Journal: Nature Neuroscience

Article Title: A CRISPRi/a platform in human iPSC-derived microglia uncovers regulators of disease states

doi: 10.1038/s41593-022-01131-4

Figure Lengend Snippet: a , Strategies for constitutive and inducible CRISPRi/CRISPRa in iTF-Microglia. Top, for constitutive CRISPRi, a dCas9-BFP-KRAB construct (catalytically dead Cas9 (dCas9) fused to BFP and the KRAB transcriptional repressor domain) is expressed from the constitutive CAG promotor integrated into the CLYBL safe-harbor locus. Middle, for inducible CRISPRi, dCas9-BFP-KRAB is tagged with ecDHFR degrons. Bottom, for inducible CRISPRa, CAG promotor-driven ecDHFR-dCas9-VPH was stably integrated into the CLYBL locus. VPH, activator domains containing 4× repeats of VP48, P65 and HSF1. Addition of TMP stabilizes the inducible CRISPRi/a machineries. b , c , Functional validation of constitutive ( b ) or inducible ( c ) CRISPRi activity via flow cytometry of TFRC surface protein level stained iTF-Microglia expressing a TFRC-targeting sgRNA or an NTC sgRNA at different days of differentiation (mean ± s.d., n = 3 biological replicates; P values from two-tailed Student’s t -test). c , TMP was added to induce CRISPRi activity where indicated. d , Functional validation of inducible CRISPRi activity via TFRC immunofluorescence (IF) microscopy on day 8. Top row, NTC sgRNA. Bottom row, sgRNA targeting TFRC . TFRC, red; F-actin, green; nuclei, blue. Scale bar, 100 μm. e , Functional validation of inducible CRISPRa activity via flow cytometry of CXCR4 surface protein level staining in iTF-Microglia expressing CXCR4 sgRNA or NTC sgRNA (mean ± s.d., n = 3 biological replicates; P values from two-tailed Student’s t -test). TMP was added to induce CRISPRa activity where indicated.

Article Snippet: Brownjohn iPSC-Microglia (Brownjohn-iMG) were differentiated from dCas9-KRAB iPSCs (AICS-0090, Coriell Cat. No. AICS-0090-391) using the published protocol with minor modifications.

Techniques: Construct, Stable Transfection, Functional Assay, Activity Assay, Flow Cytometry, Staining, Expressing, Two Tailed Test, Immunofluorescence, Microscopy

Journal: Bioengineering

Article Title: Effect of Rho–Associated Kinase Inhibitor on Growth Behaviors of Human Induced Pluripotent Stem Cells in Suspension Culture

doi: 10.3390/bioengineering9110613

Figure Lengend Snippet: The effect of Rho–associated protein kinase (ROCK) inhibitor on aggregate morphology, cell density, and aggregate number of two different hiPSC lines (( A ), Tic; ( B ), 1383D2) in suspension culture with short- and long-term exposure to ROCK inhibitor. ( A1 , B1 ) Aggregate morphology. Scale bars, 200 μm. ( A2 – 5 , B2 – 5 ) Cell density, aggregate number, apparent specific growth rate, and apparent specific decreasing rate of aggregates. Statistical significance was analyzed by two-tailed Student’s t -tests; * p < 0.05 ( n = 3 per culture). Error bars represent standard deviation. Open circle and bars, short-term exposure to ROCK inhibitor; Closed circle and bars, long-term exposure of ROCK inhibitor.

Article Snippet: The hiPSC lines (Tic and 1383D2) were obtained from the Japanese Collection of Research Bioresources Cell Bank and the Center for iPS Cell Research and Application at the Kyoto University [ ], respectively. hiPSCs were cultured on polystyrene plates coated with laminin-511 E8 fragments (iMatrix-511; Nippi, Inc., Tokyo, Japan) in specific medium (StemFit AK02N medium; Ajinomoto, Tokyo, Japan).

Techniques: Suspension, Two Tailed Test, Standard Deviation

Journal: Bioengineering

Article Title: Effect of Rho–Associated Kinase Inhibitor on Growth Behaviors of Human Induced Pluripotent Stem Cells in Suspension Culture

doi: 10.3390/bioengineering9110613

Figure Lengend Snippet: Distribution of projected area of two different hiPSC lines (( A ), Tic; ( B ), 1383D2) in suspension culture with short- and long-term exposure to ROCK inhibitor at 24, 72, and 120 h. Cell aggregates with 0–300 μm 2 were not considered as aggregates. All distributions followed non-normal nature based on the Shapiro-Wilk test ( p < 0.05).

