Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage Yechiel Elkabetz, 1,2 Georgia Panagiotakos, 2 George Al Shamy, 2 Nicholas D. Socci, 3 Viviane Tabar, 2 and Lorenz Studer 1,2,4 1 Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA; 2 Division of Neurosurgery, Sloan-Kettering Institute, New York, New York 10021, USA; 3 Computational Biology Center, Sloan-Kettering Institute, New York, New York 10021, USA Neural stem cells (NSCs) yield both neuronal and glial progeny, but their differentiation potential toward multiple region-specific neuron types remains remarkably poor. In contrast, embryonic stem cell (ESC) progeny readily yield region-specific neuronal fates in response to appropriate developmental signals. Here we demonstrate prospective and clonal isolation of neural rosette cells (termed R-NSCs), a novel NSC type with broad differentiation potential toward CNS and PNS fates and capable of in vivo engraftment. R-NSCs can be derived from human and mouse ESCs or from neural plate stage embryos. While R-NSCs express markers classically associated with NSC fate, we identified a set of genes that specifically mark the R-NSC state. Maintenance of R-NSCs is promoted by activation of SHH and Notch pathways. In the absence of these signals, R-NSCs rapidly lose rosette organization and progress to a more restricted NSC stage. We propose that R-NSCs represent the first characterized NSC stage capable of responding to patterning cues that direct differentiation toward region-specific neuronal fates. In addition, the R-NSC-specific genetic markers presented here offer new tools for harnessing the differentiation potential of human ESCs. [Keywords: Human embryonic stem cells; neural patterning; neural stem cells; neuronal specification] Supplemental material is available at http://www.genesdev.org. Received September 17, 2007; revised version accepted November 21, 2007. Neural stem cells (NSCs) are defined by their ability to clonally give rise to the three major CNS lineages: neu- rons, astrocytes, and oligodendrocytes. The in vitro iso- lation and propagation of NSCs from the developing and adult CNS has provided an essential tool to study neural precursor biology and lineage differentiation potential (Gage 2000). Much attention has been focused on the factors directing the expansion and differentiation of NSCs in vitro. These studies demonstrated that single factors act instructively to specify neuronal versus glial fate choice (Johe et al. 1996). Two major challenges have limited the use of NSCs to study neural differentiation and to develop cell-based strategies for brain repair: First, under most growth conditions, NSCs show increased gliogenic bias and concomitant loss of neurogenic poten- tial in culture. Second, in vitro expanded NSCs cannot be regionally specified in response to developmental pat- terning cues. For example, SHH/RA or SHH/FGF8 treat- ment induce motoneuron and midbrain dopamine neu- rons, respectively, in neural plate explants (Roelink et al. 1995; Ye et al. 1998) and embryonic stem cell (ESC) prog- eny (Wichterle et al. 2002; Barberi et al. 2003) but not in cultured NSCs (Caldwell et al. 2001; Jain et al. 2003). Recent work has shown improved neurogenic poten- tial of NSCs after long-term culture (Conti et al. 2005), although potential for regional specification was not ad- dressed. Studies in mouse ESCs suggested the existence of FGF2/LIF-responsive primitive NSCs (Tropepe et al. 2001). However, these cells exhibit ESC-like differentia- tion potential in chimeric mice and cannot be main- tained without progressing toward a “definitive” NSC stage with limited differentiation potential. Therefore, despite the contribution of these recent studies the iso- lation of NSCs with true self-renewal and full patterning and differentiation potential has remained elusive. During neural differentiation human ESCs (hESCs) un- dergo morphogenetic events characterized by the forma- tion of radially organized columnar epithelial cells termed “neural rosettes” (Zhang et al. 2001; Perrier et al. 2004). These structures comprise cells expressing early neuroectodermal markers such as Pax6 and Sox1 and are capable of differentiating into various region-specific neuronal and glial cell types in response to appropriate developmental cues (Perrier et al. 2004; Li et al. 2005). 4 Corresponding author. E-MAIL studerl@mskcc.org; FAX (212) 717-3642. 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