Journal: Nature medicine

Article Title: Modeling Smith-Lemli-Opitz syndrome with iPS cells reveals a causal role for Wnt/β-catenin defects in neuronal cholesterol synthesis phenotypes

doi: 10.1038/nm.4067

Figure Lengend Snippet: ( a ) Example immunocytochemistry images depicting expression of pluripotency markers SSEA4 and OCT4 in control (line BJ 3F-1) and SLOS (line A2 3F-2) iPSCs. Scale bars, 200 μm in phase contrast images and 100 μm in fluorescence images. ( b–c ) Quantification of relative DNMT3B and NANOG transcript expression in SLOS fibroblasts, hESCs, control iPSCs, and SLOS iPSCs. n = 3 for all cell lines except H1, CW 3F-1 ( DNMT3B , n = 4) and H1, H9, BJ 3F-1, CW 3F-1, CW 3F-2, CWI 4F-2, A2 4F-5 ( NANOG , n = 4); data represent mean ± s.e.m. ( d ) Example images of gross and stained (hematoxylin and eosin) teratomas obtained 4–8 weeks after injection of SLOS iPSCs into NOD/SCID immunocompromised mice. Stained panels, from left to right, depict the formation of neuroectoderm, glandular endothelium, and bone. Scale bars, 1 cm (left panel) and 100 μm (remaining panels). Representative images from three injected mice for BJ 3F-1 control and A2 3F-2 SLOS iPSCs are shown. ( e–f ) Example gas chromatography/mass spectrometric analysis of extracted lipids from control ( e ) and SLOS ( f ) iPSCs cultured in cholesterol deficient mTesR1 media for 7 days. Stars at 14.8 – 15.0 min peaks indicate the internal standard coprostanol; Arrows at 16.5 – 16.6 min peaks indicate cholesterol. Arrowheads in ( f ) at 16.74 min and 17.49 min indicate 8DHC and 7DHC, respectively. ( g–h ) Quantification of cholesterol ( g ) and DHC ( h ) levels for SLOS fibroblasts cultured in cholesterol replete media and hESCs, control iPSCs, and SLOS iPSCs cultured for 3 – 7 d in cholesterol deficient mTesR1 media. SLOS fibroblasts in cholesterol replete medium n = 5, H1 ES n = 6, hDF control n = 7, BJ control n = 5, MN 4F-1 n = 8, MN 4F-2 n = 3, CW 3F-1 n = 9, CW 3F-2 n = 11, CWI 4F-1 n = 3, A2 3F-1 n = 14, A2 3F-2 n = 7 per cell line; unpaired, two-tailed student’s t test against H1 ES cells; data represent mean ± s.e.m. * P < 0.05 and ** P < 0.01.

Article Snippet: Dispase passage of cells growing in mTeSR1 utilized ROCK inhibitor supplementation (Y27632; 10 μM; Reagents Direct) for 24 h. Though the complete formulation of mTeSR1 designates it as maintaining low cholesterol content (1.12E-03 mM; 0.433 μg cholesterol/mL), mTeSR1 was batch tested for maintenance of hESC pluripotent morphology and analyzed by GC/MS for μg cholesterol/mL media.

Techniques: Immunocytochemistry, Expressing, Control, Fluorescence, Staining, Injection, Gas Chromatography, Cell Culture, Two Tailed Test

Journal: Nature medicine

Article Title: Modeling Smith-Lemli-Opitz syndrome with iPS cells reveals a causal role for Wnt/β-catenin defects in neuronal cholesterol synthesis phenotypes

doi: 10.1038/nm.4067

Figure Lengend Snippet: ( a ) Representative neural rosette formation in controls (line H1 ES) and SLOS iPSCs (line A2 3F-1). PAX6 (green) and SOX2 (red) label neural rosettes; βIII-tubulin (green) labels neurons. Hoechst is blue; scale bars, 100 μm. ( b ) Quantification of relative transcript expression of lineage markers PAX6 (neural progenitors), MAP2 (neuronal), SOX10 (neural crest), and ALDHL1 (glial) in rosette formation assays in control and SLOS (lines A2 3F-1, A2 3F-2) iPSCs. n = 3 biological replicates per cell line; unpaired, two-tailed student’s t test compared to H1 ES cell expression (dashed line); data represent mean ± s.e.m. ( c ) Representative images of control (top panels, line BJ 3F-1) and SLOS (bottom panels, line A2 3F-2) iPSCs cultured in cholesterol replete (0 h) versus cholesterol deficient (24 h, 48 h, and 7 d) conditions; scale bars, 200 μm. Representative images from n = 10 biological replicates. ( d ) Electron microscopic analysis of controls (line H1 ES) and SLOS iPSCs (line A2 3F-1) in cholesterol deficient conditions. Loss of gap and tight junctions (arrows, top left panel) and formation of intermediate filaments (arrows, top right panel) are indicated. Controls (bottom left panel) and LDL supplemented SLOS iPSCs (bottom right panel) maintain gap and tight junctions (arrows, bottom panels; dashed lines identify cell-cell border maintenance); top right scale bar, 0.5 μm, other scale bars, 2 μm. H1 ES n = 10 cells, A2 3F-1 n = 12 cells, A2 3F-1+LDL n = 21 cells. Representative images are shown. ( e ) Pluripotent TRA-1-81 expression and phase contrast images of SLOS iPSCs (line A2 3F-1) cultured in cholesterol deficient (left panels) or LDL supplemented (right panels) conditions; scale bars, 100 μm. ( f ) Quantitation of TRA-1-81 + SLOS iPSC (line A2 3F-1) colonies cultured in cholesterol deficient mTeSR1 (untreated), mTeSR1 + LDL (10 mcg/mL medium), mTeSR1 + HDL (10 mcg/mL medium), or XF-KSR medium (1.85 mcg cholesterol/mL medium). n = 3 biological replicates per treatment, 250 – 300 colonies per replicate; unpaired, two-tailed student’s t test compared to untreated; data represent mean ± s.e.m. ( g ) Representative images of rosette formation by SLOS iPSCs (line A2 3F-2) with or without lipid supplementation (left panels); scale bars, 100 μm. Quantitation of rosette formation with or without lipid supplementation. H1 ES n = 5, BJ 3F-1 n = 5, NL5 n = 4, A2 3F-1 n = 4, A2 3F-2 n = 5, CWI 4F-5 n = 5 biological replicates; unpaired, two-tailed student’s t test compared to untreated; data represent mean ± s.e.m. ( h ) Neural progenitor (SOX2, red; hNESTIN, green) and neuronal (βIII-tubulin, green) marker expression in expandable SLOS neural progenitors; Hoechst is blue; scale bars, 100 μm. Representative images from n = 3 biological replicates for A2 3F-2 are shown. * P < 0.05; ** P < 0.01.

Article Snippet: Dispase passage of cells growing in mTeSR1 utilized ROCK inhibitor supplementation (Y27632; 10 μM; Reagents Direct) for 24 h. Though the complete formulation of mTeSR1 designates it as maintaining low cholesterol content (1.12E-03 mM; 0.433 μg cholesterol/mL), mTeSR1 was batch tested for maintenance of hESC pluripotent morphology and analyzed by GC/MS for μg cholesterol/mL media.

Techniques: Expressing, Control, Two Tailed Test, Cell Culture, Quantitation Assay, Marker

Journal: Nature medicine

Article Title: Modeling Smith-Lemli-Opitz syndrome with iPS cells reveals a causal role for Wnt/β-catenin defects in neuronal cholesterol synthesis phenotypes

doi: 10.1038/nm.4067

Figure Lengend Snippet: ( a ) Representative images of pluripotent (NANOG, red) and neural progenitor (PAX6, green) marker expression in SLOS (line A2 3F-1) and control (line BJ 3F-1) iPSCs cultured in mTeSR1 cholesterol deficient pluripotent media. Hoechst is blue; scale bars, 100 μm. ( b ) Quantification of POU5F1 (pluripotent), PAX6 (neuroectoderm), SNAI2 (neural crest), VIM (mesoderm/mesenchymal) and AFP (endoderm) transcript expression in control (line BJ 3F-1) and SLOS iPSCs cultured in cholesterol deficient pluripotent conditions. CWI 4F-1, CW 3F-2 n = 4, Control iPS, A2 3F-1, A2 3F-2, A2 4F-5 n = 3 biological replicates compared to H1 ES n = 3; data represent mean ± s.e.m. ( c ) hNestin (green) expression in SLOS iPSCs (line A2 3F-1) and controls (line H1 ES) after 7 d cholesterol deficient culture. Hoechst is blue; scale bars, 100 μm. ( d ) FACS quantitation of hNestin in SLOS iPSCs (lines CW 3F-2, A2 3F-1, A2 3F-2) and H1 ES cells after 7 d cholesterol deficient culture. Representative data from n = 3 biological replicates is shown. ( e ) Schematic representation of a microarray experiment to compare expression patterns in control (line BJ 3F-1) and SLOS (line A2 3F-2) iPSCs grown in cholesterol deficient conditions. n = 4 biological replicates were collected and analyzed at all time points. (f) Venn diagram highlights the number of genes differentially expressed between control and SLOS iPSCs on Days 2 and 3 of cholesterol deficient culture. ( g ) Heat map shows the relative intensity for selected statistically significant genes at the indicated time points.

Article Snippet: Dispase passage of cells growing in mTeSR1 utilized ROCK inhibitor supplementation (Y27632; 10 μM; Reagents Direct) for 24 h. Though the complete formulation of mTeSR1 designates it as maintaining low cholesterol content (1.12E-03 mM; 0.433 μg cholesterol/mL), mTeSR1 was batch tested for maintenance of hESC pluripotent morphology and analyzed by GC/MS for μg cholesterol/mL media.

Techniques: Marker, Expressing, Control, Cell Culture, Quantitation Assay, Microarray

Journal: eLife

Article Title: A human ESC-based screen identifies a role for the translated lncRNA LINC00261 in pancreatic endocrine differentiation

doi: 10.7554/eLife.58659

Figure Lengend Snippet:

Article Snippet: Undifferentiated hESCs were aggregated by preparing a single cell suspension in mTeSR1 media (STEMCELL Technologies; supplemented with 10 μM Y-27632) at 1 × 10 6 cells/mL and overnight culture in six-well ultra-low attachment plates (Costar) with 5.5 ml per well on an orbital rotator (Innova2000, New Brunswick Scientific) at 100 rpm.

Techniques: Flow Cytometry, Control, Recombinant, Cloning, Plasmid Preparation, Expressing, Transfection, TA Cloning, Purification, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Software, Membrane, Hybridization, DNA Extraction, Modification