Article Snippet: The hiPSC lines (Tic and 1383D2) were obtained from the Japanese Collection of Research Bioresources Cell Bank and the Center for iPS Cell Research and Application at the Kyoto University [ ], respectively. hiPSCs were cultured on polystyrene plates coated with laminin-511 E8 fragments (iMatrix-511; Nippi, Inc., Tokyo, Japan) in specific medium (StemFit AK02N medium; Ajinomoto, Tokyo, Japan).

Techniques: Suspension

Journal: Bioengineering

Article Title: Effect of Rho–Associated Kinase Inhibitor on Growth Behaviors of Human Induced Pluripotent Stem Cells in Suspension Culture

doi: 10.3390/bioengineering9110613

Figure Lengend Snippet: The effect of ROCK inhibitor on collagen type I localization in cell aggregates of two different hiPSC lines (( A ), Tic; ( B ), 1383D2) under short- and long-term exposure conditions. Florescent images of collagen type I at 120 h showing nuclei (red) and collagen type I (green) ( A1 – B4 ). Scale bars, 100 μm.

Article Snippet: The hiPSC lines (Tic and 1383D2) were obtained from the Japanese Collection of Research Bioresources Cell Bank and the Center for iPS Cell Research and Application at the Kyoto University [ ], respectively. hiPSCs were cultured on polystyrene plates coated with laminin-511 E8 fragments (iMatrix-511; Nippi, Inc., Tokyo, Japan) in specific medium (StemFit AK02N medium; Ajinomoto, Tokyo, Japan).

Techniques:

Journal: Bioengineering

Article Title: Effect of Rho–Associated Kinase Inhibitor on Growth Behaviors of Human Induced Pluripotent Stem Cells in Suspension Culture

doi: 10.3390/bioengineering9110613

Figure Lengend Snippet: The effect of ROCK inhibitor on phosphorylated myosin (pMLC) and F-actin localization in the aggregates of two different hiPSC lines (( A ), Tic; ( B ), 1383D2) under short- and long-term exposure conditions. Florescent images of pMLC and F-actin at 120 h showing nuclei (blue), pMLC (green), and F-actin (red) ( A1 – B6 ). Scale bars, 100 μm.

Article Snippet: The hiPSC lines (Tic and 1383D2) were obtained from the Japanese Collection of Research Bioresources Cell Bank and the Center for iPS Cell Research and Application at the Kyoto University [ ], respectively. hiPSCs were cultured on polystyrene plates coated with laminin-511 E8 fragments (iMatrix-511; Nippi, Inc., Tokyo, Japan) in specific medium (StemFit AK02N medium; Ajinomoto, Tokyo, Japan).

Techniques:

Journal: Bioengineering

Article Title: Effect of Rho–Associated Kinase Inhibitor on Growth Behaviors of Human Induced Pluripotent Stem Cells in Suspension Culture

doi: 10.3390/bioengineering9110613

Figure Lengend Snippet: Schematic drawing of our hypothesis that cell-derived collagen type I regulates cell division and coalescence between aggregates of two different hiPSC lines (( A ), Tic; ( B ), 1383D2) by inhibiting actomyosin contraction in suspension culture with prolonged ROCK inhibition. Panels ( A1 – B2 ) show magnified views of the boxed areas, respectively.

Article Snippet: The hiPSC lines (Tic and 1383D2) were obtained from the Japanese Collection of Research Bioresources Cell Bank and the Center for iPS Cell Research and Application at the Kyoto University [ ], respectively. hiPSCs were cultured on polystyrene plates coated with laminin-511 E8 fragments (iMatrix-511; Nippi, Inc., Tokyo, Japan) in specific medium (StemFit AK02N medium; Ajinomoto, Tokyo, Japan).

Techniques: Derivative Assay, Suspension, Inhibition

Journal: Stem cell research

Article Title: Genomic integrity of human induced pluripotent stem cells across nine studies in the NHLBI NextGen program

doi: 10.1016/j.scr.2020.101803

Figure Lengend Snippet: Genomic structural integrity of the NextGen hiPSC lines. Out of 506 hiPSC lines, 149 lines acquired CNVs during reprogramming/passaging.

Article Snippet: Reprogramming methods and maintenance of the NextGen hiPSC lines have been previously published ( and reference therein) and also available on the Wicell website ( www.wicell.org ; NHLBI NextGen collection).

