Abstract
In vitro generation of functional neurons from embryonic stem (ES) cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP) and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP+ neurons in culture display functional neuronal properties even at early stages of differentiation.
Highlights
Embryonic stem (ES) cells retain the ability to provide an unlimited supply of all cell types
green fluorescent protein (GFP)+ neurons were abundant within the cultures and most had small cell bodies
In this study we have shown that TK23 ES cells expressing tauGFP can differentiate into cells that exhibit functional neuronal properties
Summary
Embryonic stem (ES) cells retain the ability to provide an unlimited supply of all cell types. Much research has focused on transplanting ES cell-derived neural cells in animal models of neurological diseases and examining their potential in expressing appropriate regional markers characteristic to the surrounding host tissue. These studies indicate that ES cell-derived neural progeny are able to integrate into the host brain and undergo further differentiation [1]. The unambiguous identification of ES cell-derived neurons in the above studies often employ genetic marking of ES cell lines with a reporter, such as beta-gal or green fluorescent protein (GFP) expressed constitutively and ubiquitously [4,5,6], or restrictively in ES cell-derived neurons [7,8,9]. Various studies have utilized the targeted tau-GFP ES cell lines in order to gain further understanding the fundamentals of neuronal development as well as the intricacies involved in cell replacement therapies [1,2,3,7,8]
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