Journal: Stem Cell Reports

Article Title: Deterministic HOX Patterning in Human Pluripotent Stem Cell-Derived Neuroectoderm

doi: 10.1016/j.stemcr.2015.02.018

Figure Lengend Snippet: Wnt/β-Catenin and FGF Signaling Synergistically Coordinate HOX Activation during hPSC Differentiation (A) HOX paralog expression in the hindbrain and spinal cord color-coded to the region where its expression is first detected (modified from a previous report; ). (B) Timeline of H9 hESC differentiation corresponding to (C)–(E). (C) RT-PCR analysis of cultures at days 2–5. (D) qPCR analysis at day 5. ∗ p < 0.005; ∗∗ p < 0.001. (E) OTX2 and SOX2 expression at day 5 after RA or CHIR treatment. Scale bars, 100 μm. Adjacent images are the same field. (F) qPCR analysis at day 2 using the H9 ishcat2 line with or without doxycycline treatment. ∗ p < 0.02; ∗∗ p < 0.005. (G) qPCR analysis at day 5 using the H9 ishcat2 line with or without doxycycline treatment. ∗ p < 0.02; ∗∗ p < 0.0001. All qPCR data are presented as mean ± SD calculated from independent biological triplicates. H9 data are normalized to the condition yielding maximum expression and ishcat2 data are normalized to each doxycycline-free condition. Statistical significance was calculated using the Student’s unpaired t test.

Article Snippet: After collection by centrifugation, cells were gently resuspended in E6 medium containing 200 ng/ml FGF8b and CHIR99021 (CHIR, 3 μM for H9 hESCs and 2 μM for IMR90-4 iPSCs; R&D Systems) and re-seeded on VTN-NC-coated plates at a density of 1.5 × 10 5 cells/cm 2 .

Techniques: Activation Assay, Expressing, Modification, Reverse Transcription Polymerase Chain Reaction

Journal: Stem Cell Reports

Article Title: Deterministic HOX Patterning in Human Pluripotent Stem Cell-Derived Neuroectoderm

doi: 10.1016/j.stemcr.2015.02.018

Figure Lengend Snippet: Colinear HOX Expression in hPSC-Derived NMPs (A) Analysis of the NMP phenotype by flow cytometry and immunocytochemistry. Gray histogram, immunoglobulin G (IgG) control; red histogram, label of interest. Scale bars, 100 μm. (B) Optimized NMP propagation scheme. The addition of CHIR is denoted as t = 0 hr in all panels. Purity of NMPs was assessed by flow cytometry in the presence or absence of GDF11 (data are presented as mean ± SD). qPCR analysis of HOXD10 and HOXD11 was conducted in the presence or absence of GDF11 (expression normalized to the time point of maximum expression). (C) Schematic for HOX induction in NMPs. “t” indicates time under Wnt, FGF, and GDF signaling. (D) qPCR analysis of colinear HOX expression normalized to the time point of maximum expression. GDF11 was included according to (B). qPCR data in all panels are presented as mean ± SD calculated from technical duplicates. Full profiles for all analyzed genes can be found in Figure S2 A. For all flow cytometry data, a minimum of two biological replicates was used to calculate mean ± SD.

Article Snippet: After collection by centrifugation, cells were gently resuspended in E6 medium containing 200 ng/ml FGF8b and CHIR99021 (CHIR, 3 μM for H9 hESCs and 2 μM for IMR90-4 iPSCs; R&D Systems) and re-seeded on VTN-NC-coated plates at a density of 1.5 × 10 5 cells/cm 2 .

