Journal: bioRxiv

Article Title: Modeling motor neuron resilience in ALS using stem cells

doi: 10.1101/399659

Figure Lengend Snippet: (A) Time line depicting in vitro protocols for resistant oculomotor neurons (OMN) and vulnerable spinal motor neuron (SC MN) generation. ESC column reports the four cell lines used in this study. Patterning was performed for 5 days after ESC expansion and EB formation. mRNA-seq coupled with FACS was performed on EB dissociation day (day 9 of the protocol). Survival assay was performed after five days from EB dissociation. (B-C) SC MNs generated from E14.1 ESCs express Hb9, Islet1 and NF200, OMNs generated from E14.1 ESCs over-expressing the transcription factor Phox2a under the Nestin enhancer co-express Islet1 and NF200 in absence of Hb9. Scale bar in c 60 μm. (D) Percentage of Hb9+/Islet1+ cells in SC MN (64.1±5.2%, mean±SEM, n=4) and OMN (6.3±1.3%, mean±SEM) cultures respectively. (For quantification, experiments were run in quadruplicates including two technical replicates per experiment, with at least 120 Islet1+ cells counted per condition). (E) Microphotographs showing Islet1+/Tuj1+ cells in OMN cultures, (F) OMNs also express the specific marker Phox2a as indicated by asterisks. Scale bars in e and g 100 μm. (G) Quantification of Islet1+ over Tuj1+ cells demonstrates that half neuronal population appears to be Islet1+ (47.5±5.9%, mean±SEM, experiments performed in quadruplicates with two technical replicates each, total number of Tuj1+ cells counted: 1325). Quantification of Phox2A+ over Islet1+ cells (experiments performed in quadruplicates with two technical replicates each, total number Islet1+ cells counted: 746) indicates that 62±5.2% (mean±SEM) of the Islet1+ population is also Phox2a+. All quantifications were performed 5 days after EB dissociation (mean ± SEM). (H) PCA of OMN and SC MN samples based on all genes expressed confirmed cell differential identities. mRNA-seq analysis of OMNs and SC MNs isolated by FACS was performed after 5 days of patterning (day 9 of the protocol). (I) 1,017 differentially expressed genes were found between the two different cell types (adjusted P<0.05). Heatmap shows the top 500 most significant differentially expressed genes by adjusted p-value. (J) Heatmap of expected progenitors and motor neuron transcripts but also OMN and SC MN specific transcripts obtained in the two generated motor neuron populations. (K) Using the top 100 DEGs obtained from our RNAseq analysis we could separate datasets originating from in vivo microarray studies on early postnatal (J) and adult (L) rodent OMNs and SC MNs. (M) Venn diagram showing gene sets enriched either in brain stem cultures or OMN-specific as revealed by PAGODA analysis, (N) gene sets preferentially found in spinal cord cultures or MN-specific.

Article Snippet: The following primary antibodies were used: NF200 (1:1000, #AB5539 Millipore), Tuj1 (1:1000, MRB-435P Covance), Hb9 (1:10, #81.5C10 DSHB), Islet1 (1:500, #ab20670 abcam; 1:100, #40.2D6 DSHB), Phox2A (1:1000, gift of Prof JF Brunet), GFP (1:1000, #ab13970 abcam), phospho-AKT (Ser473) (1:50, #3787 Cell Signaling), activated beta-catenin (1:1000, #05-665 Millipore), alpha-catenin (1:1000, #AB153721 AbCam), ESYT1 (1:100, #HPA016858), BrdU (1:10, #G3G4 DSHB).

Techniques: In Vitro, Clonogenic Cell Survival Assay, Generated, Expressing, Marker, Isolation, In Vivo, Microarray

Journal: bioRxiv

Article Title: Modeling motor neuron resilience in ALS using stem cells

doi: 10.1101/399659

Figure Lengend Snippet: (A) Oculomotor neurons (OMNs) and spinal motor neurons (SC MNs) express similar mRNA levels of glutamate ionotropic receptor AMPA, NMDA and kainate type subunits. The heatmap shows log2 RPKM values of these subunits and no separate clustering of the two cell types is observed. (B-C) Immunohistochemistry performed on generated OMNs and SC MNs at D1 in vitro in control conditions; similar levels of glutamate ionotropic receptor kainate type subunit 5 (Grik5) are found in both cell types. Scale bar in d 60 μm. (D-E) Microphotographs presenting SC MN and OMN response to kainic acid induced toxicity (20 μM) for a week. Scale bars in f = 100 μm. (F) Curves represent percentages of MN survival over time in OMN and SC MN cultures. OMNs were visualized as NF200+Islet1+Hb9- clls, while SC MNs as NF200+Islet1+Hb9+ cells. OMNs show increased survival to KA toxicity at D7 (mean ± SEM, 2way ANOVA and Tukey’s multiple comparison test, F(9, 56)=2.333, *P=0.0261, SC MNs n=4; OMNs n=5) when compared to SC MNs (experiments were performed at least in quadruplicates, with technical replicates and with at least 130 motor neurons counted per condition in each experiment). Analysis of the length of neuronal processes in both oculomotor and spinal motor neuron cultures exposed to kainic acid for seven days. showed that oculomotor neurons were unaffected by kainic acid while spinal motor neurons displayed a shortening of neurites (G). (H-K) Sholl analysis was performed on OMN at D7 survival assay in control and KA20 conditions to further assess individual MN arborization complexity during toxicity. Scale bar = 100 μm. (I) Sholl mask was applied to individual OMNs after specifying the radius from the center of the soma of the neuron and created concentric circles every 25 μm of increasing radius. (J) Comparison of average number of neurite intersections of OMN in control and KA20 toxicity conditions with radial step size of 25 μm. OMNs did not show reduction in arborization (multiple t test, n = 10 per condition). (K) Schematic depicting identification of neurite segments by Sholl analysis. Color code is assigned depending on arbor localization from the soma in an inside-out manner following the given radius. Multiple intersections within the same segment display the same colour.

Article Snippet: The following primary antibodies were used: NF200 (1:1000, #AB5539 Millipore), Tuj1 (1:1000, MRB-435P Covance), Hb9 (1:10, #81.5C10 DSHB), Islet1 (1:500, #ab20670 abcam; 1:100, #40.2D6 DSHB), Phox2A (1:1000, gift of Prof JF Brunet), GFP (1:1000, #ab13970 abcam), phospho-AKT (Ser473) (1:50, #3787 Cell Signaling), activated beta-catenin (1:1000, #05-665 Millipore), alpha-catenin (1:1000, #AB153721 AbCam), ESYT1 (1:100, #HPA016858), BrdU (1:10, #G3G4 DSHB).

Techniques: Immunohistochemistry, Generated, In Vitro, Clonogenic Cell Survival Assay