A non-invasive platform for functional characterization of stem-cell-derived cardiomyocytes with applications in cardiotoxicity testing.

We present a non-invasive method to characterize the function of pluripotent stem-cell-derived cardiomyocytes based on video microscopy and image analysis. The platform, called Pulse, generates automated measurements of beating frequency, beat duration, amplitude, and beat-to-beat variation based on motion analysis of phase-contrast images captured at a fast frame rate. Using Pulse, we demonstrate recapitulation of drug effects in stem-cell-derived cardiomyocytes without the use of exogenous labels and show that our platform can be used for high-throughput cardiotoxicity drug screening and studying physiologically relevant phenotypes

A non-invasive platform for functional characterization of stem-cell-derived cardiomyocytes with applications in cardiotoxicity testing.

We present a non-invasive method to characterize the function of pluripotent stem-cell-derived cardiomyocytes based on video microscopy and image analysis. The platform, called Pulse, generates automated measurements of beating frequency, beat duration, amplitude, and beat-to-beat variation based on motion analysis of phase-contrast images captured at a fast frame rate. Using Pulse, we demonstrate recapitulation of drug effects in stem-cell-derived cardiomyocytes without the use of exogenous labels and show that our platform can be used for high-throughput cardiotoxicity drug screening and studying physiologically relevant phenotypes

Genomic Interaction Profiles in Breast Cancer Reveal Altered Chromatin Architecture

<div><p>Gene transcription can be regulated by remote enhancer regions through chromosome looping either in <i>cis</i> or in <i>trans</i>. Cancer cells are characterized by wholesale changes in long-range gene interactions, but the role that these long-range interactions play in cancer progression and metastasis is not well understood. In this study, we used <i>IGFBP3</i>, a gene involved in breast cancer pathogenesis, as bait in a 4C-seq experiment comparing normal breast cells (HMEC) with two breast cancer cell lines (MCF7, an ER positive cell line, and MDA-MB-231, a triple negative cell line). The <i>IGFBP3</i> long-range interaction profile was substantially altered in breast cancer. Many interactions seen in normal breast cells are lost and novel interactions appear in cancer lines. We found that in HMEC, the breast carcinoma amplified sequence gene family (<i>BCAS</i>) 1–4 were among the top 10 most significantly enriched regions of interaction with <i>IGFBP3.</i> 3D-FISH analysis indicated that the translocation-prone <i>BCAS</i> genes, which are located on chromosomes 1, 17, and 20, are in close physical proximity with <i>IGFBP3</i> and each other in normal breast cells. We also found that epidermal growth factor receptor (<i>EGFR),</i> a gene implicated in tumorigenesis, interacts significantly with <i>IGFBP3</i> and that this interaction may play a role in their regulation. Breakpoint analysis suggests that when an <i>IGFBP3</i> interacting region undergoes a translocation an additional interaction detectable by 4C is gained. Overall, our data from multiple lines of evidence suggest an important role for long-range chromosomal interactions in the pathogenesis of cancer.</p></div

<i>IGFBP3</i> interacts with <i>BCAS</i> genes.

<p>A, Representative triple labeled 3D-FISH, z-axis projection images of <i>IGFBP3, BCAS3, BCAS4</i> (left) and <i>IGFBP3, BCAS3, BCAS1</i> (right). Scale bar  = 10 µm. B, Percentage of nuclei with the listed pair of gene loci within 1 micron of each other. Distances were measured between the closest two foci in each nucleus.</p

Intrachromosomal interaction profile of <i>IGFBP3</i>.

<p>A, Spider plot showing the significant long-range interactions of the <i>IGFBP3</i> enhancer across chromosome 7 for a window size of 100 consecutive restriction fragments in HMEC (blue), MDA-MB-231 (red), and MCF7 (green). Mb position is plotted. Tick marks on chromosome 7 represent gene locations with positive strand genes on top and negative strand genes on bottom. B, Domainograms illustrating the significance of intrachromosomal interactions for window sizes ranging from 3 to 200 consecutive fragments for each cell line. The color represents −log(p-value) of the calculated significance score ranging from black (not significant) to white (most significant). The gray region corresponds to the centromere of chromosome 7, which lacks HindIII cut sites.</p

Common <i>trans</i> interactions among all samples.

<p>Common <i>trans</i> interactions among all samples.</p

Interaction frequency of <i>IGFBP3</i> with the breast cancer related gene <i>EGFR</i> by 3D-FISH.

<p>A, 3D-FISH labeling of breast cancer related loci in HMEC, MCF7, MDA-MB-231. BAC probe combinations: <i>IGFBP3</i> (green) and <i>EGFR</i> (red) n = 50, DAPI DNA stain (blue), boxes in lower right corner contain a magnified view of each interaction. Scale bar  = 10 µm. B, Cumulative percentage of distances between <i>IGFBP3</i> and <i>EGFR</i> loci. Distances were measured between the closest two foci in each nucleus. C, qRT-PCR: RNA levels of <i>EGFR</i> measured in MCF7, MDA-MB-231 and HMEC cells. Expression in cancer lines plotted as fold change relative to HMEC. Data represent the SEM of three independent biological replicates.</p

Interchromosomal interaction profile of <i>IGFBP3</i>.

<p>A, Circos plots showing the distribution of significant interchromosomal interactions involving <i>IGFBP3</i> in HMEC, MCF7 and MDA-MB-231. Grey lines in MCF7 plot represent interchromosomal translocations, adapted from Hampton <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073974#pone.0073974-Hampton1" target="_blank">[31]</a>, falling within windows of significant 4C interactions. B, Venn diagram showing the number of unique and overlapping significant interchromosomal interactions for a window size of 200 consecutive restriction fragments.</p

Distribution of MCF7 translocation breakpoints.

<p>Distribution of MCF7 translocation breakpoints.</p

Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions.

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes