In human development OPCs are characterized by

In human development, OPCs are characterized by PDGFRα and NG2 expression, followed by expression of O4 (Jakovcevski et al., 2009). Under our culture conditions, by day 75, most of the O4+ cells had lost PDGFRα, but retained NG2 expression. At this stage, we did not observe any residual pluripotent cells in culture. Our study differs from recent work in that our iPSC lines were derived from PPMS patients and the cells used for in vivo transplantation were sorted using the late-OPC marker O4 to maximally restrict the differentiation potential. Despite these differences, the PPMS-derived, O4+-sorted OPCs exhibited a similar engraftment efficiency, mitotic fraction, and proportion of host ensheathed fk 506 while generating fewer GFAP+ astrocytes compared with the unsorted iPSC-derived OPCs reported previously. Taken together, our data suggest that PPMS-derived OPCs performed in vivo at least as efficiently as healthy iPSC-derived cells (Wang et al., 2013). iPSC technology is also emerging as a tool for developing new drugs and gaining insight into disease pathogenesis (Han et al., 2011). Our differentiation protocol will aid the development of high-throughput in vitro screens for compounds that promote myelination (Lee et al., 2013). Furthermore, the PPMS iPSC lines described here provide an additional resource for investigating the process of neurodegeneration in MS. Future studies comparing PPMS iPSCs and appropriate healthy control iPSC lines will be needed to shed light on the potential intrinsic differences among patient-derived oligodendrocytes.
Experimental Procedures
Acknowledgments
Introduction Cardiomyocytes (CMs) derived from human pluripotent stem cells (hPSCs) have been found in preclinical studies to prevent the progression of heart failure and function as a biological pacemaker, and therefore are a promising cell source for regenerative medicine to treat cardiovascular diseases (Burridge et al., 2012; Maher and Xu, 2013; Mummery et al., 2012). Extensive engraftment of hPSC-CMs and electromechanical coupling of these cells with the host have been demonstrated in a nonhuman primate model (Chong et al., 2014). An area of great interest to the field of stem cell research is engineering tissue constructs from hPSC-CMs, with the aim of providing better transplantable constructs for regenerative cardiac therapy as well as in vitro models to study human cardiac development, health, and disease. Many current approaches often require a great deal of effort to prepare enriched CMs from differentiation cultures; for example, CMs can be enriched by a mitochondrial dye (Hattori et al., 2010) or metabolic selection (Tohyama et al., 2013) and then aggregated, or by fluorescence-activated cell sorting based on surface markers for the generation of tissue patches (Zhang et al., 2013). Genetically modified hPSCs have also been used to select cardiac progenitors or CMs for the production of tissue-engineered cardiac constructs (Emmert et al., 2013; Thavandiran et al., 2013). Several strategies to generate tissue-engineered cardiac constructs have been considered, including the self-assembly of 3D cell aggregates. Such aggregates offer several advantages and can be easily generated by forced aggregation and maintained in a rotary orbital suspension culture (Kinney et al., 2011). Microscale technologies allow for the generation of size-controlled 3D multicellular aggregates (Khademhosseini et al., 2006) that can promote cell-cell and cell-matrix interactions analogous to those observed among cells in vivo, which cannot be achieved in traditional 2D cultures. Furthermore, in contrast to macrotissue constructs, microtissue constructs can obviate limitations of oxygen and nutrient transport, do not require additional matrix or scaffold materials, and are suitable for scale-up suspension production, and thus represent a robust method for cardiac tissue engineering (Kinney et al., 2014). Therefore, we produced and characterized scaffold-free 3D cardiospheres from 2D differentiation cultures of hPSCs using microscale technologies. The combined technique of forced aggregation and 3D suspension culture is capable of robustly and rapidly enriching CMs from heterogeneous differentiation cultures, and also promotes enhanced structural maturation of CMs compared with parallel 2D cultures.