Techniques: Expressing, Derivative Assay, Flow Cytometry, Immunocytochemistry, Control

Journal: Stem Cell Reports

Article Title: Deterministic HOX Patterning in Human Pluripotent Stem Cell-Derived Neuroectoderm

doi: 10.1016/j.stemcr.2015.02.018

Figure Lengend Snippet: RA Induces a Neuroectodermal Fate and Halts Colinear HOX Activation (A) NMPs exposed to RA or CHIR acquire a neuroectodermal or mesodermal fate as assessed by flow cytometry at day 6 (gray histogram, IgG control; red histogram, antigen of interest). (B) Schematic for transition from NMPs to neuroectoderm by RA treatment. “t” indicates time under Wnt, FGF, and GDF signaling prior to RA treatment, which halts HOX progression to yield a defined rostrocaudal identity. (C and D) Representative hindbrain (C) and spinal cord (D) cultures assessed by qPCR, immunocytochemistry, and flow cytometry. For all plots, flow cytometry data are presented as mean ± SD from biological duplicates, and qPCR data are mean ± SD from technical duplicates normalized to the time point of maximum expression for each gene. For hindbrain cultures, HOXB1 and HOXB4 were quantified by immunocytochemistry relative to DAPI + nuclei (technical triplicates per time point, minimum 10,000 cells counted, and relative HOX expression patterns were qualitatively verified across biological duplicates), while SOX2 and PAX6 were quantified by flow cytometry. For spinal cord cultures, HOXD10 was quantified by immunocytochemistry relative to DAPI + nuclei (technical duplicates, >2,000 cells counted), while SOX2, PAX6, and HOXB4 were quantified by flow cytometry. Scale bars, 100 μm. For (D), dorsomorphin was included with GDF11 and throughout RA treatment (further details in Figure S3 ). (E) Mass spectrometry comparison of HOX profiles in cervical, thoracic, and lumbar neuroectoderm cultures. Cervical differentiation: 1 day FGF8b, 2 days FGF8b/CHIR, 4 days RA; thoracic differentiation: 1 day FGF8b, 6 days FGF8b/CHIR, 4 days RA; lumbar differentiation: 1 day FGF8b, 4 days FGF8b/CHIR, 2 days FGF8b/CHIR/GDF11, 4 days RA. Biological triplicates (neuroepithelium isolated individually from three separate culture wells) were used to calculate statistical significance between cervical, thoracic, and lumbar samples. Data are presented as mean ± SD and statistical significance was calculated using the Student’s unpaired t test. ∗ p < 0.01; ∗∗ p < 0.002; ∗∗∗ p < 0.0001.

Article Snippet: After collection by centrifugation, cells were gently resuspended in E6 medium containing 200 ng/ml FGF8b and CHIR99021 (CHIR, 3 μM for H9 hESCs and 2 μM for IMR90-4 iPSCs; R&D Systems) and re-seeded on VTN-NC-coated plates at a density of 1.5 × 10 5 cells/cm 2 .

Techniques: Activation Assay, Flow Cytometry, Control, Immunocytochemistry, Expressing, Mass Spectrometry, Comparison, Isolation

Journal: Stem Cell Reports

Article Title: Deterministic HOX Patterning in Human Pluripotent Stem Cell-Derived Neuroectoderm

doi: 10.1016/j.stemcr.2015.02.018

Figure Lengend Snippet: Derivation of Region-Specific MNs (A) Neuronal maturation from various hindbrain locales. NMPs were propagated for 4, 12, or 24 hr before conversion to ventralized neuroectoderm and differentiation to neurons. DAPI (blue) is overlaid in most images. Scale bars, 20 μm. Quantified data are presented as mean ± SD (four technical replicates, >100 HB9 + cells counted per sample). (B) Evaluation of FOXP1 columnar identity in ISL1 + and HB9 + MNs. Cervical differentiation (C): 1 day FGF8b, 2 days FGF8b/CHIR; thoracic differentiation (T): 1 day FGF8b, 5 days FGF8b/CHIR; lumbar differentiation (L): 1 day FGF8b, 4 days FGF8b/CHIR, 2 days FGF8b/CHIR/GDF11; prior to en bloc passage and further differentiation as indicated. NTFs, neurotrophic factors (described in ). For plotted data, open circles are percentages from individual fields and bars indicate the mean. Statistical significance was calculated using the Student’s unpaired t test (three to five fields were counted across three biological replicates for each condition, minimum 2,500 cells counted). n.s., p > 0.02; ∗∗ p < 0.000001. Scale bars, 100 μm. (C) Neuronal maturation from cervical, thoracic, and lumbar patterned neuroectoderm corresponding to the NMP state described in (B). DAPI is overlaid in all images. Scale bars, 20 μm. RT-PCR (50 cycles) demonstrates maintenance of regional identity.

