It was recently shown that hPSCs can

It was recently shown that hPSCs can be expanded as cell-only dnmt in serum-free suspension culture irrespective of matrix supplementation (Amit et al., 2011; Olmer et al., 2010; Singh et al., 2010), which is mandatory for conventional surface-attached propagation of hPSCs. In contrast to matrix-attached 2D conditions, suspension culture (3D) provides a straightforward strategy for process upscaling, including cell cultivation in stirred tank bioreactors (Couture, 2010). Stirred tank reactors represent a universal, well-established vessel type for the production of recombinant proteins in industrial biotechnology (Carrondo et al., 2012) and allow for cost-effective, multiparametric monitoring and optimization of mammalian cell culture processes (Bulnes-Abundis et al., 2013). Once established, relative linear process upscaling is feasible since reactors from 0.1 to >1.000 l culture scale are available. However, the application of stirred bioreactors to hPSC expansion and their differentiation is still in its infancy. Single cell-based inoculation of suspension cultures establishes a well-controlled starting point at every passage (Zweigerdt et al., 2011). The inoculation density as well as the physical properties of the culture system (such as the reactor design and the stirring speed) can then be used to control formation of PSC aggregates and their subsequent growth (Olmer et al., 2012; Schroeder et al., 2005). Importantly, when utilizing appropriate media such as mTeSR, hPSCs remain pluripotent over multiple passages in aggregate culture (Olmer et al., 2010; Zweigerdt et al., 2011), thus providing the attractive option of directly switching from hPSC expansion to lineage-specific differentiation in a continuous suspension process. Recent work has demonstrated that Wnt pathway modulation by small molecules is an efficient strategy for hPSC cardiomyogenic induction, resulting in ∼60%–80% CMs content in defined media (Gonzalez et al., 2011; Lian et al., 2012; Minami et al., 2012). A common feature of these protocols is the activation of the Wnt pathway at early stages of differentiation by the GSK3 inhibitor CHIR99021 (CHIR) aiming at enhanced mesoderm induction. Following cues from developmental biology, Wnt pathway activity is then inhibited using inhibitors such as IWP (inhibitor of Wnt production) or IWR (inhibitor of Wnt response). This later step aims at specifying cardiac differentiation of the mesoderm-directed cells (Hudson et al., 2012; Lian et al., 2012; Ren et al., 2011; Willems et al., 2011). However, these protocols rely on confluent monolayer cultures limiting straightforward industrial scale production. In this study, we aimed at directly combining hPSC expansion with cardiomyogenic differentiation in suspension culture. Taking advantage of a NKX2.5-GFP reporter line (Elliott et al., 2011), a multiwell screening assay was established to develop Wnt modulator-based CMs differentiation of hPSC aggregates in static suspension culture. By scaling up to rotated Erlenmeyer flasks and ultimately to fully equipped stirred tank bioreactors, we show the robustness of the method, as well as its applicability to dynamic suspension culture. The work provides insights on critical cellular and molecular process parameters and a straightforward strategy for the scalable mass production of CMs at up to 85% purity, which predominantly displayed ventricular-like action potentials (APs). Moreover, bioreactor-derived embryoid bodies (EBs) were directly applicable for the generation of human bioartificial cardiac tissue (BCT) (Kensah et al., 2011), a promising strategy for heart repair and novel in vitro drug discovery and drug safety assays.
Results
Discussion Chemical Wnt pathway modulators were recently applied to direct cardiomyogenic differentiation of hPSCs seeded on matrigel or the synthetic matrix Synthemax (Gonzalez et al., 2011; Lian et al., 2012; Lian et al., 2013). Disregarding upscaling limitations of monolayer cultures, respective matrices might be a prerequisite for these protocols to work. By successful transition to free floating aggregates, we demonstrate that matrices and direct cell-to-substrate contacts are dispensable, stripping away another (costly) layer toward fully defined conditions.