The polymer polydimethylsiloxane (PDMS) is widely used to build microfluidic devices compatible with cell culture. Whilst convenient in manufacture, PDMS has the disadvantage that it can absorb small ...molecules such as drugs. In microfluidic devices like “Organs-on-Chip”, designed to examine cell behavior and test the effects of drugs, this might impact drug bioavailability. Here we developed an assay to compare the absorption of a test set of four cardiac drugs by PDMS based on measuring the residual non-absorbed compound by High Pressure Liquid Chromatography (HPLC). We showed that absorption was variable and time dependent and not determined exclusively by hydrophobicity as claimed previously. We demonstrated that two commercially available lipophilic coatings and the presence of cells affected absorption. The use of lipophilic coatings may be useful in preventing small molecule absorption by PDMS.
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•Binding of different compounds to PDMS varies greatly.•Previous reported correlations of absorption and LogP values could not be repeated.•Topological polar surface area possibly related to compound absorption.•A lipid based coating partially obviates compound absorption.•Presence of cultured cells affects free drug concentration, but less than substrate.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Technical advances in generating and phenotyping cardiomyocytes from human pluripotent stem cells (hPSC-CMs) are now driving their wider acceptance as in vitro models to understand human heart ...disease and discover therapeutic targets that may lead to new compounds for clinical use. Current literature clearly shows that hPSC-CMs recapitulate many molecular, cellular, and functional aspects of human heart pathophysiology and their responses to cardioactive drugs. Here, we provide a comprehensive overview of hPSC-CMs models that have been described to date and highlight their most recent and remarkable contributions to research on cardiovascular diseases and disorders with cardiac traits. We conclude discussing immediate challenges, limitations, and emerging solutions.
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EMUNI, FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Multi electrode arrays (MEAs) are increasingly used to detect external field potentials in electrically active cells. Recently, in combination with cardiomyocytes derived from human (induced) ...pluripotent stem cells they have started to become a preferred tool to examine newly developed drugs for potential cardiac toxicity in pre-clinical safety pharmacology. The most important risk parameter is proarrhythmic activity in cardiomyocytes which can cause sudden cardiac death. Whilst MEAs can provide medium- to high- throughput noninvasive assay platform, the translation of a field potential to cardiac action potential (normally measured by low-throughput patch clamp) is complex so that accurate assessment of drug risk to the heart is in practice still challenging. To address this, we used computational simulation to study the theoretical relationship between aspects of the field potential and the underlying cardiac action potential. We then validated the model in both primary mouse- and human pluripotent (embryonic) stem cell-derived cardiomyocytes showing that field potentials measured in MEAs could be converted to action potentials that were essentially identical to those determined directly by electrophysiological patch clamp. The method significantly increased the amount of information that could be extracted from MEA measurements and thus combined the advantages of medium/high throughput with more informative readouts. We believe that this will benefit the analysis of drug toxicity screening of cardiomyocytes using in time and accuracy.
•Computational model for the translation of field potential to action potential.•Validation of the model using patch clamp and multi electrode array.•Quantification of INA modulation, prolongation and triangulation from MEA data.•Validation of the correlations by measurements with known cardioactive drugs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Human heart tissues grown as three-dimensional spheroids and consisting of different cardiac cell types derived from pluripotent stem cells (hiPSCs) recapitulate aspects of human physiology better ...than standard two-dimensional models in vitro. They typically consist of less than 5000 cells and are used to measure contraction kinetics although not contraction force. By contrast, engineered heart tissues (EHTs) formed around two flexible pillars, can measure contraction force but conventional EHTs often require between 0.5 and 2 million cells. This makes large-scale screening of many EHTs costly. Our goals here were (i) to create a physiologically relevant model that required fewer cells than standard EHTs making them less expensive, and (ii) to ensure that this miniaturized model retained correct functionality. We demonstrated that fully functional EHTs could be generated from physiologically relevant combinations of hiPSC-derived cardiomyocytes (70%), cardiac fibroblasts (15%) and cardiac endothelial cells (15%), using as few as 1.6 × 104 cells. Our results showed that these EHTs were viable and functional up to 14 days after formation. The EHTs could be electrically paced in the frequency range between 0.6 and 3 Hz, with the optimum between 0.6 and 2 Hz. This was consistent across three downscaled EHT sizes tested. These findings suggest that miniaturized EHTs could represent a cost-effective microphysiological system for disease modelling and examining drug responses particularly in secondary screens for drug discovery.
