Induced Pluripotent Stem Cells (iPSCs): An Emerging Model System for the Study of Human Neurotoxicology
This chapter describes the materials and methods necessary to generate human induced pluripotent stem cells (iPSCs) from primary human fibroblasts and direct their differentiation into neural progenitor cells. Application of such methods is an emerging model for the study of neurotoxicity focused on human neurons and glia derived from specific patients. The techniques described here include primary human fibroblast culture, lentiviral/retroviral-mediated iPSC inductions, iPSC clonal expansion and maintenance, validation of pluripotency markers, and neuronal differentiation of iPSCs. Methods and applications using iPSCs are rapidly changing: here we describe the current methods used in our laboratories. The iPSC induction method featured in this chapter is based on a two-step viral transduction approach described by Dr. Shinya Yamanaka and colleagues (Cell 131:861–872, 2007) modified following the protocol of Dr. Sheng Ding and collaborators (Nat Methods 6:805–808, 2009). The neuralization method featured in this chapter is based on the method described by Lorenz Studer and colleagues (Nat Biotechnol 27:275–280, 2009). Maintenance and cryostorage methods were developed in our lab by optimizing a combination of approaches described in the literature. This chapter is not meant to be comprehensive, but instead focuses on the core competencies needed to begin working with human iPSCs and neuralization of these cells for toxicological studies.
Key wordsInduced pluripotent stem cells Human models of neurotoxicity Neuronal differentiation Patient-derived fibroblasts
We would like to thank Angela Ellen Tidball for the artwork and illustration of Fig. 1 of this chapter. Also, we are grateful to Dr. Lorenz Studer and Stuart Chambers for personal communications on implementing their published neuralization protocols. AMT was supported by the Vanderbilt Brain Institute. KCE was supported by a Doris Duke Clinical Scientist Development Award, NIH/NINDS K08NS050484, and the Tuberous Sclerosis Alliance. This work was further supported by a Hobbs Discovery Award from the Vanderbilt Kennedy Center (KCE, ABB), an equipment grant from the Vanderbilt Institute for Clinical and Translational Research 1UL 1RR024975 NCRR/NIH (KCE, ABB), core support from NIH NICHD grant P30HD15052 (ABB), and research support from the NIH/NIEHS grant RO1ES016931 (ABB).
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