supplementary materials for€¦ · sareen et al, page 6 fig. s4. rna-seq analysis of snp rs1075766...

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Supplementary Materials for

Targeting RNA Foci in iPSC-Derived Motor Neurons from ALS Patients with a C9ORF72 Repeat Expansion

Dhruv Sareen, Jacqueline G. O’Rourke, Pratap Meera, A. K. M. G. Muhammad,

Sharday Grant, Megan Simpkinson, Shaughn Bell, Sharon Carmona, Loren Ornelas, Anais Sahabian, Tania Gendron, Leonard Petrucelli, Michael Baughn, John Ravits,

Matthew B. Harms, Frank Rigo, C. Frank Bennett, Thomas S. Otis, Clive N. Svendsen, Robert H. Baloh*

*Corresponding author. E-mail: [email protected]

Published 23 October 2013, Sci. Transl. Med. 5, 208ra149 (2013)

DOI: 10.1126/scitranslmed.3007529

The PDF file includes:

Fig. S1. Characterization of C9ORF72 patient and control iPSCs. Fig. S2. Confirmation of the absence of exogenous pluripotency genes in C9ORF72 patient iPSCs and generation of motor neuron precursors. Fig. S3. Generation of control subject motor neuron cultures from iPSCs. Fig. S4. RNA-seq analysis of SNP rs1075766 for identification of wild-type and expansion alleles, and differential C9ORF72 transcript analysis from Ref-seq and Ensembl annotations. Fig. S5. C9ORF72 protein and antibody characterization. Fig. S6. 5′RACE and qRT-PCR analysis of C9ORF72 in human ALS patient spinal cords. Fig. S7. RNA foci quantitation in iPSC-derived motor neuron cultures from individual patients. Fig. S8. Characterization of binding of GGGGCC RNA foci to known hnRNPs and ALS-related factors in C9-ALS motor neuron cultures by confocal imaging. Fig. S9. C9RANT and p62 inclusions were not observed in C9-ALS motor neuron cultures. Fig. S10. Summary of RNA-seq analysis in iPSC-derived motor neurons from C9-ALS patients versus controls. Fig. S11. Hierarchical clustering analysis of RNA-seq data in iPSC-derived motor neurons from C9-ALS patients versus controls. Fig. S12. Electrophysiological properties of control and C9-ALS patient–derived motor neurons separated by individual subject. Fig. S13. Absence of any ASO toxicity in motor neuron cultures and reversal of transcription profiles by RNA-seq in iPSC-derived motor neurons.

Table S1. Clinical information on subjects with C9ORF72 hexanucleotide expansions and controls used for iPSC lines in this study. Table S2. Functional pathway analysis of differentially expressed genes in iPSC-derived motor neuron cultures from C9-ALS patients versus controls.

Sareen et al, Page 1

Fig. S1: Characterization of C9ORF72 patient and control iPSCs. (A) Generation of feeder-free non-integrating unaffected control and C9ORF72 repeat expansion ALS iPSC lines. Four healthy control and four C9ORF72 repeat expansion ALS patient fibroblasts were reprogrammed to iPSCs using episomal reprogramming plasmids. All iPSC lines were maintained using feeder-free methods on Matrigel and mTeSR1 defined media. Bright-field images of the reprogrammed iPSC colonies lines maintained in Matrigel/mTeSR1 show high nuclear-to-cytoplasmic ratio, typical of standard pluripotent stem cells (hESCs and hiPSCs). Successfully reprogrammed iPSC lines show positive staining for alkaline phosphatase. (B) Flow cytometric analysis with pluripotency marker stains shows that the control and C9-ALS iPSC cells routinely maintain greater than 80% SSEA4 and OCT3/4 double positive population. (C) Gene-chip and bioinformatics based PluriTest characterization of new C9-ALS iPSC lines. The novelty score is a score based on well-characterized pluripotent samples in the stem-cell model matrix. Samples


