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  • hexokinase inhibition We next aimed to test the differentiat

    2018-11-12

    We next aimed to test the differentiation capabilities of the EZ spheres. We found that at any passage for all cell lines regardless of cryopreservation, EZ spheres can be placed in specific neural induction media for terminal neuronal or glial cell differentiation using protocols adapted from those reported previously (Aubry et al., 2008; Lee et al., 2010; Li et al., 2005; Nistor et al., 2005; Schneider et al., 2007; Zhang et al., 2001) (Fig. S3). Methods in which EB formation is the initial step in differentiation, EZ spheres were substituted for EBs. Using the EZ sphere technique for both hESCs and iPSCs, we show the generation of various neuronal hexokinase inhibition (Fig. 5A), including dopamine neurons (Fig. 5B), motor neurons (Fig. 5C), striatal neurons (Fig. 5D), neural crest progenitor cells (Fig. 5E), and peripheral sensory neurons (Fig. 5F). Using glial differentiation protocols (Fig. 5G), we were able to generate oligodendrocytes and astrocytes from EZ spheres. Importantly, we have previously shown that neurons derived from EZ spheres are electrophysiologically active (HD iPSC Consortium, 2012). Similarly, EZ sphere derived astrocytes express typical potassium channel properties (Fig. S4). Differentiations presented in Fig. 5 are from passages ranging from 5 to 36 with no variation in differentiation efficiency (data not shown). Previous studies have demonstrated increased neural differentiation following inhibition of TGFβ, activin, and BMP (Chambers et al., 2009). We therefore tested whether this dual SMAD inhibition would increase nestin expression in p3 H9 hESC EZ spheres. However, we found no difference in nestin expression between treated and untreated EZ spheres using both immunocytochemistry and PCR (Fig. S5). Finally, we wanted to directly compare the differentiation efficiency between the traditional EB method and the EZ sphere method. Using H9 hESCs, we performed simultaneous directed differentiation toward tyrosine hydroxylase (TH) positive neurons by exposing EB and EZ spheres to FGF-8 and sonic hedgehog as described previously (Schneider et al., 2007). Importantly, EZ spheres do not generate TH dopamine neurons when cultured only in the presence of EGF and FGF-2 (Fig. 6B). However, treatment of floating EZ spheres with FGF-8 for two weeks, FGF-8 and sonic hedgehog for one week, and maturation of plated cells in growth factor supplemented medium (Fig. S3) generated TH/Tuj1 double positive neurons in equal numbers to those generated using the EB dopamine neuron differentiation method (Figs. 6A, B). In all the differentiations we have attempted thus far using EZ spheres, efficiencies are comparable to standard EB differentiation protocols reported in the literature, they proceed through a similar time line as neural cells derived using traditional EB methods, and display up-regulation of appropriate transcription factors and markers as would be indicative of regional specification for a particular neural cell type. For example, the ventral hindbrain/spinal cord markers ISLET1 and HB9 were detected in EZ sphere cultures undergoing motor neuron differentiation (Fig. 5C). Similarly, prior to the generation of peripherin positive sensory neurons, neural crest markers AP2 and p75 were significantly up-regulated indicating the appropriate lineage restriction process is followed (Fig. 5E). The anterior forebrain markers OTX2 and FOXG1 were highly expressed as early as 7days following transition into the striatal differentiation conditions using 21.8 iPSC EZ spheres (Fig. S6). This is particularly important because 21.8 iPSC EZ spheres did not show expression of OTX2 and FOXG1 while expanded in EGF and FGF-2 (Fig. 3B, Table S2). Finally, during glial differentiation we detected increased expression of progenitor and more mature markers including NG2, PDGFRα, S100, and ALDH1L1 (data not shown). Taken together, these data highlight that EZ spheres expanded in EGF and FGF-2 maintain multipotent neural stem cells responsive to multiple regional cues that generate differentiated cell types in equivalent efficiencies to more complex methods.