ARTICLE IN PRESS Regular Article Stromal cell-derived inducing activity does not promote dopaminergic differentiation, but enhances differentiation and proliferation of neural stem cell-derived astrocytes Laurent Roybon, Patrik Brundin, Jia-Yi Li * Neuronal Survival Unit, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, BMC A10, 221 84 Lund, Sweden Received 26 November 2004; revised 19 August 2005; accepted 22 August 2005 Abstract Previous evidence has shown that stromal cell-derived inducing activity (SDIA), produced by the mouse PA6 stromal cell line, promotes dopaminergic differentiation of mouse, monkey and human embryonic stem cells in vitro. To examine whether PA6 stromal cells can enhance the yield of dopaminergic differentiation from neural progenitors, we generated neurospheres from embryonic day 11.5 (E11.5) (midbrain and forebrain) and E14.5 (ventral mesencephalon and cortex) rat embryos and allowed them to differentiate in co-culture with PA6 cells or poly-l- lysine/laminin-coated dishes. We observed that SDIA did not promote dopaminergic differentiation of E11.5 and E14.5 neurospheres but more prominently, enhanced astrocyte differentiation, cell survival and astrocyte proliferation. Our results suggest that PA6 cells do not have a general capacity to promote differentiation into dopaminergic neurons from all types of stem cells, but that they may specifically induce dopaminergic differentiation of highly uncommitted stem cells such as embryonic stem cells. D 2005 Elsevier Inc. All rights reserved. Keywords: PA6 stromal cell; Neural stem cells; Neurosphere; Dopaminergic neurons; Astrocytes Introduction The most striking neuropathological feature of Parkinson’s disease (PD) is the loss of dopaminergic (DAergic) neurons in the substantia nigra (Lang and Obeso, 2004). One promising approach to the treatment of PD is to replace lost DAergic neurons by the transplantation of embryonic nigral tissue (Hagell et al., 2002). However, despite the reported clinical benefit of neural grafting in PD patients (Hagell and Brundin, 2001), only a relatively small number of patients has been transplanted worldwide, mainly due to shortage of suitable donor tissue (Bjorklund et al., 2003). Neural stem cells (NSC) have been considered as a potential source of donor tissue for cell replacement therapy in neurological disorders (Tai and Svendsen, 2004). They are defined as multipotent, undifferentiated cells with a high proliferative capacity (Gage, 2000), and are found in specific regions of the developing and adult mammalian brain (Brustle and McKay, 1996; Johansson et al., 1999; Kuhn et al., 2001; Svendsen et al., 1999). NSC can be cultured as a monolayer on substrate-coated dishes (McKay, 1997) or as suspended aggregates of cells known as neurospheres (Reynolds and Weiss, 1992; Svendsen et al., 1998; Weiss et al., 1996). Neurospheres contain a heterogeneous mixture of both multi- potent stem cells and more committed progenitor cells (Reynolds and Weiss, 1996; Svendsen and Caldwell, 2000) and are usually generated by supplementing the culture medium with mitogens such as epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Developing protocols to promote controlled differentiation of neurosphere- derived cells into neurons has been a major challenge that has given rise to many different experimental strategies (Caldwell et al., 2001). Ventral mesencephalic NSC can be expanded and differentiated into DAergic neurons in monolayer culture conditions using soluble growth factors such as sonic hedgehog (SHH) (Matsuura et al., 2001) and forskolin (Pliego Rivero et al., 1999). Additionally, DAergic differentiation of either NSC or embryonic stem cells (ESC) has also been achieved using 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.08.023 * Corresponding author. Fax: +46 46 2220531. E-mail address: jia-yi.li@med.lu.se (J.-Y. Li). Experimental Neurology xx (2005) xxx – xxx www.elsevier.com/locate/yexnr YEXNR-08952; No. of pages: 8; 4C: 4, 6 + model