The following are the supplementary

The following are the supplementary data related to this article.
Disclosure of conflict of interest
Acknowledgments We thank Martin Rŭtze for expert help during manuscript revision, Nicole Buchta and Eva-Maria Maurer for technical assistance and the Robert-Mathys Foundation for the donation of β-TCP granules. This project was funded by the Orthopaedic University Hospital Heidelberg and in part by grants from the German Research FoundationRI707/7-1, RI707/8-1, NE1174/8-1 and NE1174/6-1 (IMMUNOBONE).
Introduction Neuronal differentiation of embryonic stem cells (ESCs) has emerged as a potential approach to study and overcome neurodegenerative disorders, such as Alzheimer\'s and Parkinson\'s diseases (Tomaskovic-Crook and Crook, 2011; Nikoletopoulou and Tavernarakis, 2012; Visan et al., 2012). Retinoic nitric oxide (RA), a natural morphogen, is extensively used to induce differentiation of stem cells into a neuronal phenotype, but it is also well known to trigger apoptosis and cell toxicity (Sarkar and Sharma, 2002). On the other hand, viral-mediated approaches have been employed to deliver differentiation factors (Dottori et al., 2011) but they induce striking transcriptional reprogramming and might not be safe for human therapy. 2,4-Dinitrophenol (DNP) is classically known as a mitochondrial uncoupling agent that is toxic at high concentrations (De Felice and Ferreira, 2006). However, recent studies have shown that, at low micromolar concentrations, DNP is neuroprotective. Low-dose DNP protects against central and peripheral neurodegeneration (Maragos et al., 2003; Pandya et al., 2007; da Costa et al., 2010), and blocks the aggregation and neurotoxicity of Alzheimer\'s amyloid-beta peptide (De Felice et al., 2001; De Felice, 2004). In addition, DNP has been shown to promote neuronal differentiation in a neural cell line and neurite outgrowth in primary cultured neurons, an effect that is accompanied by stimulation of cAMP signaling (Wasilewska-Sampaio et al., 2005; Sebollela et al., 2009). Collectively, these studies suggest that DNP could be a useful tool in the development of novel therapeutics against neurodegeneration and brain injury (De Felice et al., 2007). Here, we asked whether DNP would induce neural differentiation in murine ESCs and investigated the mechanisms underlying this effect. We found that DNP triggered neuronal differentiation and cell migration from embryoid bodies, reduced apoptosis and caused an increase in neurite density and length in differentiated cells. We further demonstrate that DNP increases the mitochondrial ATP turnover coupled to O2 consumption and ERK activation, mechanisms likely involved in neuronal differentiation. Results indicate that, at low concentrations, DNP may be a novel, effective and safe drug to integrate disease modeling and cell-based therapies against neurodegenerative diseases.
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Discussion Development of protocols for neuronal differentiation of embryonic stem cells (ESCs) has been a focus of intense research due to their potential to integrate therapeutic approaches to prevent or treat neurodegeneration (Bull and Martin, 2011; López-González and Velasco, 2012). Although a number of molecules and strategies have been proposed to achieve neural differentiation, further optimization of existing protocols is still required to circumvent toxicity and facilitate cell survival and function. In vitro differentiation of ESCs into neurons most frequently employs the natural morphogen retinoic acid (RA), either alone or in combination with other compounds (Dhara and Stice, 2008). However, RA treatment of progenitor cells has the disadvantage of triggering apoptotic cell death and a reduction in cell viability (Sarkar and Sharma, 2002; Huang et al., 2013), prompting the development of novel neurogenic approaches (Wang et al., 2009; Paulsen et al., 2011; Wu et al., 2013). In fact, recently developed protocols attempt to enhance the efficiency of neuronal differentiation by dually targeting Wnt and BMP signaling (Osakada and Takahashi, 2011).