Article Snippet: After collection by centrifugation, cells were gently resuspended in E6 medium containing 200 ng/ml FGF8b and CHIR99021 (CHIR, 3 μM for H9 hESCs and 2 μM for IMR90-4 iPSCs; R&D Systems) and re-seeded on VTN-NC-coated plates at a density of 1.5 × 10 5 cells/cm 2 .

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: ACS chemical biology

Article Title: Targeted Elimination of Tumorigenic Human Pluripotent Stem Cells Using Suicide-inducing Virus-like Particles

doi: 10.1021/acschembio.8b00490

Figure Lengend Snippet: (a) Schematic of Qβ(ZZ)42@CD13 viral coat protein expression and particle assembly, followed by VLP labeling with SSEA-5 antibodies. (b) Electrophoretic, (c) FPLC, and (d) dynamic light scattering analyses of Qβ(ZZ)42@CD13 VLPs. (%Đ = % dispersity; Rh = hydrodynamic radius). (e) EC50 curves showing dose-dependent decreases in human induced pluripotent stem cell (hiPSC) and human embryonic stem cell (hESC) survival after 24 h treatment with both 8 nM Qβ(ZZ)42@CD13+SSEA-521 VLPs in the presence of the prodrug 5-fluorocytosine (5-FC) or the cytotoxin 5-fluorouracil (5-FU). (f) EC50 curves show dose-dependent decreases in human dermal fibroblast (hDF) survival percentages after 24 h treatment with 5-FU, but negligible cell death after treatment with 8 nM Qβ(ZZ)42@CD13+SSEA-521 VLPs in the presence of 5-FC. (g) Representative microscopy images show co-cultures of hPSC colonies and MEFs that were treated for 15 h with 8 nM of unlabeled, IgG1-labeled (isotype control), and SSEA-5-labeled Qβ(ZZ)42@CD13 VLPs in the presence of 100 μM 5-FC as test conditions, and 100 μM 5-FU as a positive control. Red fluorescence indicates dead cells and green fluorescence indicates expression of the pluripotent stem cell-specific TRA-1–60 marker.

Article Snippet: Human induced pluripotent stem cells [iPS(IMR90)-1, WiCell] 29 and human embryonic stem cells (H7, WiCell) 30 were cultured either on a feeder layer of irradiated mouse embryonic fibroblasts (MEFs), where they were maintained in hPSC medium (KnockOut DMEM, 20% KnockOut Serum Replacement, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 0.1 mM β-mercaptoethanol, 100 U/mL Penicillin-Streptomycin) supplemented with 8 ng/mL human basic fibroblast growth factor (bFGF), or in feeder-free hPSC conditions as previously described.

Techniques: Expressing, Labeling, Microscopy, Positive Control, Fluorescence, Marker

Journal: PLoS ONE

Article Title: Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation

doi: 10.1371/journal.pone.0178821

Figure Lengend Snippet: ( A ) Distribution of NADs along human autosomes. NADs are indicated by red rectangles over the ideograms of the chromosomes. Note that the p-arms of the five acrocentric chromosomes (13, 14, 15, 21 and 22), centromeres and some pericentromeric regions were not analysed because they are not present in the current human genome assembly. ( B ) Histogram of NAD sizes. Median = 361kb, a total of 1,646 NADs were identified. ( C ) 3D immuno-FISH analysis of NAD and inter-NAD regions (iNADs) in IMR90 cells. Nucleolus association of a chromosomal domain is illustrated by showing the Z-projection of an IMR90 nucleus on the left and the corresponding single light optical sections with the associated and non-associated allele on the right. BAC hybridization signals are shown in green, nucleolar staining in red and DAPI counterstain in blue (scale bar: 1.6 μm). ( D ) Hybridization signals (percentage of nucleolus-associated alleles) are plotted against the according microarray signals (average log2-fold difference of the nucleolar signal over the background). Red and grey circles indicate genomic regions that reside in NADs and iNADs, respectively (see for further details). The positions of the BAC clones used in 3D immuno-FISH experiments to monitor NADs and iNADs are shown also in ( A ) by red and grey circles, respectively.

