One of the major transduction pathways for

One of the major transduction pathways for apelin signal depends on the interaction with a Gi-protein coupled to the APJ receptor, independently of Ras protein; although dependent on Protein Kinase C (PKC) (Masri et al., 2005). In addition to the adenyl cyclase inhibition pathway, apelin activates ERK pathways through a PTX (pertussis toxin) sensitive Gαi protein, in a PKC-dependent process (Masri et al., 2005). Endothelial cell proliferation control is activated by apelin through two mechanisms: one is ERK-dependent and the other is PI3K-dependent (Masri et al., 2004). Some of these events may be conducive to enhance cell survival. Our current data demonstrated that apelin/APJ exerts its anti-apoptotic effects in BMSCs through ERK and Akt pathways. We showed that inhibition of PI3K with the specific blocker wortmannin or blocking ERK with UO126 effectively reversed the protective effects of apelin-13. These observations are consistent with previous investigations on cardiomyocytes and neuronal order Bestatin from our and other groups (Zeng et al., 2009, 2010; Masri et al., 2004; O\'Donnell et al., 2007). In our experiments, apelin-13 did not stimulate proliferation of BMSCs. The regulatory mechanism for proliferation of BMSCs is not well defined. Our data implies that proliferation of BMSCs is not under direct regulation of apelin/APJ signaling.
Materials and methods
Introduction In most neurological disorders, neurogenesis is insufficient to replenish lost neuronal populations. Endogenous stem cell populations are hindered by limited numbers, variable proliferation in response to disease, and in some cases, differentiation into glia rather than neurons (Barnabe-Heider et al., 2010; Meletis et al., 2008; Yang et al., 2006; Baker et al., 2004). Embryonic stem cells (ESCs) can be differentiated into specific neuronal subtypes and may be useful for cell replacement strategies in the central nervous system (Sonntag et al., 2007). Transplantation of ESC-derived dopaminergic neurons and cholinergic motoneurons (MNs) has been shown to promote partial recovery from Parkinson\'s-like symptoms and spinal cord injury, in rodent models (Roy et al., 2006; Erceg et al., 2010). Heterogeneous populations arising from differentiation of ESCs, however, currently limit the efficacy of such treatments (Gogel et al., 2011). Strategies for controlled differentiation of ESCs and the subsequent enrichment ESC-derived cells types are therefore critical to the development of ESC-based therapies and diagnostic screening tools. Directed differentiation of ESCs into spinal MNs can be achieved following exposure to retinoic acid (RA) and sonic hedgehog (Shh) (Wichterle and Peljto, 2008; Wichterle et al., 2002). During this process, ESCs first differentiate into progenitor motor neurons (pMNs) expressing the basic helix–loop–helix transcription factor Olig2 (Mizuguchi et al., 2001; Novitch et al., 2001). These cells can commit to the MN fate by downregulating Olig2 and expressing the homeodomain (HD) transcription factors Islet 1 (Isl1) and Hb9, also known as Mnx1 (Mizuguchi et al., 2001; Novitch et al., 2001; Arber et al., 1999; Pfaff et al., 1996). Despite optimization, differentiation protocols for pMNs result in a heterogeneous population of cells including other ventral spinal progenitor cells (Wichterle et al., 2002). Hb9+-committed MNs compose only 15–50% of the total culture after differentiation of ESCs (Wichterle and Peljto, 2008; Deshpande et al., 2006). Low-purity cultures give rise to multiple types of spinal interneurons, therefore subsequent enrichment may be necessary (Deshpande et al., 2006). Greater pMN purity can be obtained by fluorescence-activated cell sorting (FACS) of a transgenic ESC line that expresses GFP under the Olig2 gene regulatory elements (GRE) (Xian et al., 2005; Xian and Gottlieb, 2004; Xian and McNichols, 2003). This method, however, requires expensive equipment and must be performed at a centralized facility, risking contamination. Gradient centrifugation can enrich spinal MNs from the mouse embryonic lumbar spinal cord and human ESCs, but has not been optimized for mouse ESC-derived MNs (Wada et al., 2009; Wiese et al., 2010). Transgenic selection may provide a low-cost alternative and can be performed directly in the culture dish. Puromycin resistance through expression of the enzyme puromycin N-acetyl-transferase (PAC) has been shown to allow enrichment of ESC-derived cardiomyocytes and endothelial cells in transgenic lines (Marchetti et al., 2002; Kim and von Recum, 2009; Kolossov et al., 2006; Anderson et al., 2007), but has not been used to enrich specific neural populations.