Techniques:

Journal: Stem cell research

Article Title: Genomic integrity of human induced pluripotent stem cells across nine studies in the NHLBI NextGen program

doi: 10.1016/j.scr.2020.101803

Figure Lengend Snippet: Characteristics of the Copy Number Variants in NextGen hiPSC lines. [A] Histogram showing the number of hiPSC lines with number of detected CNVs. [B] Histogram showing the cumulative size of CNVs in mega base pairs (Mb) per hiPSC line. Black dashed line shows that cumulative CNV coverage for 85% hiPSC lines was less than 2 Mb. [C] Distribution of 258 CNVs acquired by hiPSC lines across the genome. The chromosomal regions harboring cluster of CNVs are indicated.

Article Snippet: Reprogramming methods and maintenance of the NextGen hiPSC lines have been previously published ( and reference therein) and also available on the Wicell website ( www.wicell.org ; NHLBI NextGen collection).

Techniques:

Journal: Stem cell research

Article Title: Genomic integrity of human induced pluripotent stem cells across nine studies in the NHLBI NextGen program

doi: 10.1016/j.scr.2020.101803

Figure Lengend Snippet: Gene set enrichment analysis for the set of 1,395 genes mapping to 258 CNVs from 149 hiPSC lines for enrichment with regards to biological processes. [A] Fold enrichment of the 22 GO Terms with an FDR of <5% ( q < 0.05) are displayed, q values <0.01 and <0.001 are shown as “*” and “**”, respectively. [B] Sequential Gene Set Enrichment of 4 biological process: I-kappaB kinase/NF-kappaB signaling cascade (GO:0007249), JNK cascade (GO:0007254), Cytokine production (GO:0001816) and IL-6 production (GO:0032635). Input set 1/2/3/4 and 5 include gene sets mapping to regions ≥1, ≥2, ≥3, ≥4 and ≥5 acquired CNVs in lines. The FE and FDR- P values increase markedly from input set1 through to input set5. [C] Sequential GSEA for gene sets mapping to regions of acquired Dels. Input set 1/2/3 and 4 include gene sets mapping to regions of ≥1, ≥2, ≥3 and ≥4 acquired Dels in hiPSCs. The FE and FDR- P values are higher for gene sets mapping to regions of Dels than all CNVs combined.

Article Snippet: Reprogramming methods and maintenance of the NextGen hiPSC lines have been previously published ( and reference therein) and also available on the Wicell website ( www.wicell.org ; NHLBI NextGen collection).

Techniques:

Journal: Stem cell research

Article Title: Genomic integrity of human induced pluripotent stem cells across nine studies in the NHLBI NextGen program

doi: 10.1016/j.scr.2020.101803

Figure Lengend Snippet: Chromosomal regions harboring clusters of CNVs. The chromosomal position of each CNV cluster is shown in a red box over the ideogram. The amplifications are represented as blue bars and deletions are shown in red bars. Dashed vertical lines delineate the core overlap regions, and in some cases nearby CNVs within the same genomic locus are also shown. The bottom panel shows the genes lying in the overlap region that could be potentially affected by the CNV in the hiPSC lines.

Article Snippet: Reprogramming methods and maintenance of the NextGen hiPSC lines have been previously published ( and reference therein) and also available on the Wicell website ( www.wicell.org ; NHLBI NextGen collection).

Techniques:

Journal: Stem cell research

Article Title: Genomic integrity of human induced pluripotent stem cells across nine studies in the NHLBI NextGen program

doi: 10.1016/j.scr.2020.101803

Figure Lengend Snippet: Expression levels of the transcripts mapping to the core overlap region of chr20 amplification. The chromosomal position of the amplification is shown in a red box over the ideogram. hiPSC lines harboring chr20 amplification are shown as blue bars and dashed vertical lines delineate the core overlap region. The bottom panel shows the fold changes comparing carriers to non-carries of the genes mapping to the overlap region, and their respective 95% confidence interval. Statistically significant up-regulation is observed for the genes marked in red. The expression of each gene is drawn positionally to the center of the gene.

Article Snippet: Reprogramming methods and maintenance of the NextGen hiPSC lines have been previously published ( and reference therein) and also available on the Wicell website ( www.wicell.org ; NHLBI NextGen collection).

Techniques: Expressing, Amplification

Journal: Stem cell research

Article Title: Genomic integrity of human induced pluripotent stem cells across nine studies in the NHLBI NextGen program

doi: 10.1016/j.scr.2020.101803

Figure Lengend Snippet: Details of sibling hiPSC lines. 25 hiPSC lines acquired CNVs as compared to their corresponding donor cell.

Article Snippet: Reprogramming methods and maintenance of the NextGen hiPSC lines have been previously published ( and reference therein) and also available on the Wicell website ( www.wicell.org ; NHLBI NextGen collection).

Techniques: