iPSC models are useful INCB024360 in vitro for studying human disease pathogenesis and could serve as a powerful human and allele-specific

tool to evaluate therapeutics. To study the pathology of the C9ORF72 repeat expansion, we isolated fibroblasts from unrelated C9ORF72 ALS patients whose repeat expansion was confirmed by repeat-primed PCR (Renton et al., 2011) and Southern blot analysis (Figures 1A and 1B; for demographic information on all cell lines see Table S1 available online), reprogrammed them to TRA-1-60+ iPSCs (Dimos et al., 2008), and differentiated them to Tuj-1+ iPS-derived neurons (Figure S1A). iPSC lines were generated from fibroblasts reprogramed using Sox2, Oct4, Klf4, and c-Myc encoding vectors (data not shown). All iPSC lines were validated

via strict quality control profiling including expression of pluripotency markers as well as normal karyotyping C646 nmr (data not shown). The iPSN cultures are composed of a heterogeneous neuronal cell population, of which about 30%–40% stained positive for motor neuron marker HB9 (Figure S1B). It is widely known that not only motor neurons, but also cortical neurons, interneurons, and glia are pathologically injured in ALS (Morrison et al., 1998, Kang et al., 2010 and Reis et al., 2011), which is why studies were carried out using a mixed neuronal cell population. Southern blot analysis revealed that the GGGGCC expansion is maintained in all lines after reprogramming and for differentiation from fibroblast to iPSC neurons or astroglia (Figure 1A) with little or minor changes in expansion size, which is likely to be reflected by clonal selection of fibroblasts. No expansion size instability was observed after increasing cell passage numbers in vitro (>50; Figure 1B). Earlier studies have shown that ALS and FTD patients exhibit decreased C9ORF72 RNA levels in patient tissue as measured by real-time PCR (Ciura et al., 2013, DeJesus-Hernandez et al., 2011 and Gijselinck et al., 2012). Therefore, to determine whether

patient fibroblasts and iPSNs exhibit similar in vivo C9ORF72 RNA variant expression patterns, we quantified C9ORF72 RNA levels in C9ORF72 ALS fibroblasts, in iPSNs, and in human CNS regions from multiple unrelated patients (Table S2) using the highly sensitive, probe-based nanostring RNA detection system (probe sequences in Table S3). This method is ideal for screening human tissue due to the lack of any nucleotide amplification step. There are three validated mRNA products transcribed from the C9ORF72 gene, C9ORF72 variant 1, 2, and 3 (NM_145005.5, NM_018325.3, and NM_001256054.1, respectively) with variant 1 and 3 containing open reading frames (ORFs) upstream of the expanded GGGGCC repeat.