Article Snippet: Human IMR90 embryonic fibroblasts were obtained from Coriell Repositories (Cat. No. I90-79) and cultivated in DMEM (Gibco Cat. No. 21885–025 supplemented with 10% v/v Foetal Calf Serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin) at 37°C in humidified, 5% CO 2 atmosphere and regularly tested for mycoplasma contamination.

Techniques: Hybridization, Staining, Microarray, Clone Assay

Journal: PLoS ONE

Article Title: Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation

doi: 10.1371/journal.pone.0178821

Figure Lengend Snippet: ( A ) Venn diagrams and Jaccard coefficients show the extent of overlap between NADs and LADs. LAD1: LADs of Tig3 cells , LAD2 and LAD3: LADs of IMR90 cells [ , ]. ( B ) Bar graphs show Gencode v19 and UniProt gene frequencies in NADs (red), iNADs (grey), LADs (black), and iLADs (white) based on UCSC Table Browser data. ( C ) RefSeq gene (ZNF, OR and DEF indicate zinc finger, olfactory receptor and defensin gene families, respectively) frequencies in NADs, iNADs, LADs, and iLADs. ( D ) Non-coding RNA gene (‘RNA genes’) and ( E ) repeat frequencies in NADs, iNADs, LADs, and iLADs. The SINE repeat bars are divided with a horizontal line into MIR (bottom) and Alu (top) sub-groups.

Article Snippet: Human IMR90 embryonic fibroblasts were obtained from Coriell Repositories (Cat. No. I90-79) and cultivated in DMEM (Gibco Cat. No. 21885–025 supplemented with 10% v/v Foetal Calf Serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin) at 37°C in humidified, 5% CO 2 atmosphere and regularly tested for mycoplasma contamination.

Techniques:

Journal: PLoS ONE

Article Title: Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation

doi: 10.1371/journal.pone.0178821

Figure Lengend Snippet: ( A ) Bar graph of nucleolus number in young, proliferating (‘Y’) and senescent (‘S’) IMR90 cells. Proliferating cells have 3.0±1.2 and senescent cells 1.7±1.1 nucleoli per nucleus. Z projections of mid-sections of representative confocal microscopy images are shown on the top. Nucleolar staining is shown in red and DAPI counterstain in blue (scale bars: 1.6 μm). ( B ) Maps of NADs on chromosome 5 from young and senescent cells. Genomic regions associated with nucleoli only in young (¬S) or senescent (¬Y) cells are shown also as individual tracks. ( C ) Y-only and S-only NADs are enriched in protein-coding genes compared to all NADs and the genome. RefSeq gene data were obtained from the UCSC Table Browser. ( D ) Boxplots show positive correlation of senescence-related loss of nucleolus association and gene activation. Global gene expression changes (log2 fold change in senescent versus young cells) in constitutive (Y∧S), Y-only and S-only NAD genes are shown. The notches are defined as +/-1.58*IQR/sqrt(n) and represent the 95% confidence interval for each median. Group means are significantly different for all comparisons (p-value < 0.05, Tukey HSD). ( E ) Stacked columns show that the association frequency of five selected genomic regions is similar in young and senescent cells. Nucleolus-association data were collected from 50 cells for each category. BAC clones 1 to 5: RP11-44B13, RP11-173M10, RP11-828F4, RP11-125O21, RP11-81M8.

