iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types.

Large-scale collections of induced pluripotent stem cells (iPSCs) could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants) as determined using high-throughput RNA-sequencing and genotyping arrays, respectively. Using iPSCs from a family of individuals, we show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines

Functional and Molecular Characterization of the Role of CTCF in Human Embryonic Stem Cell Biology

<div><p>The CCCTC-binding factor CTCF is the only known vertebrate insulator protein and has been shown to regulate important developmental processes such as imprinting, X-chromosome inactivation and genomic architecture. In this study, we examined the role of CTCF in human embryonic stem cell (hESC) biology. We demonstrate that CTCF associates with several important pluripotency genes, including <em>NANOG, SOX2</em>, <em>cMYC</em> and <em>LIN28</em> and is critical for hESC proliferation. CTCF depletion impacts expression of pluripotency genes and accelerates loss of pluripotency upon BMP4 induced differentiation, but does not result in spontaneous differentiation. We find that CTCF associates with the distal ends and internal sites of the co-regulated 160 kb <em>NANOG-DPPA3-GDF3</em> locus. Each of these sites can function as a CTCF-dependent enhancer-blocking insulator in heterologous assays. In hESCs, CTCF exists in multisubunit protein complexes and can be poly(ADP)ribosylated. Known CTCF cofactors, such as Cohesin, differentially co-localize in the vicinity of specific CTCF binding sites within the <em>NANOG</em> locus. Importantly, the association of some cofactors and protein PARlation selectively changes upon differentiation although CTCF binding remains constant. Understanding how unique cofactors may impart specialized functions to CTCF at specific genomic locations will further illuminate its role in stem cell biology.</p> </div

CTCF sites within the <i>NANOG</i> locus can function as CTCF-dependent enhancer-blocking insulators.

<p>A. Luciferase reporter assay measuring enhancer-blocking activity of CTCF sites from the <i>NANOG</i> locus. Enhancer-blocking plasmids contained a CMV enhancer, a CMV promoter-driven luciferase reporter, and 1.2 kb of DNA encompassing CTCF binding sites from relevant regions were cloned into either XhoI or Pst1 restriction enzyme sites. <i>Luciferase</i> activity was normalized to that of a co-transfected <i>renilla-luciferase</i> and normalized relative to pELuc (empty vector) set to 100%. 5′HS4 from the chicken β-globin locus was used as a positive control. Mean ± SEM represented. B. Same as above except cells were transfected with either a scrambled siRNA control or siCTCF before transfection of XhoI luciferase reporter constructs. **represents p<0.01. C. Same as panel B except cells were transfected with PstI luciferase reporter constructs. **represents p<0.01, *represents p<0.05.</p

Differential association of CTCF interacting partners and protein poly(ADP- ribosyl)ation within the <i>NANOG</i> locus in pluripotent and differentiated hESCs.

<p>A. CTCF ChIP as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042424#pone-0042424-g003" target="_blank">Figure 3</a> in H9 hESCs (black bars) and H9 hESCs differentiated with BMP4 for 5 days (Diff. hESC). The α-IgG (control) immunoprecipitates significantly lower levels of DNA relative to input (data not shown). Mean ± SEM represented. B. Western analyses of indicated proteins in H9 hESCs and 5 day BMP4-treated H9 hESCs (Diff. hESC). Mean ± SEM represented. C. Top panel: Immunoprecipitation of CTCF followed by Western blot analysis of indicated proteins. Bottom panel: Immunoprecipitation using an anti-PAR antibody followed by Western blot analysis with CTCF or Nucleolin. 1% Input was used in both top and bottom panels. D–F. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042424#pone-0042424-g003" target="_blank">Figure 3</a>, except ChIP analysis of indicated proteins or modification. *represents p<0.05, **represents p<0.01. Mean ± SEM represented.</p

Effect of CTCF depletion on hESCs.

<p>A. RT-PCR (left) and Western analysis (right) showing CTCF mRNA and protein levels, respectively, in H9 hESCs transfected with a scrambled siRNA (control si) or siCTCF. mRNA levels are normalized to <i>GAPDH</i> and represented relative to pluripotent hESC set to 1. B. BrDU proliferation assay in H9 showing that CTCF knockdown impairs proliferation. A₄₅₀ for siCTCF is normalized to that of control si (scrambled control) set to 1. Mean ± SEM represented. * represents p<0.05. C. Schematic of experimental design for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042424#pone-0042424-g002" target="_blank">Figures 2D, E and F</a>. Each vertical line represents a 24 hr period. D. mRNA levels of indicated genes at 48 hrs and 96 hrs after CTCF knockdown in H9 and H1 hESCs. mRNA levels of indicated genes in control si and siCTCF were normalized to respective <i>GAPDH</i> levels. Subsequently, mRNA levels of siCTCF were normalized to control si set to 1. Mean ± SEM represented. E. RT-PCR analysis of indicated genes upon control si (black line) or siCTCF (red line) transfection followed by BMP4 treatment for 5 days in H9. mRNA levels were analyzed at the indicated days and normalized to <i>GAPDH</i>. Mean ± SEM represented. F. Immunofluorescence of NANOG and OCT4 proteins upon control si or siCTCF transfection followed by BMP4 treatment at indicated days in H9. Scale bar represents 360 µm.</p

Characterization of the hESC differentiation model.

<p>A. 10X Phase contrast images of H9 pluripotent hESCs and H9 hESCs induced to differentiate with BMP4 for 5 days. 200 ng/ml BMP4 in TeSR was used in all panels. B. RT-PCR analysis showing decrease in mRNA levels of indicated genes upon BMP4 treatment of H9 cells. Expression levels are normalized to <i>GAPDH</i> and represented relative to pluripotent ESCs set to 1. C. Western analysis showing decrease in expression levels of indicated proteins upon BMP4 treatment of H9 cells. D. hCG ELISA from supernatant of BMP4-treated (yellow bars) and control-treated H9 (black bars hCG levels in control cells (hESCs) were barely detectable. E. RT-PCR analysis showing increase in mRNA levels of indicated genes upon BMP4 treatment of H9. Expression levels are normalized to <i>GAPDH</i>.</p

Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells

Although adipose tissue is an expandable and readily attainable source of proliferating, multipotent stem cells, its potential for use in regenerative medicine has not been extensively explored. Here we report that adult human and mouse adipose-derived stem cells can be reprogrammed to induced pluripotent stem (iPS) cells with substantially higher efficiencies than those reported for human and mouse fibroblasts. Unexpectedly, both human and mouse iPS cells can be obtained in feeder-free conditions. We discovered that adipose-derived stem cells intrinsically express high levels of pluripotency factors such as basic FGF, TGFβ, fibronectin, and vitronectin and can serve as feeders for both autologous and heterologous pluripotent cells. These results demonstrate a great potential for adipose-derived cells in regenerative therapeutics and as a model for studying the molecular mechanisms of feeder-free iPS generation and maintenance