Our work extends these efforts

Our work extends these efforts by directly targeting transgenes to inducible, endogenous loci using the efficient and highly specific CRISPR/Cas9 genome-engineering technology (Maeder and Gersbach, 2016; Jinek et al., 2012). In this manner, our strategy forgoes limitations associated with predicting regulatory features in a genetic locus such as distal enhancers. In addition, this approach abrogates the need to consider limitations on packaging efficiency, as the entire regulatory region need not be packaged in a gene delivery vector. Moreover, by performing targeted integration, this strategy absolves concerns associated with random TIC10 manufacturer of provirus within the host genome. By performing targeted integration to the Ccl2 locus, we rewired the transcriptional circuitry associated with inflammatory signaling in iPSCs. Additional strategies for coupling input/output relationships in cell populations are rapidly emerging in synthetic biology. Continued development of toggle switches (Gardner et al., 2000; Greber et al., 2008; Kobayashi et al., 2004; Kramer et al., 2004), microRNA classifiers (Wroblewska et al., 2015; Xie et al., 2011), and synthetic transcription regulators (Liu et al., 2001; Perez-Pinera et al., 2013a, 2013b; Qi et al., 2013) will facilitate the generation of complex circuits capable of integrating multi-input cues and may enable a cell to discern and respond specifically to not only varying degrees of inflammation, but also a particular type of inflammation. Furthermore, the autoregulated nature of this approach may allow for therapeutic delivery during early, possibly pre-symptomatic stages of diseases, effectively providing the potential for biologic cell-based vaccines for autoimmune diseases. The customization of intrinsic cellular signaling pathways in therapeutic stem cell populations, as demonstrated in this work, opens innovative possibilities for safer and more effective treatments applicable to a wide variety of diseases.
Experimental Procedures
Author Contributions
Acknowledgments The authors would like to thank Dr. David Pisetsky for important discussions on this project and Sameer Chervu for technical assistance. Supported by the Nancy Taylor Foundation for Chronic Diseases, the Arthritis Foundation, NIH grants AR061042, AR50245, AR48852, AG15768, AR48182, AR067467, AR065956, OD008586, NSF CAREER AwardCBET-1151035, and the Collaborative Research Center of the AO Foundation, Davos, Switzerland.
Introduction Mesenchymal stem/stromal cell (MSC)-based therapeutic intervention has become an emerging strategy for immune modulation, and therefore MSCs have been exploited in a variety of clinical trials for immune-mediated disorders, including autoimmune diseases. Although the exact mechanisms underlying the immunomodulatory functions of MSCs remain largely unknown, MSCs have shown suppressive effects on many types of immune cells in vitro and in vivo. For example, it has been demonstrated that MSCs directly suppress T cell activation/proliferation and induce T cell apoptosis by expressing nitric oxide (NO), indoleamine 2,3, dioxygenase (IDO), programmed death ligand 1 (PD-L1), or Fas ligand (Abdi et al., 2008; Akiyama et al., 2012; Jurewicz et al., 2010; Lee et al., 2011; Lenardo et al., 1999; Meisel et al., 2004; Sato et al., 2007; Wei et al., 2013). Also, MSCs have been shown to affect differentiation, maturation, and function of antigen-presenting cells (APCs), including dendritic cells (DCs) and macrophages, which results in conversion of APCs into a suppressive or tolerogenic phenotype (Aldinucci et al., 2010; Beyth et al., 2005; Chiesa et al., 2011; Jiang et al., 2005; Kronsteiner et al., 2011; Liu et al., 2013; Spaggiari et al., 2009; Zhang et al., 2004, 2009). Although MSC therapies are safe compared with embryonic stem cells or induced pluripotent stem cells, which have tumorigenic potential, there are still concerns that MSCs might trigger tumorigenicity, allo-immune responses, and pulmonary embolism in a clinical setting (Ankrum et al., 2014; Barkholt et al., 2013; Boltze et al., 2015; Heslop et al., 2015; Isakova et al., 2014; Jung et al., 2013). In line with these clinical findings, we and others observed that intravenous administration of MSCs caused embolism and death in several mice (Furlani et al., 2009; Lee et al., 2009b; Tatsumi et al., 2013). Therefore, the long-term safety of MSC administration in human patients requires further investigation.