to provide robust in vitro models for applications in drug discovery, neurological disease modeling, and cell therapy. Toward this goal, the objective of this study is to construct 3-D neural models from hiPSCs through the scalable embryoid body-based suspension culture which can generate cortical glutamatergic neurons and motor neurons by tuning the sonic hedgehog (SHH) signaling. 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 SHH antagonist) or purmorphamine (the SHH agonist) along with other factors and further maturation. The neural cells 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 and HB9 (>40%) with the purmorphamine treatment. The cells also expressed pre- and post-synaptic markers (Synapsin I and PSD95), and generated action potentials in response to depolarizing current injections and spontaneous excitatory post-synaptic currents after maturation. To assess the cellular responses, three classes of small molecules/drugs were investigated: (1) N-methyl-D-aspartate to induce general neural toxicity; (2) matrix metalloproteinases inhibitors to affect matrix remodeling; (3) amyloid β (1-42) oligomers to induce disease-specific neural toxicity. Differential responses to various treatments were observed for different neuronal subtypes. Overall, this study can provide a transformative approach to establish 3-D neural models for neurological disease modeling (e.g., Alzheimer’s disease), drug discovery, and cell therapy. Figure 1 – Characterizations of neural spheres derived from human iPSK3 cells. (i) Neural spheres derived from iPSK3 cells based on embryoid body (EB) formation; (ii) cyclopamine-treated cells (day 35) and purmorphamine-treated cells (day 35). At day 35, cyclopamine-treated cells have more cortical glutamatergic (Glut) neurons while purmorphamine-treated cells have more motor neurons expressing Islet-1 (Ist-1) and HB9. β-tub III indicates β-tubulin III. Both populations express pre-synaptic marker synapsin I (Syn I). Scale bar: 100 μm. (iii) Action potentials in response to depolarizing current injections and at the end of hyperpolarizing current injections (“rebound” action potentials) (day 30 cells); (iv) spontaneous excitatory post-synaptic currents (day 30 cells).