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

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 1 | Identification of CD177+ and CD275+ ADE subpopulations. a, Schematic representation of hESC differentiation toward DE. b,c, Representative FACS plots of apparently homogeneous FOXA2+/SOX17+ DE (b) showing a heterogenous population marked by CXCR4+/CD117+ cells (c) (n = 3 (b), n = 6 (c) biologically independent experiments). d–g, Gene expression profiles of CXCR4+/CD117−, CXCR4high/CD117high, CXCR4mid/CD117mid and CXCR4low/CD117low cells for FOXA2 (d), SOX17 (e), CER1 (f) and HHEX (g) (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h, Summary of the antibody screen identifying and isolating CD177 and CD275 as markers of ADE subpopulations. CXCR4 and FOXA2 are used as controls to identify the whole DE. i, hPSCs and hPSC-derived DE stained for CXCR4, CD177 and CD275 as shown by live-cell FACS (n = 10 biologically independent experiments). AA, activin A; D, day.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Gene Expression, Derivative Assay, Staining

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 2 | Molecular profiling of CD177+, CD275+ and CXCR4+ DE subpopulations reveals distinct signatures. a, Summary of differentiation protocol toward DE/ADE followed by MACS to enrich for CD177, CD275 and CXCR4 populations. b, Principal component analysis showing that mRNA-derived transcriptome profiles are characteristic of different DE/ADE subpopulations (n = 3 biologically independent experiments). c–e, Bar graphs of selected and significantly enriched gene ontology terms in CD275+ versus CXCR4+ (c), CD177+ versus CD275+ (d) and CD177+ versus CXCR4+ (e) DE populations (n = 3 biologically independent experiments). Enrichment P values are calculated by HOMER findGO.pl based on the cumulative hypergeometric distribution. f,g, Validation of the microarray analysis by qPCR for noncanonical WNT/PCP components and ligands (f) and canonical WNT components and ligands (g). Data were normalized to 18S (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h,i, Western blot analysis (h) and quantification (i) of WNT/PCP components such as p-JNK and DVL2 in ADE subpopulations (n = 3 biologically independent experiments). GAPDH is used as a loading control. Data are represented as mean ± s.e.m. j, Immunofluorescence analysis validated the exclusive localization of β-catenin in the membrane in CD177+ ADE cells and in the cytoplasm and nucleus in CD275+ ADE and CXCR4+ DE cells (n = 3 biologically independent experiments). FOXA2 is used as a nuclear marker. Scale bars, 20 µm and 10 µm in inset. PC1/2, principal component 1/2.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Derivative Assay, Biomarker Discovery, Microarray, Western Blot, Control, Immunofluorescence, Membrane, Marker

Journal: Cancer Research

Article Title: The Myc–miR-17∼92 Axis Blunts TGFβ Signaling and Production of Multiple TGFβ-Dependent Antiangiogenic Factors

doi: 10.1158/0008-5472.can-10-2412

Figure Lengend Snippet: Figure 1. Clusterin inhibits tumor growth and angiogenesis. A, growth of subcutaneous tumors derived from RasMyc cells transduced with a retrovirus expressing murine clusterin and/or the puromycin resistance gene. Inset, immunoblot of clusterin levels in Ras and RasMyc colonocytes. B, growth of subcutaneous tumors derived from HCT116 cells transduced with an empty vector or the retrovirus expressing human clusterin. Regression lines represent average rates of growth. The P value refers to the difference in regression coefficients. Inset, immunoblot of clusterin levels in transduced cells. C, in vivo bioluminescent imaging of representative animals from the experiment in B. Mice were photographed at days 3 and 21 after injection. No less than five animals per group were used in all these experiments. D, immunohistochemical staining of Ki-Ras and Ki-Ras/Myc tumors with an anti-clusterin antibody. Cytoplasmic staining for clusterin is depicted in brown whereas nuclei are counterstained in blue. E, alignment of the second TSR of murine thrombospondin-1 with the C9 protein and clusterin. F, hemoglobin content of Matrigel pellets 7 d after injection. Matrigels were admixed with p53-null mouse colonocytes transduced with either empty vector (blue bar) or clusterin retrovirus (red bar) and injected s.c. into syngeneic host animals. G, H&E staining of RasMyc/vector (top) and RasMyc/clusterin (bottom) tumor sections. Perfused blood vessels contain numerous RBC, which could be clearly seen under higher magnification (inset). The scatter plot represents densities of perfused blood vessels in four individual tumors. H, immunohistochemical staining of control (top) and clusterin-overexpressing (bottom) HCT116 tumor sections with antibody recognizing the endothelial cell surface antigen CD31. The scatter plot represents the densities of CD31-positive blood vessels in four individual tumors.

