Our data related to C orf mRNA

Our data related to C9orf72 mRNA levels conflict with the reports of others, who demonstrate a general reduction in C9orf72 in diseased adenosine receptor (Ciura et al., 2013; Cooper-Knock et al., 2013; Donnelly et al., 2013; Waite et al., 2014; Xi et al., 2013). These discrepancies may stem from the different types of samples (single versus mixed type of cells) employed. Nevertheless, even if our mutant NPCs do not reflect the physiological levels observed in fully mature disease-relevant cells, this model system could help to interpret the role of methylation in disease pathogenesis because it features both an extreme C9 hypermethylation state and an extreme C9 hypomethylation state. To summarize, this study clearly demonstrates how reprogramming excessively hypermethylates the C9 expanded locus, and how the C9 mutation alters C9orf72 variant transcription and processing. In addition, the current work highlights the importance of neural differentiation in the pathogenesis of ALS-FTD and points to the potential role of C9 hypermethylation as a neuroprotective mechanism that attenuates the accumulation of potentially toxic repeat-containing mRNAs in C9 neurons.
Experimental Procedures
Author Contributions
Acknowledgments We thank the family who donated the C9 embryos for hESC line derivation, and the C9 mutation carriers (patients H and M) who made donations of skin for the generation of C9 iPSCs. We would also like to thank Dr. David Zeevi for critical reading of the manuscript and Tamar Golan-Lev for the graphic illustrations. This research was partly supported by the ALS Association initiator grant (grant no. 16-IIP-256, R.E. and S.K.), the Legacy Heritage Biomedical program of the Israel Science Foundation (grant no. 0621/02), Mirsky foundation (S.E.-L.), the Canadian Consortium on Neurodegeneration in Aging and Weston Brain Institute (E.R., M.Z.).
Introduction To fully apply human pluripotent stem cells (hPSCs) to the study of neural development and disease, it is crucial to build efficient neural differentiation protocols with targeted regional identity. The embryoid body (EB) formation and dual-Smad inhibition-based adherent culture (AD) paradigms are the two most frequently applied neural differentiation systems for hPSCs (Chambers et al., 2009; Zhang et al., 2001). The EB formation method models the gastrulation period by suspension of detached hPSCs in the hPSC culture medium followed by the neural medium for neural lineage enrichment. The AD paradigm keeps hPSCs adherent, but triggers neural induction by applying inhibitors of both transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways. With the EB protocol, hPSCs have been efficiently patterned to medial ganglionic eminence (MGE) and lateral ganglionic eminence (LGE) (Li et al., 2009; Liu et al., 2013; Ma et al., 2012). Functional basal forebrain cholinergic neurons and medium spiny γ-aminobutyric acid (GABA) neurons have been efficiently generated from these regional progenitors, which show remarkable therapeutic potentials in correcting behavioral abnormalities resembling those of Alzheimer\'s disease and Huntington disease (Liu et al., 2013; Ma et al., 2012; Yue et al., 2015). hPSCs has also been efficiently differentiated to floor plate (FP) and then midbrain dopamine (DA) neurons through the AD protocol, and the yielded DA neurons hold a promising role in correcting the phenotypes of Parkinson\'s disease (Fasano et al., 2010; Kriks et al., 2011; Steinbeck et al., 2015). Although both EB and AD differentiation paradigms have been reported to be equally efficient in differentiating certain neuronal subtypes, such as spinal motor neurons and GABA interneurons (Chen et al., 2014; Du et al., 2015; Li et al., 2005, 2008; Liu et al., 2013; Maroof et al., 2013; Qu et al., 2014; Wang et al., 2013; Xu et al., 2016), it remains unclear whether both EB and AD neural differentiation protocols are suitable for specifying all types of regional progenitors or whether they have any bias. Here, we systematically compare both protocols and offer a reproducible way to generate various ventral neuroprogenitors and related neuronal subtypes through selection of appropriate differentiation paradigms and signaling inhibitors.