br Experimental Procedures br Author

Experimental Procedures
Author Contributions
Acknowledgments The research leading to these results has received funding from the European Community’s Seventh Framework Programme under grant agreement n.602300 (SYBIL) to A.V., PRIN Project 20102M7T8X_003 to A.V., Ricerca Finalizzata from Ministero della salute RF-2009-1499,542 to A.V., Programma Nazionale per la Ricerca-Consiglio Nazionale delle Ricerche “Aging Program 2012-2014” to A.V. and P.V., PE-2011-02347329 to P.V., Lombardia Region - National Research Council of Italy (16/7/2012) Project “MbMM - Basic methodologies for innovation in the diagnosis and therapy of multi factorial diseases” (signed 25/7/2013) to P.V., Telethon (TIGET grant D2) EU (FP7 601958 SUPERSIST, ERC Advanced Grant 249845 TARGETINGGENETHERAPY) and the Italian Ministry of Health to L.N. F. Ficara was supported by a Marie Curie International Reintegration Grant (grant number PIRG6-GA-2009-256452). We thank Barbara Tondelli for the generation of the oc/oc mouse strain in the Ly5.1 genetic background.
Introduction Brown adipocytes (BAs) have unique roles in Dig-11-utp expenditure and non-shivering thermogenesis, which is contrary to white adipocytes (WAs) that store excess energy obtained from food in the form of lipids (Gesta et al., 2007; Chechi et al., 2013). The BAs specifically express uncoupling protein 1 (UCP1) at the mitochondrial inner membrane to dissipate the electrochemical proton gradient as heat, playing a pivotal role in regulation of body temperature, energy balance, and adiposity. In rodents, BAs prevent diet-induced obesity, insulin resistance, and type II diabetes (Kontani et al., 2005). Until recently, however, the significance of BAs in human remained controversial, because BAs are rarely detectable in the adult human. Since 2009, positron emission tomography (PET) using 18F-fluorodeoxyglocose (FDG) has clearly visualized metabolically active brown adipose tissue (BAT) in supraclavicular, mediastinal, paravertebral, and perirenal regions characterized by robust uptake of 18F-FDG upon acute cold exposure (Saito et al., 2009; van Marken Lichtenbelt et al., 2009; Cypess et al., 2009). Interestingly, the BA activity considerably varies from person to person and correlates inversely with age, fasting plasma glucose levels, and BMI (Saito et al., 2009; Ouellet et al., 2011, 2012). These findings strongly suggest the importance of BAs in physiological regulation of the whole-body energy balance and body fat accumulation, as well as the pathophysiology of obesity and metabolic diseases in humans. If human BAs with high energy expenditure activity can be artificially generated, the technology may greatly facilitate basic as well as applied research of these BAs. Especially, transplantation of such BAs may provide a regenerative therapy to control metabolic diseases, including diabetes mellitus. Yamanaka’s group demonstrated that mammalian somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) with embryonic-stem-cell-like characteristics by transducing genes for transcription factors, Octamer-binding transcription factor 3/4 (OCT4), SOX2, KLF-4, and C-MYC (so called reprogramming factors; Takahashi and Yamanaka, 2006; Takahashi et al., 2007), strongly suggesting that a small number of key transcription factors may globally change epigenetic programming in somatic cells to convert their fates (Meissner, 2010; Papp and Plath, 2011). More recently, somatic cells were redirected into another differentiated cell lineage, without passing an intermediate pluripotent stage, by transducing a set of transcription factors that play crucial regulatory roles in the differentiation of the destination cell; direct conversion, or direct reprogramming, of murine fibroblasts into cardiomyocytes (Ieda et al., 2010; Inagawa et al., 2012; Inagawa and Ieda, 2013), neurons (Kim et al., 2011, 2012; Caiazzo et al., 2011; Han et al., 2012), chondrocytes (Hiramatsu et al., 2011), and hepatocytes (Sekiya and Suzuki, 2011; Huang et al., 2011), as well as of human fibroblasts into cardiomyocytes (Wada et al., 2013; Nam et al., 2013), neurons (Pang et al., 2011; Caiazzo et al., 2011; Kim et al., 2012), and hematopoietic cells (Szabo et al., 2010) have been reported. Although the efficiencies of direct conversion were generally low (0.005%–30% of fibroblasts were successfully converted into the cells of interest; Thier et al., 2012; Sekiya and Suzuki, 2011; Pang et al., 2011; Nam et al., 2013; Kim et al., 2011, 2012; Inagawa et al., 2012; Inagawa and Ieda, 2013; Ieda et al., 2010; Huang et al., 2011; Han et al., 2012; Caiazzo et al., 2011), these technologies may be quite valuable for generation of the desired cells that could be used in basic research as well as in regenerative therapy for various human disorders. We have recently succeeded in directly reprogramming human fibroblasts into osteoblasts that massively produced bone matrix and contributed to bone regeneration with an efficiency as high as 80% (Yamamoto et al., 2015).