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    • In our static wells the

    2018-10-31

    In our static 12 wells, the average aggregate diameter was ∼210 μm, representing a relatively large spread of ∼100–400 μm in each well, typical of random aggregation. However, at 7.5 μM CHIR, glycogen synthase kinase 3 of all shapes and sizes differentiated into contracting, GFP+ EBs, whereas, at suboptimal CHIR concentrations, GFP expression was reduced or failed in respective wells, again without apparent aggregate/EBs size correlation. In stirred Erlenmeyer Flasks, the mean aggregate diameter was ∼400 μm (∼300–600 μm), and in bioreactors, at C-perfusion, it was ∼450 μm (∼350–600 μm), representing about double the mean diameter in 12 wells. These culture-dependent disparities were still compatible with efficient CM formation applying the same preoptimized differentiation protocol. In contrast, following batch expansion, cardiomyogenesis completely failed in repeated bioreactor runs, although the mean aggregate diameter of ∼300 μm (∼150–450 μm) was within the range of successful conditions. In the bioreactor setup the spatiotemporal pattern of aggregate development was the major determinant of subsequent differentiation results. We and others have shown that the pattern of PSC aggregation and further sphere development can be controlled well in stirred bioreactors by means of the inoculation density, the impeller design, and the stirring speed (Hunt et al., 2014; Olmer et al., 2012; Schroeder et al., 2005), including the on/off patterns applied in this study. In addition to the feeding-dependent differences of the culture milieu, the striking distinction was the continuous growth of initially small aggregates at batch, whereas at C-perfusion relative large CPAs were formed at 24 hr with a minor increase in size thereafter. Although global profiling revealed rather uniform gene expression patterns typical of pluripotent cells under both expansion conditions, expression of the key morphogen BMP2 was highly upregulated in C-perfusion cultures. Modest BMP2 and BMP4 expression is suggested to act as an endogenous prodifferentiation signal in hESC, although these levels are insufficient to promote differentiation at pluripotent culture conditions (Teo et al., 2012). However, upon differentiation, the dominant role of BMP signaling on posterior primitive streak and subsequently on mesoderm formation in hPSCs was recently underscored (Loh et al., 2014). The upregulation of BMP2 as well as CDX4 (involved in anteroposterior axis specification regulated by Wnt activity; Hikasa et al., 2010) in CPAs might suggest the priming of these cells for mesoderm differentiation ahead of CHIR supplementation. Loh et al. (2014) further noted the necessity to neutralize endogenous BMP to eliminate mesoderm induction of hPSCs. The upregulation of BMPER (BMP endothelial cell precursor derived regulator), an established antagonist of BMP2, BMP4, and BMP6 (Moser et al., 2003), in BAs (bottom panel in Figure 3C) might act as a neutralizing signal and, at least in part, explain the entire lack of cardiomyogenesis upon differentiation of these cells. The control of cardiac differentiation by BMP signaling is well established (Laflamme and Murry, 2011). However, the role of autocrine/paracrine hPSC-derived BMP agonists and antagonists in modulating WNT pathway controlled differentiation is not well studied. Although future investigations on this are necessary, our data indicate the importance of this interplay. We further found that the expression of 6 of 13 tested subtypes of the major metallothionein isoforms 1 and 2 (MT1, MT2) was upregulated in BAs. Since MTs bind to metal ions, a role of these proteins in hematopoietic cell proliferation and differentiation was suggested, but regulation of MTs expression by numerous stimuli, including oxidative stress (Takahashi, 2012), has been described. In mouse ESCs, upregulation of MT1 was discovered after addition of the p38 mitogen-activated protein kinase inhibitor PD169316 at LIF starvation-induced stress conditions (Duval et al., 2006). Overexpression of MT1 was notably sufficient to protect mESC from differentiation-induced apoptosis. To date, little is known about the functional role of MT in human PSCs, but we hypothesize that the feeding-induced fluctuation of the culture environment at batch conditions induces MT expression, as compared with more homogeneous conditions at C-perfusion. Although further investigation is required, metallothionein might thus present an interesting marker of stress response in hPSC culture. In this context, it is noteworthy that the stress-related genes HSPA1A and HSPA1B (HSP70 protein) were both found to be ∼2-fold upregulated in BAs compared with CPAs (data not shown).