are color coded, green (pluripotent) and red (not pluripotent). Red samples are more dissimilar to the pluripotent samples in the model matrix than other pluripotent samples. The Pluripotency score gives an indication if a sample contains a pluripotent signature, but not necessarily if the cell preparation is a normal, bona-fide hESC or iPSC. Chart combines pluripotency score on y-axis and novelty score on x-axis. The red and blue background hint to the empirical distribution of the pluripotent (red) and non-pluripotent samples (blue) in Müller FJ et al. test data set. Hierarchical Clustering of the ALS and Control iPSCs based on PluriTest Gene Expression profile. (D) Spontaneous embryoid-body (EB) formation assay on C9-ALS iPSCs. Spontaneous in vitro EB differentiation of C9-ALS iPSCs and germ-layer specific gene expression analysis confirms that these cells are pluripotent and capable of differentiating into the three germ layers. This is determined by positive expression for markers of ectoderm (MSX1 and PAX6), mesoderm (HAND1and MSX1), and endoderm (CTNNB1) at 14 & 28 days post-EB formation. Pluripotency gene (TDGF) expression decreases as EB differentiation progresses from iPSC stage. GAPDH is a housekeeping control gene.


Fig. S2. Confirmation of the absence of exogenous pluripotency genes in C9ORF72 patient iPSCs and generation of motor neuron precursors. (A) Lack of exogenous plasmid gene expression in most C9-ALS iPSCs. Quantitative RT–PCR analyses of OCT4, SOX2, KLF4, LIN28, and L-MYC, genes relative to an H9 hESC line. “CDS” indicates that primers designed for the coding sequence measure expression of the total endogenous gene expression only, whereas “Pla” indicates that primers for the transgene expression are not detected. Fibroblasts transfected with the plasmids were collected after 6 days as a positive control to detect plasmid-based gene expression. Vertical solid black lines on the graph demarcates expression changes for each reprogramming factor gene and vertical dotted black lines separates normalized expression levels between “CDS” and “Pla”-specific primers for that gene. (B) Absence of episomal plasmid gene expression in C9-iPSCs. OriP/EBNA1 reprogramming plasmids


replicate extra-chromosomally once per cell cyle. Expression of EBNA1, a gene present on all reprogramming plasmids was monitored over multiple passages. C9-ALS and control iPSCs eliminated the plasmids post-reprogramming, confirming their integration-free status. C, Schematic depicting our protocol for production of motor neuron precursors and mature motor neurons from healthy control and ALS C9-iPSCs. Timeline is in DIV (days in vitro) post-iPSC stage. (D) Motor neuron precursors can be generated with equal efficiencies from CTR or C9-ALS iPSCs using our protocol as shown by Hb9 and Olig2 immunostaining.


Fig. S3. Generation of control subject motor neuron cultures from iPSCs. (A) Efficient production of mature motor neurons from healthy control and ALS C9-iPSCs, as demonstrated by SMI32+ and ChAT+ immunostaining. TuJ1 is pan-neuronal marker utilized for normalization for total production of neurons. (B) Graphs showing consistency in cellular composition for all cell lines (4 control subjects and 4 C9-ALS patients) SMI32+ motor neurons and TuJ1+ neurons over 4 and 7 weeks of differentiation. Data are represented as mean ± SEM from n=3 independent experiments.


Fig. S4. RNA-seq analysis of SNP rs1075766 for identification of wild-type and expansion alleles, and differential C9ORF72 transcript analysis from Ref-seq and Ensembl annotations. (A) Sequence reads in exon 2 from RNA-seq of motor neuron cultures from C9-ALS lines 29i and 30i (antisense strand shown), the two lines which carry SNP rs1075766. These data indicate a roughly equal number of reads coming from the wild-type (A) and expanded (G) alleles. (B) Differential transcript analysis of RNA-seq data between control and C9-ALS derived motor neuron cultures. The various annotated transcripts (Ensembl build 71 above, Ref-seq build 5-10-13 below) are shown with graphical comparison of transcript utilization prediction shown below. An estimate of individual transcript utilization was generated via alignments that are contained entirely within the exon of a specific isoform or was aligned to a splice junction unique to that isoform, and represented as RPKM (for algorithm see http://www.partek.com/Tutorials/microarray/User_Guides/RNASEQ.pdf). No differences were observed between control derived motor neuron cultures (n=4) and C9-ALS derived motor neuron cultures (n=4).


