Cardiac spheroids as promising in vitro models to study the human heart microenvironment.

Three-dimensional in vitro cell systems are a promising alternative to animals to study cardiac biology and disease. We have generated three-dimensional in vitro models of the human heart ("cardiac spheroids", CSs) by co-culturing human primary or iPSC-derived cardiomyocytes, endothelial cells and fibroblasts at ratios approximating those present in vivo. The cellular organisation, extracellular matrix and microvascular network mimic human heart tissue. These spheroids have been employed to investigate the dose-limiting cardiotoxicity of the common anti-cancer drug doxorubicin. Viability/cytotoxicity assays indicate dose-dependent cytotoxic effects, which are inhibited by the nitric oxide synthase (NOS) inhibitor L-NIO, and genetic inhibition of endothelial NOS, implicating peroxynitrous acid as a key damaging agent. These data indicate that CSs mimic important features of human heart morphology, biochemistry and pharmacology in vitro, offering a promising alternative to animals and standard cell cultures with regard to mechanistic insights and prediction of toxic effects in human heart tissue

Stem Cell-Derived Cardiac Spheroids as 3D In Vitro Models of the Human Heart Microenvironment.

Our laboratory has recently developed a novel three-dimensional in vitro model of the human heart, which we call the vascularized cardiac spheroid (VCS). These better recapitulate the human heart's cellular and extracellular microenvironment compared to the existing in vitro models. To achieve this, human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes, cardiac fibroblasts, and human coronary artery endothelial cells are co-cultured in hanging drop culture in ratios similar to those found in the human heart in vivo. The resulting three-dimensional cellular organization, extracellular matrix, and microvascular network formation throughout the VCS has been shown to mimic the one present in the human heart tissue. Therefore, VCSs offer a promising platform to study cardiac physiology, disease, and pharmacology, as well as bioengineering constructs to regenerate heart tissue

Tailoring an in-silico model to the electrophysiology of individual iPSC-derived caradiomyocyte lines: one size fits all?

Poster given at the Safety Pharmacology Society Annual Meeting in Berlin, September 2017.<br

Ruffles are a site of cooperative invasion.

<p>(A) 3D-reconstruction of ruffles on HeLa cells infected for 6 min with <i>S</i>.Tm^SopE(pGFP) at an m.o.i. of 250. The actin channel (phalloidin stain) is shown in grey, <i>S.</i> Typhimurium in green (GFP) and extracellular bacteria in red (anti-<i>Salmonella</i> LPS stain before permeabilization). 3 views of a typical, large ruffle (ruffle 1) and details of another ruffle (ruffle 2) are depicted. Scale bar: 10 µm. (B) HeLa cells were infected with a 1∶1 mixture of <i>S</i>.Tm^SopE and <i>S</i>.Tm^Δ4(pGFP)at an m.o.i. of 5 and a movie was acquired via DIC imaging (see also supplementary VideoS4). A snapshot of the movie is depicted. The tracks of 2representative bacteria and their estimated position in the z-axis are indicated by the colored ellipsoids. Both bacteria stop at the ruffle and stay for the remaining observation time (blue track: 4.95 s, green track: 1.24 s). Scale bar: 9 µm. (C) Quantification strategy for analyzing <i>S</i>. Typhimurium docking onto ruffles. HeLa cells were infected for 6 min with a 1∶1 mixture <i>S</i>.Tm^Δ4(pGFP) (green; reporter strain), shown in green and the <i>S</i>.Tm^SopE as a helper strain which did not express <i>gfp</i> at an m.o.i. of 62.5 for each strain. After infection, cells were fixed and stained for actin (TRITC-phalloidin, red) and extracellular <i>S.</i> Typhimurium (indirect immunofluorescence using an anti-<i>Salmonella</i> antibody; blue; stained before permeabilization). Three types of <i>S.</i> Typhimurium can be distinguished: Extracellular reporter <i>S</i>.Tm^Δ4 (labeled green and blue), extracellular helper <i>S</i>.Tm^SopE (only blue), and intracellular reporter <i>S</i>.Tm^Δ4 (only green). Intracellular helper <i>S</i>.Tm^SopE is non-fluorescent and cannot be detected. Scale bar: 10 µm.(D, E) HeLa cells were infected for 6 min with a 1∶1 mixture of a helper strain (either <i>S</i>.Tm^Δ4 or <i>S</i>.Tm^SopE) and the reporter strain <i>S</i>.Tm^Δ4(pGFP) at the indicated m.o.i.. Cells were stained for actin and extracellular bacteria were stained with anti-LPS antibodies. In the control scenario (helper strain <i>S</i>.Tm^Δ4, only non-ruffling cells) bound bacteria were quantified for the area of a whole cell (grey bars); in the ruffling scenario (helper strain <i>S</i>.Tm^SopE) bacteria were quantified over the area of a ruffle as explained in panel (B,C).Even with a complex 3D structure, the surface of a ruffle should be much smaller than the surface of a whole cell. Therefore, if anything, our approach should underestimate the specific recruitment of bacteria onto ruffles. Extracellular bacteria of the reporter strain and the helper strain were quantified separately ((D): reporter strain, expresses <i>gfp</i>; (E): helper strain; no <i>gfp</i>). The bars summarize 170–220 cells/ruffles from two independent experiments. ***: p<0.0001.</p

Cellular morphology affects docking of <i>S</i>. Typhimurium.

<p>(A) Cells were incubated with the indicated concentration of cytochalasin D for one hour prior to infection with<i>S</i>.Tm^Δ4(pGFP) at an m.o.i. of 125 for 10 min. Afterwards, cells were fixed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002810#ppat-1002810-g006" target="_blank">Fig. 6</a> and stained with DAPI and TRITC-phalloidin (stains actin). Stacks of confocal images were acquired in the actin channel (grey) and the bacteria channel (green). An extended focus projection and a reconstruction of a zx-layer are shown. Scale bar, upper images: 20 µm, zx-layer: 5 µm. (B)Cells were pre-treated with the indicated concentration of cytochalasin D for 1 hour and infected with the respective <i>S.</i> Typhimurium strain for 12 min. After washing, fixing and staining <i>S.</i> Typhimurium docking was quantified by an automated microscopy based docking assay <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002810#ppat.1002810-Misselwitz1" target="_blank">[9]</a>. The curves shown summarize 3 independent experiments. Error bars: standard deviation.</p

Cooperative invasion at membrane ruffles.

<p>(A) Scheme explaining our quantification strategy for cooperative invasion. (B) HeLa cells were incubated with <i>S</i>.Tm^SopE(pGFP) at the indicated m.o.i. for 9 min, followed by washing, fixation and staining of actin and extracellular bacteria (anti-LPS antibody; staining before permeabilization). Left panel: quantification of the fraction of cells carrying ruffles. Middle panel: quantification of the number of “inside” and “outside” <i>S</i>.Tm^SopE(pGFP) per individual ruffle. Right Panel: number of invaded <i>S</i>.Tm^SopE(pGFP) per cell, calculated by multiplying the number of intracellular bacteria per ruffle with the fraction of ruffling cells. The black line indicates the estimated number of invaded <i>S</i>.Tm^SopE(pGFP) assuming independent invasion events, extrapolated from experimental data of ruffles with only one associated bacterium at the lowest m.o.i.. Each data point summarizes 4×150 cells for the analysis of cellular ruffling and 4×25 cells for inside and outside bacteria from 2 independent experiments. Error bars: standard deviation.</p