Article Snippet: Human IMR90 embryonic fibroblasts were obtained from Coriell Repositories (Cat. No. I90-79) and cultivated in DMEM (Gibco Cat. No. 21885–025 supplemented with 10% v/v Foetal Calf Serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin) at 37°C in humidified, 5% CO 2 atmosphere and regularly tested for mycoplasma contamination.

Techniques: Confocal Microscopy, Staining, Activation Assay, Expressing, Clone Assay

Journal: PLoS ONE

Article Title: Nucleolus association of chromosomal domains is largely maintained in cellular senescence despite massive nuclear reorganisation

doi: 10.1371/journal.pone.0178821

Figure Lengend Snippet: ( A ) Semi-quantitative immunoblots show severely decreased H3K9me3 and Lamin B1 levels, less strongly decreased H3 levels and no detectable alterations in Lamin A/C, tubulin and GAPDH levels in senescence. The same amounts of whole cell extracts of young and senescent cells were loaded as serial two-fold dilutions on SDS-PA gels and analysed on immunoblots (see the entire dataset from three independent experiments in ). ( B ) H3K9me3 is accumulated at spatially compact satellite repeat clusters. 3D immuno-FISH shows strong co-localization of H3K9me3 and HSATII staining. Mid-section of a representative confocal microscopy image is shown. HSATII FISH signals are in red, DAPI counterstain in blue, and H3K9me3 immunofluorescence signals are in green (scale bar: 1.6 μm). ( C ) Quantitative immunofluorescence analysis of H3K9me3 distribution. The areas of interest are illustrated on a light optical section of a representative confocal microscopy image. The lamina- and nucleolus-associated areas label 240 nm distances from the edges of the DAPI and nucleolus staining, respectively. ( D ) Bee swarm plots of relative fluorescence intensities show senescence-dependent small decrease in H3K9me3 levels at the nuclear periphery and strong reduction at the perinucleolar space. Proliferating and senescent IMR90 cells were stained for H3K9me3 and the relative immunofluorescence intensities were measured at the nuclear periphery (lamina) and at the perinucleolar space (No). Values measured in proliferating cells (‘Y’) are shown in red, values measured in senescent cells (‘S’) are shown in blue. Results from individual cells are illustrated as single data points (n Y = 88, n S = 88). A solid line indicates the median, and thin lines the upper and lower quartile. Median: Y.lamina = 0.095, S.lamina = 0.084; Y.No = 0.052, S.No = 0.022. ( E ) Bee swarm plots indicate more heterogeneous H3K9me3 staining in the nucleus and at the nuclear periphery of senescent cells, but no change in the perinucleolar space. The heterogeneity of staining was calculated as coefficient of variation (C.V. = standard deviation/mean of fluorescence intensity) for the total nucleus (Nu), the nuclear periphery (lamina) and the perinucleolar space (No). Plot labels are as in ( D ). Median: Y.Nu = 0.573, S.Nu = 0.677; Y.lamina = 0.632, S.lamina = 0.709; Y.No = 0.576, S.No = 0.555, n Y = 88, n S = 88. ( F ) Bee swarm plots illustrate robust rearrangement of the most heterochromatic regions in the perinucleolar space. The distribution of the 10% brightest pixels was quantified at the nuclear periphery (lamina) and the perinucleolar space (No). Ratios were calculated compared to the whole nucleus. Plot labels are as in ( D ). Median: Y.lamina = 0.157, S.lamina = 0.137; Y.No = 0.056, S.No = 0.013. n Y = 88, n S = 88.

Article Snippet: Human IMR90 embryonic fibroblasts were obtained from Coriell Repositories (Cat. No. I90-79) and cultivated in DMEM (Gibco Cat. No. 21885–025 supplemented with 10% v/v Foetal Calf Serum, 100 U/mL Penicillin, 100 μg/mL Streptomycin) at 37°C in humidified, 5% CO 2 atmosphere and regularly tested for mycoplasma contamination.

Techniques: Western Blot, Staining, Confocal Microscopy, Immunofluorescence, Fluorescence, Standard Deviation