Fig. S5. C9ORF72 protein and antibody characterization. (A) Densitometry quantitation of C9ORF72 protein levels (isoform 1, ~50kDa and isoform 2, ~29kDa) detected in the membrane fraction of iPSC-derived motor neuron cultures in Fig. 2C. Protein levels were normalized to the membrane fraction marker EGFR. C9ORF72 protein levels in controls (CTR; n=3 – lines 00i, 03i, 83i) were not different from C9-ALS patients (ALS; n=3 – lines 28i, 29i, 52i). n.s. – not significant. (B) Western analysis of C9ORF72 protein to prove C9ORF72 antibody specificity is shown by expression in the membrane fraction of a motor neuron cultures (30iC9-ALS) that were treated with either control ASO (scramble) or C9ORF72 ASO (ASO816). ASO816 treated cultures show knockdown of C9ORF72 isoforms 1 and 2 in the MN cultures. Calreticulin is a membrane fraction protein showing equal protein loading in scramble and ASO samples.


Fig. S6. 5′RACE and qRT-PCR analysis of C9ORF72 in human ALS patient spinal cords. (A) Alignments of sequences obtained by 5′RACE on spinal cord from a normal control, sporadic ALS patient, and a patient carrying the C9ORF72 expansion (C9-ALS). Utilization of upstream exons 1a and 1b were similar to that observed in iPSC-derived motor neurons from C9ORF72 patients. Neither of the patients carried SNP rs1075766 to distinguish the wild-type vs. mutant allele. (B) Quantitative RT-PCR for mRNA expression in the post-mortem spinal cord tissue from two control (CTR), two sporadic ALS, and two C9ORF72-ALS (C9-ALS) using RPL13A as the normalizing housekeeping. While there was wide variation, overall C9ORF72 mRNA levels were not significantly diminished in C9-ALS patients.


Fig. S7. RNA foci quantitation in iPSC-derived motor neuron cultures from individual patients. Graph shows the distribution of the number of cells with nuclear foci versus the number of nuclear foci per cell separated for individual cell lines. Lines 28iC9-ALS and 50iC9-ALS (which harbor ~800 repeats) showed the most cells with foci, and many cells with high numbers of foci per cell. Line 30iC9-ALS (which harbors ~70 repeats) showed the fewer cells with nuclear foci, and no cells were observed with more than 2 foci in the nucleus.


Fig. S8. Characterization of binding of GGGGCC RNA foci to known hnRNPs and ALS-related factors in C9-ALS motor neuron cultures by confocal imaging. (A and B) Representative co-staining of GGGGCC RNA FISH with hnRNPA2/B1 in (A) shows co-localization of GGGGCC foci with hnRNPA2/B1 occasionally (14.4% ± 5.2%) in the nuclei of C9-ALS motor neurons, while more frequently in (B) hnRNPA2/B1 nuclear or cytoplasmic co-localization was not observed. (C-E) Representative images showing that frequently nuclear or cytoplasmic GGGGCC RNA foci were not observed to co-localize with (C) hnRNPA3; (D) FUS; or (E) TDP-43. White arrows point to foci where RNA foci were detected in the nuclei. All the images are in same focal plane and also shown in the adjacent panels at 100% magnification are the y-z and x-z axes where the RNA foci were observed. (F) Quantification table for percent overlap in nuclear or cytoplasmic co-staining of GGGGCC RNA FISH with Pur-α, hnRNPA1, hnRNPA2/B1, hnRNPA3, FUS and TDP-43. RNA foci co-staining were scored for four patient C9-ALS lines (28i, 29i, 30i, 52i) in multiple confocal images (~10-12 per patient cell line) and multiple nuclei with foci (60-110 GGGGCC RNA foci). Data in are represented as mean ± SEM from n=3 independent experiments.


