Potential candidates contributing to the fate

Potential candidates contributing to the fate determination in the injured spinal cord include bone morphogenetic proteins (BMPs) — in particular BMP2 and BMP4, which strongly promote astroglial differentiation at the expense of oligodendroglial and neuronal differentiation (Bonaguidi et al., 2005; Cheng et al., 2007; Grinspan et al., 2000; Gross et al., 1996; Mabie et al., 1997; Nakashima et al., 2001; Samanta and Kessler, 2004; See et al., 2004). BMPs are upregulated in oligodendrocytes, astrocytes and microglia/macrophages adjacent to a site of spinal cord injury (Ara et al., 2008; Cheng et al., 2007; Matsuura et al., 2008; Miyagi et al., 2012; See and Grinspan, 2009) and contribute to glial scar formation (Fuller et al., 2007; Setoguchi et al., 2001, 2004). Our data indicate that BMP2 and BMP4 expression is upregulated for at least 3days in the injury model used in this study. Our in vitro experiments further show that almost 90% of NPCs differentiate into astrocytes when BMP2/4 is added to NPCs differentiated with BMSC-conditioned medium or control medium, whereas very few SB 225002 express oligodendroglial differentiation marker. Moreover, the BMP antagonist Noggin overexpressed by BMSCs can, effectively prevent the BMP2/4-mediated decrease in oligodendroglial and increase in astroglial differentiation in NPC/BMSC co-cultures. On the transcriptional level, BMPs are known to upregulate the transcriptional regulator Id2, resulting in the inhibition of the pro-oligodendrogenic transcription factors Olig1 and Olig2 (Samanta and Kessler, 2004), necessary for oligodendroglial differentiation. We also found that Id2 gene expression is significantly increased in NPCs 7days after adding BMP2/4 to control medium, whereas the relative expression of Olig1 and Olig2 was increased after incubation of NPCs with BMSC-CM. Since Id2 and Id4 must reach a critical level in the cytoplasm to inhibit oligodendrogenesis (Samanta and Kessler, 2004), we determined the Olig1/Id2 and Olig2/Id2 ratio. BMP2/4 effectively prevented the BMSC-mediated increase in the ratio of Olig1 to Id2 and Olig2 to Id2, accompanying the pro-oligodendrogenic effect of BMSC-CM. Importantly these changes are observed within 3days in vitro. In vivo, BMPs are also upregulated for at least 3days, a time frame sufficient to influence cellular fate. One potential mean to increase oligodendroglial differentiation of grafted progenitor cells might be the neutralization of BMP signaling by BMP antagonists such as Noggin (Groppe et al., 2002, 2003). Overexpression of Noggin, which blocks the binding sites of BMP receptors (Groppe et al., 2003), has been shown to shift NPC differentiation from an astrogenic to an oligodendrogenic phenotype in vitro and in vivo (Mabie et al., 1999; Setoguchi et al., 2004). Our data indicate that co-cultivation of BMSCs overexpressing Noggin (BMSC-Noggin) with NPCs can reverse the BMP-mediated shift in NPC differentiation from an astrogenic to an oligodendrogenic phenotype. Thus, the pro-oligodendrogenic effect of BMSC, which was blocked by BMP2/4 can be reestablished. While some studies have suggested that intracerebroventricular infusion of Noggin or transplantation of noggin-expressing NPCs can enhance the number of Olig2-expressing intrinsic OPCs (Colak et al., 2008) or promote the neuronal and oligodendroglial differentiation of NPCs (Setoguchi et al., 2004), other studies did not find changes in astroglial or oligodendroglial differentiation after grafting of Noggin-expressing NPCs into the injured spinal cord (Enzmann et al., 2005) or after intrathecal administration of Noggin (Xiao et al., 2010). Thus, factors other than BMPs might also contribute to the limited oligodendroglial differentiation of grafted NPCs. Based on the time course of BMP expression after injury, delayed transplantation e.g. at 7days post-injury, when injury-induced BMP-levels have declined, could be an alternative strategy to increase oligodendroglial differentiation. Previous studies have shown that delayed transplantation can be beneficial, at least in terms of graft survival (Hofstetter et al., 2005; Karimi-Abdolrezaee et al., 2006; Parr et al., 2007). However, transplantation itself might result in injury responses including an increase in BMP expression. We therefore chose to graft cells into the acutely injured spinal cord to avoid additional spinal cord damage during delayed cell injections.