Article Snippet: For thrombospondin-1 expression is, either cell lysates or conditioned media were used. ranes were probed with antibodies to clusterin, , TGFBR2 (Santa Cruz Biotechnology and Abcam), 4 (Santa Cruz Biotechnology), Smad2 and Smad3 ogen), phosphorylated Smad3 (Cell Signaling), and (Ab-11, Lab Vision) according to the recommendaof the manufacturer.

Techniques: Derivative Assay, Transduction, Expressing, Western Blot, Plasmid Preparation, In Vivo, Imaging, Injection, Immunohistochemical staining, Staining, Control

Journal: Cancer Research

Article Title: The Myc–miR-17∼92 Axis Blunts TGFβ Signaling and Production of Multiple TGFβ-Dependent Antiangiogenic Factors

doi: 10.1158/0008-5472.can-10-2412

Figure Lengend Snippet: Figure 2. Clusterin is regulated by miR-17~92 via the TGFβ pathway. A, immunoblotting analysis of clusterin expression levels in the following cell lines. Left, Ras-only mouse colonocytes transduced with either empty vector (Ras/Puro) or the miR-17~92–encoding retrovirus (Ras/miR-17~92). Right, RasMyc cells transfected with scrambled or anti–miR-17~92 2′-O-methyl oligoribonucleotides. B, changes in expression levels of thrombospondin-1 (THBS1) and clusterin mRNAs in HCT116 Dicerhypo (left) and A172 (right) cells after transfection with the indicated microRNA mimics. mRNA levels in HCT116 and A172 cells were profiled using Affymetrix microarrays and qPCR as described in Materials and Methods. C, activation of TGFβ signaling in Ras colonocytes. Ras cells treated with vehicle or 10 ng/mL of TGFβ1 for 30 min were analyzed by immunoblotting for phosphorylated Smad3 (pSmad3) and total Smad3. D, measurement of TGFβ effects on TSR proteins. Ras cells were treated with increasing doses of TGFβ1 for 48 h and lysates were immunoblotted for clusterin and CTGF proteins. Tsp-1 was detected in conditioned medium. E, immunoblotting analysis of TSR proteins in Ras/vector, Ras/miR-17~92 or c-Myc cells cultured in the absence or presence of TGFβ1 (5 ng/mL) for 48 h. F, CLU mRNA levels in Ras/vector and Ras/17~92 cells before and after (24 h) stimulation with TGFβ, as measured by qPCR. Expression levels of CLU are adjusted to those of glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: For thrombospondin-1 expression is, either cell lysates or conditioned media were used. ranes were probed with antibodies to clusterin, , TGFBR2 (Santa Cruz Biotechnology and Abcam), 4 (Santa Cruz Biotechnology), Smad2 and Smad3 ogen), phosphorylated Smad3 (Cell Signaling), and (Ab-11, Lab Vision) according to the recommendaof the manufacturer.

Techniques: Western Blot, Expressing, Transduction, Plasmid Preparation, Transfection, Activation Assay, Cell Culture

Journal: Cancer Research

Article Title: The Myc–miR-17∼92 Axis Blunts TGFβ Signaling and Production of Multiple TGFβ-Dependent Antiangiogenic Factors

doi: 10.1158/0008-5472.can-10-2412

Figure Lengend Snippet: Figure 3. miR-17~92 targets endogenous TGFβ receptor II. A, luciferase sensor assay. Constructs tested were psiCHECK-2 derivatives containing a single miR-17/20a binding site from TGFBR2 3′-UTR in either wild-type (wt) or seed-mutated (mut) conformation. Cells were additionally cotransfected with miR-17 or control mimic. Results are expressed as ratios of renilla to firefly luciferase, the latter being constitutively expressed from the same vector and serving as a control for transfection efficiency. Sequence alignment corresponds to positions 268 to 274 of TGFBR2 3′-UTR and mature hsa-miR-17. Arrows indicate mutated nucleotides. B, expression levels of TGFBR2 mRNA in DLD1 Dicerhypo cells 10 h after transfection with microRNA mimics (25 nmol/L). mRNA levels were quantified by microarray. C, immunoblotting analysis of TGFBR2 and clusterin expression levels in Ras cells transfected with 10 nmol/L of nontargeting siRNA pool or siRNA pool targeting mouse TGFBR2. D and E, immunoblotting analysis of TGFBR2 and Smad3 in Ras cells 20 h after transfection with microRNA mimics (25 nmol/L). Bottom, quantitations of Western blots above.

Article Snippet: For thrombospondin-1 expression is, either cell lysates or conditioned media were used. ranes were probed with antibodies to clusterin, , TGFBR2 (Santa Cruz Biotechnology and Abcam), 4 (Santa Cruz Biotechnology), Smad2 and Smad3 ogen), phosphorylated Smad3 (Cell Signaling), and (Ab-11, Lab Vision) according to the recommendaof the manufacturer.

Techniques: Luciferase, Construct, Binding Assay, Control, Plasmid Preparation, Transfection, Sequencing, Expressing, Microarray, Western Blot