It is widely recognized that gain-and loss-of-function approaches are essential for understanding the functions of specific genes, and such approaches would be particularly valuable in studies involving human embryonic stem (hES) cells. We describe a simple and efficient approach using lipofection to transfect hES cells, which enabled us to generate hES cell lines expressing naturally fluorescent green or red proteins without affecting cell pluripotency. We used these cell lines to establish a means of diminishing gene function using small interfering (si)RNAs, which were effective at knocking down gene expression in hES cells. We then demonstrated that stable expression of siRNA could knock down the expression of endogenous genes. Application of these gain-and loss-of-function approaches should have widespread use, not only in revealing the developmental roles of specific human genes, but also for their utility in modulating differentiation.

The ability to differentiate human ESCs (hESCs) to defined lineages in a totally controlled manner is fundamental to developing cell-based therapies and studying human developmental mechanisms. We report a novel, scaleable, and widely applicable system for deriving and propagating neural stem cells from hESCs without the use of animal products, proprietary formulations, or genetic manipulation. This system provides a definitive platform for studying human neural development and has potential therapeutic implications. STEM CELLS 2007;25:731-737

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Neural stem cells have considerable therapeutic potential because of their ability to generate defined neuronal cell types for use in drug screening studies or cell-based therapies for neurodegenerative diseases. In this study, we differentiate mouse embryonic stem cells to neural progenitors with an initial forebrain identity in a defined system that enables systematic manipulation to generate more caudal fates, including motoneurons. We demonstrate that the ability to pattern embryonic stem cell-derived neural progenitors is temporally restricted and show that the loss of responsiveness to morphogenetic cues correlates with constitutive expression of the basic helix-loop-helix transcription factors Olig2 and Mash1, epidermal growth factor receptor, and vimentin and parallels the onset of gliogenesis. We provide evidence for two temporal classes of embryonic stem cell-derived putative radial glia that coincide with a transition from neurogenesis to gliogenesis and a concomitant loss of regional identity. STEM