Abstract
A major challenge in regenerative medicine is the generation of functionally effective target cells to replace or repair damaged tissues. The finding that most somatic cells can be directly converted into cells of another lineage by the expression of specific transcription factors has paved the way to novel applications. Induced neurons (iNs) represent an alternative source of neurons for disease modeling, drug screening, and potentially, for cell replacement therapy. This unit describes methods for the efficient conversion of blood cells into iNs, including protocols to isolate cord blood CD133+ cells, infect them with Sendai virus vectors that express SOX2 and c-MYC, and differentiate the infected cells (PB-MNCs) into mature neurons. A method to reprogram peripheral blood mononuclear cells into iNs is also described. Support protocols describe how to culture rat astrocytes and characterize the electrophysiology of iNs. © 2016 by John Wiley & Sons, Inc.
Highlights
Cellular reprogramming has opened new avenues to investigate human disease and identify potential targets for drug discovery (Bellin et al, 2012)
We report a fast and efficient approach for generating induced neural cells directly from human hematopoietic cells using Sendai virus
Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters
Summary
Cellular reprogramming has opened new avenues to investigate human disease and identify potential targets for drug discovery (Bellin et al, 2012). Direct lineage conversion (or transdifferentiation) of somatic cells into neurons (induced neurons [iNs]) has been achieved by forced expression of lineage-specific transcription factors and microRNAs (miRNA) (Ambasudhan et al, 2011; Caiazzo et al, 2011; Pang et al, 2011; Pfisterer et al, 2011; Vierbuchen et al, 2010; Yoo et al, 2011) Using this approach, several cell types (Giorgetti et al, 2012; Karow et al, 2012; Marro et al, 2011) have been converted into functional neurons in vitro and in vivo (Guo et al, 2014; Su et al, 2014; Torper et al, 2013). Nonintegrative methods based on Sendai virus (SeV) or chemically defined culture conditions have been described for the direct conversion of nonhuman cells into neural progenitor cells (iNPCs) (Cheng et al, 2014; Lu et al, 2013)
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