A scaleable and defined system for generating neural stem cells from human embryonic stem cells.

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.

Embryonic stem cell-derived neural progenitors display temporal restriction to neural patterning.

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.

Neural differentiation of mouse embryonic stem cells in chemically defined medium.

Directed differentiation of embryonic stem (ES) cells has enormous potential to derive a wide variety of defined cell populations of therapeutic value. To achieve this, it is necessary to use protocols that promote cell differentiation under defined culture conditions. Furthermore, understanding the mechanisms of cell differentiation in vitro will allow the development of rationale approaches to systematically manipulate cell fates. Here we have analysed the differentiation of mouse ES cells to the neural lineage under serum and feeder cell-free conditions, using a previously described chemically defined medium (CDM). In CDM, ES cell differentiation is highly neurogenic. Cell differentiation was monitored by analysis of a gene expression array (Clontech-Atlas) and by semi-quantitative RT-PCR for a panel of genes involved in cell lineage specification and patterning of the epiblast. In addition to expression of neural markers, data identified a transient expression of several genes associated with the organising activities of the embryonic node and visceral endoderm, including regulators of WNT, BMP, Hedgehog and FGF signaling pathways. Neural differentiation in CDM does not occur by a simple default mechanism, but was dependent on endogenous FGF signaling, and could be blocked by adding BMP4, and LiCl to simulate WNT activation. Neural differentiation was also inhibited by antagonising endogenous hedgehog activity. Taken together the profile of gene expression changes seen in CDM cultures recapitulates those seen in the early embryo, and is suggestive of common developmental mechanisms.

Enhancing and diminishing gene function in human embryonic stem cells.

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.