Fig. S9. C9RANT and p62 inclusions were not observed in C9-ALS motor neuron cultures. (A) Filter retardation assay analyzing the detergent soluble (S) and detergent insoluble (I) fractions of CTR (14i and 83i) and C9-ALS MN (28i and 52i) cultures for insoluble C9ORF72 RAN proteins (Blue circles). Human post-mortem brain tissue from CTR and C9-ALS was fractionated as previously reported (Ash et. al, Neuron 2013) and used as a positive control for the C9-RANT antibody (red circles). (B) EZ spheres from Huntington’s disease patients or controls expressing both unexpanded (28Q) and expanded (180Q) Huntington protein (HTT) were lysed and separated into detergent soluble and insoluble at the same time as the iPSC MNs and used as a positive control for the fractionation (green circles). As expected, HTT expressing 180 polyQs forms insolubles aggregates detected with anti-HTT (EM48). (C) Quantification of p62 inclusion positive cells (expressed as % of total cells) in CTR and C9-ALS motor neuron cultures using Image Express Micro high-content imager showed no significant difference in inclusion positive cells in controls (n=4, 00i, 03i, 14i, 83i) vs. C9-ALS (n=4, 28i, 29i, 30i, 52i) MN cultures. Greater than 100 sites at 20x magnification were acquired per well for positive cell scoring. Data are represented as mean ± SEM from n=3 independent experiments.


Fig. S10. Summary of RNA-seq analysis in iPSC-derived motor neurons from C9-ALS patients versus controls. (A) Volcano plot showing upregulated (blue) and downregulated (red) in C9-ALS motor neuron cultures (n=4, 28i, 29i, 30i, 52i) vs. controls (n=4, 00i, 03i, 14i, 83i). P-value is plotted on the y-axis, with fold change on the x-axis. A total of 68 genes showed differential expression with a p-value<0.05, and fold change greater than 2. Figure 3f shows the 20 most highly up and downregulated genes. (B) Average RPKM value for genes mutated in familial forms of ALS. No difference was observed in the expression of any of the genes listed, including SOD1, TDP-43, or FUS.


Fig. S11. Hierarchical clustering analysis of RNA-seq data in iPSC-derived motor neurons from C9-ALS patients versus controls. Differential whole gene expression and splicing analysis was performed via the Partek software package using RNA-seq data sets from four controls and four C9-ALS patient derived motor neuron cultures. After removal of genes with low level expression, hierarchical cluster analysis was performed to determine if the control or C9-ALS samples would cluster together based on either differential gene expression, or differential splicing. (A) As shown in the dendrogram on the left, the clustering algorithm did not group the C9-ALS and control samples based on genes predicted to be alternatively spliced, supporting that no consistent distinct splicing profile was present in C9-ALS patient motor neuron cultures. (B) Unsupervised hierarchical clustering analysis of whole gene expression showed that three of the four C9-ALS lines clustered together (lines 28i, 29i and 52i, each with ~800 repeats), with C9-ALS line 30i (the line with the fewest repeats, ~70) clustering separately.


Fig. S12. Electrophysiological properties of control and C9-ALS patient–derived motor neurons separated by individual subject. (A) Histogram showing the percentage of neurons in control (83i and 14i) and C9-ALS (28i and 52i) motor neuron cultures showing no spikes (n=21 for control, n=46 for ALS), one spike (n=22 for control, n=87 for ALS), or multiple spikes (n=94 for control and 51 for ALS). Recordings were performed on motor neuron cultures that were between 66-79 days of differentiation (n=321 neurons, n=2 to 3 independent culture sets for each line). Resting potential and input resistance of a subset of these cells are -69.5±2 mV with 2.3±0.28 GigaOhm for the control (n=43) and -68±1.4 mV with 2.42±0.15 GigaOhm for the C9-ALS motor neurons (n=70). (B) Graph showing data from cells with multiple spikes as in Fig. 3H, separated according to individual cell line. Mean number of action potentials elicited as a function of current injection for individual control (black) and C9-ALS (red) motor neuron cultures, with significantly reduced numbers of spikes fired in C9-ALS motor neurons as compared to CTR motor neurons (n=31 and 52 neurons for control lines 14i and 83i; n=19 and 26 neurons for ALS lines 28i and 52i respectively).


