Derivation of neural precursors from human embryonic stem cells in the presence of noggin Pavel Itsykson, a Nili Ilouz, a Tikva Turetsky, a Ronald S. Goldstein, b Martin F. Pera, c Ianai Fishbein, d Menahem Segal, d and Benjamin E. Reubinoff a, * a The Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy and The Department of Gynecology, Hadassah University Hospital, Ein-Kerem, P.O.B. 12000, Jerusalem 91120, Israel b Gonda Research Center, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel c Institute of Reproduction and Development, Monash University, Melbourne, Australia d Department of Neurobiology, The Weizmann Institute, Rehovot, Israel Received 21 October 2004; revised 13 April 2005; accepted 20 May 2005 The utilization of human embryonic stem cells (hESC) for basic and applied research is hampered by limitations in directing their differ- entiation. Empirical poorly defined methods are currently used to develop cultures enriched for distinct cell types. Here, we report the derivation of neural precursors (NPs) from hESC in a defined culture system that includes the bone morphogenetic protein antagonist noggin. When hESC are cultured as floating aggregates in defined medium and BMP signaling is repressed by noggin, non-neural differentiation is suppressed, and the cell aggregates develop into spheres highly enriched for proliferating NPs. The NPs can differ- entiate into astrocytes, oligodendrocytes, and mature electrophysiolog- ically functional neurons. During prolonged propagation, the differentiation potential of the NPs shifts from neuronal to glial fate. The presented noggin-dependent controlled conversion of hESC into NPs is valuable for the study of human neurogenesis, the development of new drugs, and is an important step towards the potential utilization of hESC in neural transplantation therapy. D 2005 Elsevier Inc. All rights reserved. Introduction Pluripotent self-renewing human embryonic stem cells (hESC) (Reubinoff et al., 2000; Thomson et al., 1998a) are expected to have far-reaching applications in basic research and cell therapy. Derivation of neural precursors (NPs) from hESC may be invaluable to the study of early human neurogenesis, the develop- ment of models of neurodegenerative disorders, screening for neuroprotective and neurotoxic compounds, and cell therapy for neurological conditions. During spontaneous differentiation of hESC, neural cells are formed within a mixture of other cell types (Itskovitz-Eldor et al., 2000; Reubinoff et al., 2000; Schuldiner et al., 2000). While retinoic acid can promote neuralization, the cultures obtained are still relatively heterogeneous (Carpenter et al., 2001; Schuldiner et al., 2001). Highly enriched cultures of NPs were recently developed by selecting NPs from spontaneously differentiating hESC and culturing them under conditions that promote their proliferation (Carpenter et al., 2001; Reubinoff et al., 2001; Zhang et al., 2001). While these approaches may reproducibly facilitate the development of enriched cultures of NPs, they depend on an initial phase of spontaneous disorganized multilineage differ- entiation. Controlled conversion into a homogeneous population of NPs would be a more desirable approach for both basic and applied scientific research. In the mouse system, in line with the default model of neural differentiation (Munoz-Sanjuan and Brivanlou, 2002), undiffer- entiated ES cells will autonomously acquire a neural fate upon removal of signals that prevent neural differentiation. Neuralization occurs following incubation in the absence of serum or added growth factors and is blocked by BMPs (Tropepe et al., 2001; Ying et al., 2003a,b). In Xenopus laevis (Munoz-Sanjuan and Brivanlou, 2002) and in low-density suspension cultures of mouse ES cells, BMP inhibitors such as noggin promote neural differentiation (Tropepe et al., 2001). We have recently reported initial evidences that BMP antagonism may also have a potential role in neuralization of hESC. Noggin treatment of hESC, when cultured on mouse feeders in the presence of serum, induced the appearance of a novel cell type that could give rise to NPs upon modification of the culture system (Pera et al., 2004). However, the culture conditions in these experiments were undefined, and it remains unclear whether the 1044-7431/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2005.05.004 * Corresponding author. Fax: +972 2 6430982. E-mail address: reubinof@md.huji.ac.il (B.E. Reubinoff). Available online on ScienceDirect (www.sciencedirect.com). www.elsevier.com/locate/ymcne YMCNE-01685; No. of pages: 13; 4C: 2, 3, 4, 8, 9 Mol. Cell. Neurosci. 30 (2005) 24 – 36