•Co-culture based on three cardiac cell types derived from hiPSCs form functional Engineered Heart Tissues (EHTs).•Miniaturized EHTs with only 16,000 cells maintain functionality.•Three-fold miniaturized EHTs maintain the same contraction kinetics as their larger counterparts.•Downscaling of EHTs does not decrease force delivered per cardiomyocyte.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We previously generated a doxycycline-inducible H2B-mTurq2 reporter in hiPSCs to track cells and study cell division and apoptosis. To improve visualization of cycling cells, we introduced a ...ubiquitously transcribed mScarletI-Geminin (GMMN) (1–110) into the previously untargeted second AAVS1 allele. Fusion to the N-terminal part of GMNN provided tightly controlled mScarletI expression during the cell cycle. mScarletI fluorescence increased gradually from the S-phase through the M-phase of the cell cycle and was lost at the metaphase-anaphase transition. The resulting hiPSC reporter line generated, which we named ProLiving, is a valuable tool to study cell division and cell cycle characteristics in living hiPSC-derived cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A number of human congenital disorders present with both heart and limb defects, consistent with common genetic pathways. We have recently shown that the LIM homeodomain transcription factor islet 1 ...(Isl1) marks a subset of cardiac progenitors. Here, we perform lineage studies with an Isl1Cre mouse line to demonstrate that Isl1 also marks a subset of limb progenitors. In both cardiac and limb progenitors, Isl1 expression is downregulated as progenitors migrate in to form either heart or limb. To investigate common heart-limb pathways in Isl1-expressing progenitors, we ablated the Type I Bmp receptor, Bmpr1a utilizing Isl1Cre/+. Analysis of consequent heart and limb phenotypes has revealed novel requirements for Bmp signaling. Additionally, we find that Bmp signaling in Isl1-expressing progenitors is required for expression of T-box transcription factors Tbx2 and Tbx3 in heart and limb. Tbx3 is required for heart and limb formation, and is mutated in ulnar-mammary syndrome. We provide evidence that the Tbx3 promoter is directly regulated by Bmp Smads in vivo.
An induced pluripotent stem cell (iPSC) line, in which a H2B-fluorescent protein fusion is temporally expressed, is a valuable tool to track cells and study cell divisions and apoptosis. To this end ...we introduced a 3rd generation “all-in-one” doxycycline-inducible H2B-mTurquoise2 vector into the AAVS1 locus of PAX3-Venus iPSCs via CRISPR/Cas9. H2B-mTurquoise2 expression is absent but readily induced by doxycycline allowing quantification of cell divisions and imaging of living cells. Besides being a universal reporter in iPSC-based differentiation and toxicity assays, the generated pluripotent and genomically normal LUMCi041-A-2 line is particularly suited to study PAX3-positive stages of development.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Unexpected induction of arrhythmias in the heart is still one of the major risks of new drugs despite recent improvements in cardiac safety assays. Here we address this in a novel emerging assay ...system. Eleven reference compounds were administrated to spontaneously beating clusters of cardiomyocytes from human pluripotent stem cells (hPSC-CM) and the responses determined using multi-electrode arrays. Nine showed clear dose-dependence effects on field potential (FP) duration. Of these, the Ca2+ channel blockers caused profound shortening of action potentials, whereas the classical hERG blockers, like dofetilide and d,l-sotalol, induced prolongation, as expected.
Unexpectedly, two potent blockers of the slow component of the delayed rectifier potassium current (IKs), HMR1556 and JNJ303, had only minor effects on the extracellular FP of wild-type hPSC-CM despite evidence of functional IKs channels. These compounds were therefore re-evaluated under conditions that mimicked reduced “repolarization reserve,” a parameter reflecting the capacity of cardiomyocytes to repolarize and a strong risk factor for the development of ventricular arrhythmias. Strikingly, in both pharmacological and genetic models of diminished repolarization reserve, HMR1556 and JNJ03 strongly increased the FP duration. These profound effects indicate that IKs plays an important role in limiting action potential prolongation when repolarization reserve is attenuated. The findings have important clinical implications and indicate that enhanced sensitization to repolarization-prolonging compounds through pharmacotherapy or genetic predisposition should be taken into account when assessing drug safety.
► Validation of hPSC-CM for predictive cardiac safety pharmacology. ► IKs plays an important role in limiting action potential prolongation. ► Repolarization reserve determines drug sensitivity. ► Diminished repolarization reserve risk factor for QT prolongation and TdP.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Endoglin is a transmembrane accessory receptor for transforming growth factor‐β (TGF‐β) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels ...in vivo. Endoglin, as well as other TGF‐β signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor‐like kinase 1 (ALK1), an endothelial specific TGF‐β type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF‐β/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF‐β/ALK1 signalling, which indirectly inhibits TGF‐β/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF‐β/ALK1 signalling is reduced and TGF‐β/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF‐β‐induced growth arrest by indirectly reducing TGF‐β/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Deletion of the transforming growth factor β1 (TGF‐β1) gene in mice has previously suggested that it regulates both hematopoiesis and angiogenesis. To define the function of TGF‐β more precisely, we ...inactivated the TGF‐β type I receptor (TβRI) gene by gene targeting. Mice lacking TβRI die at midgestation, exhibiting severe defects in vascular development of the yolk sac and placenta, and an absence of circulating red blood cells. However, despite obvious anemia in the TβRI−/− yolk sacs, clonogenic assays on yolk sac‐derived hematopoietic precursors in vitro revealed that TβRI−/− mice exhibit normal hematopoietic potential compared with wild‐type and heterozygous siblings. Endothelial cells derived from TβRI‐deficient embryos show enhanced cell proliferation, improper migratory behavior and impaired fibronectin production in vitro, defects that are associated with the vascular defects seen in vivo. We thus demonstrate here that, while TβRI is crucial for the function of TGF‐β during vascular development and can not be compensated for by the activin receptor‐like kinase‐1 (ALK‐1), functional hematopoiesis and development of hematopoietic progenitors is not dependent on TGF‐β signaling via TβRI.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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