Fig. S13. Absence of any ASO toxicity in motor neuron cultures and reversal of transcription profiles by RNA-seq in iPSC-derived motor neurons. (A) Motor neurons from C9-ALS iPSCs (n=4, 28i, 29i, 30i, 52i) untreated or treated with scramble ASO, ASO816 or ASO061 (3 µM) for 2-weeks were immuno-stained and quantified for SMI32+ and Tuj1+ neurons (expressed as % of Tuj1+ cells) using Image Express Micro high-content imager, showing no toxicity of ASOs. Greater than 100 sites at 20x magnification were acquired per well for positive cell scoring. TuJ1 is pan-neuronal marker utilized for normalization for total production of neurons. Normal numbers of motor neurons were observed after treatment with any of the ASOs, supporting that there is no non-specific (scramble) toxicity or effect of C9ORF72 knockdown on motor neuron survival. Data are represented as mean ± SEM from n=3 independent experiments. (B) Heat map representation of differentially expressed genes in C9-ALS patient motor neurons vs. controls identified in Supplemental Figure 10. Two genes were no longer


annotated, so only 66 of the original 68 genes are shown. Control (83iCTR) and C9-ALS patient (52iC9-ALS) motor neuron cultures were treated with either scrambled ASO or ASO816, which targets exon 2 to knock down all C9ORF72 isoforms. Genes identified as being differentially expressed (p<0.05, fold >2) in the initial RNA-seq analysis (C9-ALS vs. control, n=4 cell lines of each, shown in boxed region in Fig. S10) were used for the heat map representation and generated by hierarchical clustering. The dendrogram indicated that ASO816 treated C9-ALS samples clustered apart from scramble treated cultures, and were closer to control cultures although the ASOs did not completely reverse the transcriptional changes. Genes with one asterisk (*) indicate they repeated the original observation of being either up or downregulated in C9-ALS patients vs. controls (p<0.05), showing a high correlation between the two independent experiments. Genes with two asterisks (**) also showed statistically significant (p<0.05) reversal of the transcriptional abnormality toward that seen in the control neuron cultures after treatment with ASO816 to knock down C9ORF72. The reversal was more pronounced in the upregulated genes (14/45 = 31% of genes) compared with those that were downregulated (3/21 = 14% of genes) in C9-ALS patient motor neuron cultures compared to controls. (C) Quantitative RT-PCR for genes which showed aberrant upregulation in C9-ALS motor neuron cultures (CBLN1, DPP6, and SLITRK2) after treatment with scrambled ASO or ASO061. After ASO061 treatment, a subset of the most highly upregulated genes in C9-ALS motor neuron cultures (shown in Figure 3G, 4E) were partially reversed to the level of control motor neurons. Error bars are mean ± sem. *** = p<0.001; ** = p<0.01; unpaired t-test (two-tailed), scramble vs. ASO061 from n=3 independent experiments. (D) Analysis of 1a vs. 1b upstream exon utilization assessed by 5′RACE analysis of scrambled ASO vs. ASO061 treated motor neuron cultures from C9-ALS patient 52i from two independent experiments. Treatment with ASO061 led to a significant increase in the utilization of exon 1b. Error bars are mean ± s.d. * = p<0.05; t-test (one-tailed), Scrambled vs. ASO061.


Table S1. Clinical information on subjects with C9ORF72 hexanucleotide expansions and controls used for iPSC lines in this study. In duration column, * indicates duration at the time of this study, subject is still living. Abbreviations: CAUC – Caucasian; AA – African American; N/A – not applicable.


Table S2. Functional pathway analysis of differentially expressed genes in iPSC-derived motor neuron cultures from C9-ALS patients versus controls, p<0.05 and fold>2, using the DAVID functional annotation tool (http://david.abcc.ncifcrf.gov/home.jsp). Annotation clusters showing significant enrichment from functional categories (SP_PIR_KEYWORDS, UP_SEQ_FEATURE), gene ontology (GOTERM_BP_FAT, GOTERM_CC_FAT, GOTERM_MF_FAT), protein domains (INTERPRO, PIR_SUPERFAMILY, SMART) are shown. Functional clusters included cell adhesion and synaptic transmission, while structural clusters included complement-like proteins (from all three cerebellin family members) and GPI-anchored proteins.

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