casin It has previously been reported

It has previously been reported that differentiation of hESCs without the embryonic body stage led to an increase in the capacity of hESCs to differentiate into osteogenic progenitor casin (Karp et al., 2006). Consistent with these findings, our differentiation culture method resulted in cells with increased mesodermal and endodermal lineage marker gene expression, while the expression of the ectodermal lineage marker, PAX6, was unchanged. These results differ distinctly from embryonic bodies in which the inhibition of NF-κB promotes ectodermal lineage alone. Such discrepancies may stem from differences in culturing conditions including dosing. Yang et al. (2010) used a 20 μM dose of IKKi daily for 6 days, which is significantly higher than the concentration used in this study, 1 μM. This super-physiological concentration of IKKi may be responsible for the significant cell death and morphology changes that Yang et al. (2010) observed in hESCs. During our dosing optimization studies, we initially experienced massive cell death with higher doses, leading us to decide on a 1 μM dose. While IKKi at this low dose not only induces minimal cell death and proliferation impairment, it is sufficient to significantly suppress IKK activity, as demonstrated by a reduction in the S536 phosphorylated form of p65. As such, evaluating the role of NF-κB signaling by directly comparing different culturing conditions warrants closer examination.
Experimental Procedures
Author Contributions
Acknowledgments This work was supported by the UCLA Eli & Edythe Broad Center of Regenerative Medicine and Stem Cell Research Innovation Award and NIH/NIDCR grant R01DE016513. We thank Dr. Jinghua Tang for technical support on cell culture.
Introduction Mesenchymal stem/stromal cells (MSCs) are attractive for different cell-based therapies, from bone regeneration to treatment of autoimmune diseases (Singer and Caplan, 2011). However, tissue regeneration therapies that involve injection/implantation of MSCs have not been fully successful, and strategies that use soluble mediators produced by MSCs or that attract endogenous stem cells and regulate its behavior are appealing, as recruitment, and not only proliferation and differentiation of progenitor cells, is important for effective repair/regeneration (Wei et al., 2013). In this context, it is important to know which factors regulate MSC recruitment. Mobilization and recruitment of MSCs to a bone injury has been correlated with repair (Granero-Molto et al., 2009; Kumar and Ponnazhagan, 2012). Inflammatory mediators can lead to increased MSC migration (Ren et al., 2010; Tondreau et al., 2009), and thus immune cells such as macrophages and natural killer (NK) cells can stimulate MSC recruitment (Almeida et al., 2012; Anton et al., 2012). While monocytes/macrophages can also stimulate MSC differentiation along the osteoblastic lineage (Champagne et al., 2002; Ekstrom et al., 2013), NK cells do not interfere with MSC differentiation capacity (Almeida et al., 2012). This may be of interest as cell differentiation into specific lineages can then be orchestrated by other cues from the microenvironment. Macrophages can recruit MSC by producing the chemokine RANTES (Anton et al., 2012), which is involved in recruitment of MSCs in the degenerated intervertebral disc (Pattappa et al., 2014). However, the chemokines behind NK cell-mediated MSC recruitment are still unknown. NK cells are one of the first immune cell populations to arrive at an injury site (Agaiby and Dyson, 1999), are involved in uterine tissue remodeling in pregnancy (Moffett and Colucci, 2014), may contribute to wound healing (Liippo et al., 2009), and can trigger differentiation of monocytes into osteoclasts (Soderstrom et al., 2010). NK cells are capable of recognizing cells in different stages of the cell cycle (Nolte-\'t Hoen et al., 2006), and their activation by target cells depends on the target cells activating/inhibitory ligands ratio and distribution in the cell membrane (Almeida and Davis, 2006; Endt et al., 2007; Kaplan et al., 2011). Activation of NK cells by different ligands or in different contexts may lead to degranulation of lytic granules, or cytokine or chemokine secretion (Almeida et al., 2011; Fauriat et al., 2010). NK cells produce several chemokines (Fauriat et al., 2010; Robertson, 2002), which can stimulate MSC recruitment (Anton et al., 2012; Ponte et al., 2007).