Compared with D surfaces three dimensional D stem cell

Compared with 2D surfaces, three-dimensional (3D) stem cell culture can facilitate substantially higher yields (Lei and Schaffer, 2013; Serra et al., 2012; Zweigerdt et al., 2011). For example, using a 3D thermoresponsive material under completely chemically defined conditions, we previously achieved 20-fold expansion of hPSCs every 5 days for a 1072-fold increase in yield during the course of a 280-day culture (Lei and Schaffer, 2013). Notably, this thermoresponsive material had a 2D footprint ∼35-fold lower than the equivalent 2D culture platform. In addition, 3D matrices have the potential to be engineered to more closely resemble embryonic tissue (Kraehenbuehl et al., 2011) and can conceivably promote more rapid and higher efficiency differentiation (Adil et al., 2017; Wu et al., 2014). Furthermore, a defined biomaterial offers the possibility of avoiding undefined components, such as Matrigel and/or fetal bovine serum, which are currently used for hPSC differentiation into OPCs (Douvaras et al., 2014; Stacpoole et al., 2013; Wang et al., 2013).
Results
Discussion OPCs are strong candidates for cell therapy (Lebkowski, 2011). This is particularly evident given their ability to rescue p2x receptors function through remyelination in a mouse model of congenital hypomyelination (Wang et al., 2013), promote functional recovery in a rat model of radiation-induced brain trauma (Piao et al., 2015), and yield encouraging initial clinical results for cervical SCI (Priest et al., 2015). Over the past decade, several methods have been established for differentiating human OPCs from hPSCs (Douvaras et al., 2014; Stacpoole et al., 2013). As a step toward their clinical manufacture and translation, the development of a scalable and chemically defined platform for producing OPC would be desirable. Here, we demonstrate that transplantation-quality OPCs can be differentiated from hPSCs using a fully defined and scalable 3D biomaterial system. The NKX2.2-EGFP hESC reporter line described here allowed for optimization of early OPC differentiation. By measuring NKX2.2 expression in real time, we found that synchronous addition of SAG and RA, combined with dual-SMAD inhibition, was sufficient to promote OPC patterning in a PNIPAAm-PEG hydrogel, a robust result validated using three different hPSC lines. Interestingly, while most of the conditions that contained RA yielded similar levels of OLIG2+ and NKX2.2+ cells, our best method for early OPC differentiation in the 3D biomaterial later generated at least 2-fold more O4+ cells than the other conditions tested upon late OPC maturation. This suggests that precise dorsal and ventral commitment of early OPCs can have a profound effect on late-stage commitment. Another factor that enabled rapid OPC generation was our use of OPC maturation medium earlier in the differentiation protocol. Because the relative decrease in OPC marker expression that was observed previously (Douvaras et al., 2014) was seen here after day 14 (Figure S3), we added OPC maturation medium earlier than in other reports (Douvaras et al., 2014; Keirstead et al., 2005; Nistor et al., 2005; Stacpoole et al., 2013). In addition, because MBP expression was observed as early as day 72 using our differentiation protocol (Figure S4) versus day 95 (Douvaras et al., 2014), or even day 140 (Stacpoole et al., 2013; Wang et al., 2013), 3D culture possibly offers the means to enhance and accelerate the generation of oligodendrocytes. OPC differentiation efficiencies approaching ∼90% could obviate the need for cell-purification strategies before the in vitro use of hPSC-derived oligodendrocytes (Najm et al., 2015). Future work may thus explore additional optimization to further enhance maturation efficiency in 3D culture, such as the use of patterning factors that have been suggested to promote efficient oligodendrocyte maturation (Figure S5) (Barratt et al., 2016; Lourenço et al., 2016; Stacpoole et al., 2013).