Accelerated neural induction to create hiPSC-derived peripheral neuron progenitors in vitro via an intermediate neural crest stage

University of Minnesota M.S. thesis. December 2018. Major: Stem Cell Biology. Advisor: James Dutton. 1 computer file (PDF); v, 71 pages.Pluripotent stem cells (PSCs) have the potential to differentiate into any cell type of the body, including neurons of the central and peripheral nervous system. (Takahashi, K. et. al., 2006). Current protocols for differentiating human pluripotent stem cells into posterior ectoderm-derived tissues in vitro are time consuming, however, due to an initial, protracted, rate-limiting step called neuroepithelialization. This problem of protracted timing in differentiation has hindered development of neuron differentiation protocols, limiting their usefulness in biomedical applications, drug toxicity studies or cell transplantation and/or therapies. The Dutton Lab has reduced the rate-limiting step for neural induction of human induced Pluripotent Stem Cells (hiPSCs) from 7 days to 24 hours, which has enabled rapid protocol development to explore neuronal diversity. The current work published in this thesis has been built on these findings to define an efficient, systematic step-wise differentiation protocol for the production of hiPSC-derived vagal neural crest in 4 days, which can be further differentiated into peripheral neuron progenitors and Schwann cell precursors by day 7. The peripheral neurons derived from this protocol express characteristic proteins Peripherin, Brn3A, and Islet1, while the Schwann cells express Egr1 and Etv5. This vagal crest protocol is the fastest and most efficient protocol described in the literature and has been validated on a panel of three characterised iPSC lines. Peripheral neurons derived from this protocol have been further used to study neuro-hepato interactions in an in vitro co-culture system