br Materials and methods br Results br

Materials and methods
Results
Discussion riPSCs are typically generated and cultured on MEF feeders (Liu et al., 2008). Feeders are sufficient to support rhesus iPSC long-term maintenance. However, this approach is too labor intensive for large-scale culture. Optimization of this culture system would therefore benefit future research. Although the regulation cascade was conserved between species (Han et al., 2007; Khaitovich et al., 2005), the medium which maintains human ESCs and iPSCs undifferentiated state failed to support riPSCs self-renewal. The most likely reason for this is that not all the important signaling pathways to support riPSC self-renewal are activated properly when cultured in human ESC/iPSC media. The new culture system we established maintained the self-renewal of riPSCs and generated monkey iPSCs. Three signaling pathways support pluripotent stem cell self-renewal, including WNT, FGF, and TGF-β/activin/nodal. The bFGF signaling pathway support human ES cell self-renewal by suppressing bone morphogenetic protein (BMP) signals, which promote differentiation into trophoblasts (R. H. Xu et al., 2005). Activin A signaling can maintain stemness by inducing Oct4 and Nanog, and also by inhibiting BMP4 signal (Xiao et al., 2006). CHIR/IWR-1 retains β-catenin in the ML 00253764 hydrochloride but not in the nucleus, and prevents differentiated gene expression through an unknown mechanism (Kim et al., 2013; Zwaka, 2012). Even though both bFGF/Activin A (Xiao et al., 2006), and CHIR/IWR-1(Kim et al., 2013) are well known to individually support hiPSC self-renewal, both systems were required for riPSC self-renewal in the feeder-free culture condition. The canonical Wnt signal pathway has biphasic and stage-specific effects on human and mouse CM differentiation (Cao et al., 2013; Lian et al., 2012; Mignone et al., 2010; Naito et al., 2006; Tran et al., 2009; Yang et al., 2008). GSK-3β inhibitor CHIR99021 and Wnt inhibitor IWR-1 are efficacious to induced human cardiac lineage cell differentiation (Lian et al., 2012; Willems et al., 2011). Previous studies demonstrate that RA signal induces human atrial CMs, whereas ventricular CMs can be differentiated by RA antagonists (Zhang et al., 2011). Consistent with these previous studies on human CM differentiation, we obtained large numbers of pure functional rhesus atrial and ventricular CMs by temporally manipulating Wnt and RA pathways. The emergence of spontaneously contracting riPSC-CMs was earlier than hiPSC-CMs. Rhesus CMs possessed characteristics similar with human CMs in many aspects, including morphology, gene expression and electrophysiological properties. These riPSC-CMs exhibited classical APs, whose shapes and properties were similar to those of human CMs (Fig. S4) (Zhang et al., 2011). Like human CMs, rhesus CMs also possess typical Ca images (Zhang et al., 2011), and their major ion currents, including INa, ICa and Ik are sensitive to canonical cardiac drugs (TTX, nifedipine and E4031). Our results demonstrated that riPSC-CMs have similar characteristics with hiPSC-CMs in gene expression and electrophysiology, and would be an authentic reflection of human physiology in medical research. Rhesus monkeys are important models for medical research owing to their closely relationship with humans (Chong and Murry, 2014; Khaitovich et al., 2005). Transplanting hiPSC-CMs into animals, including rats (Caspi et al., 2007), mice (van Laake et al., 2007), guinea pigs (Shiba et al., 2012), and monkeys (Chong et al., 2014), has been shown to induce arrhythmias. One possible cause for these arrhythmias is differences in species-specific cardiac physiology. Transplanting riPSC-CMs into corresponding monkeys could not only avoid these species differences, but also minimize the immune-response reactions that impede the majority of transplantation research.
Introduction Throughout life, a homeostatic mechanism within the marrow cavity regulates fat and bone tissue formation. Both tissues originate from the same bone marrow progenitor cells; these are known as skeletal stem cells (SSCs), bone marrow-derived multipotent stromal cells, or mesenchymal stem cells (Bianco and Robey, 2004; Caplan, 1991; Friedenstein, 1976, 1968; Friedenstein et al., 1966; Owen and Friedenstein, 1988). SSCs are multipotent stromal cells that can differentiate into adipocytes, osteoblasts, or chondrocytes in response to various microenvironmental stimuli, including growth factors, cytokines, and epigenetic regulators (Beresford et al., 1992; Gimble and Nuttall, 2004; Gimble et al., 1996). An imbalance between osteogenic and adipogenic lineage commitment and differentiation has been implicated as a cause of age-related impairment of bone formation. Thus, some therapeutic interventions have been proposed that aim to enhance bone mass by targeting SSCs and improving their functions (Yokota et al., 2002, 2003).