Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture Yirui Sun, a,b Steven Pollard, a Luciano Conti, c Mauro Toselli, d Gerardo Biella, d Georgina Parkin, e Lionel Willatt, e Anna Falk, a Elena Cattaneo, c and Austin Smith a, ⁎ a Wellcome Trust Centre for Stem Cell Research and Department of Biochemistry, University of Cambridge, Cambridge, UK b The Institute for Stem Cell Research, University of Edinburgh, Edinburgh, UK c Department of Pharmacological Sciences and Centre for Stem Cell Research, University of Milano, Milan, Italy d Institute of Physiological and Pharmacological Sciences, University of Pavia, Pavia, Italy e Cytogenetics Laboratories, Addenbrooke's Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK Received 12 November 2007; revised 15 February 2008; accepted 26 February 2008 Available online 18 March 2008 Stem cell lines that provide a renewable and scaleable supply of central nervous system cell types would constitute an invaluable resource for basic and applied neurobiology. Here we describe the generation and long-term expansion of multiple human foetal neural stem (NS) cell lines in monolayer culture without genetic immortalization. Adherent human NS cells are propagated in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), under which conditions they stably express neural precursor markers and exhibit negligible differentiation into neurons or glia. However, they produce astrocytes, oligodendrocytes, and neurons upon exposure to appro- priate differentiation factors. Single cell cloning demonstrates that human NS cells are tripotent. They retain a diploid karyotype and constant neurogenic capacity after over 100 generations. In contrast to human neurospheres, we observe no requirement for the cytokine leukaemia inhibitory factor (LIF) for continued expansion of adherent human NS cells. Human NS cells can be stably transfected to provide reporter lines and readily imaged in live monolayer cultures, creating the potential for high content genetic and chemical screens. © 2008 Elsevier Inc. All rights reserved. Introduction Cultured neural stem cells are attracting increasing interest from neuroscientists as a powerful tool for basic and applied neurobiol- ogy. In vitro expanded human neural stem cells in principle provide an accessible model system to investigate human neurodevelopment and cell biology. They also offer a renewable resource for neuro- degenerative disease studies and would be suitable for pharmaceu- tical and neurotoxicology screening. In addition, scaleable production of in vitro human neurons from stem cell lines is the first step towards their use in regenerative medicine. Until the late 1990s, the only cell line that could consistently generate human neuronal cells in vitro was the teratocarcinoma derived NTERA-2. This is a transformed aneuploid cell line that requires complicated manipulations to induce differentiation (Andrews, 1984; Pleasure et al., 1992). These limitations led to the exploration of alternative sources and approaches to produce human neurons in vitro. Foetal brain and spinal cord contain pro- liferating neural progenitor cells, and are potential sources for deriving in vitro cell lines. In 1997, Sah et al. established the first immortalized adherent human foetal neural precursor cell line using retrovirally expressed avian v-myc (Sah et al., 1997). Subsequent independent reports used similar strategies (Flax et al., 1998; Villa et al., 2000; De Filippis et al., 2007). Several groups have also explored the possibility of expanding human foetal neural precursors in suspension cultures (Svendsen et al., 1998; Carpenter et al., 1999; Riaz et al., 2002), under which conditions neural precursors form floating aggregates termed neurospheres (Reynolds and Weiss, 1992). However, neurosphere cultures are often accompanied by progressive loss of self-renewal and differentiation capacity (Ostenfeld et al., 2000; Reynolds and Rietze, 2005). In addition, since the cell populations in neurosphere are heterogeneous, it is hard to determine the quantity and identity of neurosphere- forming cells (Suslov et al., 2002; Reynolds and Rietze, 2005; Singec et al., 2006). Other researchers have explored derivation of human neural precursor cells using adherent cultures without genetic immortalization (Palmer et al., 2001; Yan et al., 2007). However, characterization of these monolayer human neural precursors is limited to primary cultures. Their long-term stability and tripotent differentiation capacity have not been demonstrated. We have reported the establishment of clonogenic mouse neural stem (NS) cell lines derived from both ES cells and foetal CNS. www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 38 (2008) 245 – 258 ⁎ Corresponding author. Fax: +44 1223 760241. E-mail address: ags39@cscr.cam.ac.uk (A. Smith). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2008.02.014