Emily Dick

Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a long QT syndrome type 2 mutation

Aims Congenital long QT syndromes (LQTSs) are associated with prolonged ventricular repolarization and sudden cardiac death. Limitations to existing clinical therapeutic management strategies prompted us to develop a novel human in vitro drug-evaluation system for LQTS type 2 (LQT2) that will complement the existing in vitro and in vivo models. Methods and results Skin fibroblasts from a patient with a KCNH2 G1681A mutation (encodes IKr potassium ion channel) were reprogrammed to human induced pluripotent stem cells (hiPSCs), which were subsequently differentiated to functional cardiomyocytes. Relative to controls (including the patients mother), multi-electrode array and patch-clamp electrophysiology of LQT2–hiPSC cardiomyocytes showed prolonged field/action potential duration. When LQT2–hiPSC cardiomyocytes were exposed to E4031 (an IKr blocker), arrhythmias developed and these presented as early after depolarizations (EADs) in the action potentials. In contrast to control cardiomyocytes, LQT2–hiPSC cardiomyocytes also developed EADs when challenged with the clinically used stressor, isoprenaline. This effect was reversed by β-blockers, propranolol, and nadolol, the latter being used for the patients therapy. Treatment of cardiomyocytes with experimental potassium channel enhancers, nicorandil and PD118057, caused action potential shortening and in some cases could abolish EADs. Notably, combined treatment with isoprenaline (enhancers/isoprenaline) caused EADs, but this effect was reversed by nadolol. Conclusions Findings from this paper demonstrate that patient LQT2–hiPSC cardiomyocytes respond appropriately to clinically relevant pharmacology and will be a valuable human in vitro model for testing experimental drug combinations.

Evaluating the utility of cardiomyocytes from human pluripotent stem cells for drug screening

Functional cardiomyocytes can now be derived routinely from hPSCs (human pluripotent stem cells), which collectively include embryonic and induced pluripotent stem cells. This technology presents new opportunities to develop pharmacologically relevant in vitro screens to detect cardiotoxicity, with a view to improving patient safety while reducing the economic burden to industry arising from high drug attrition rates. In the present article, we consider the need for human cardiomyocytes in drug-screening campaigns and review the strategies used to differentiate hPSCs towards the cardiac lineage. During early stages of differentiation, hPSC-cardiomyocytes display gene expression profiles, ultra-structures, ion channel functionality and pharmacological responses reminiscent of an embryonic phenotype, but maturation during extended time in culture has been demonstrated convincingly. Notably, hPSC-cardiomyocytes have been shown to respond in a highly predictable manner to over 40 compounds that have a known pharmacological effect on the human heart. This suggests that further development and validation of the hPSC-cardiomyocyte model as a tool for assessing cardiotoxicity is warranted.

Directed Differentiation of Human Embryonic Stem Cells to Interrogate the Cardiac Gene Regulatory Network

The limited ability of the heart to regenerate has prompted development of new systems to produce cardiomyocytes for therapeutics. While differentiation of human embryonic stem cells (hESCs) into cardiomyocytes has been well documented, the process remains inefficient and/or expensive, and progress would be facilitated by better understanding the early genetic events that cause cardiac specification. By maintaining a transgenic cardiac-specific MYH6-monomeric red fluorescent protein (mRFP) reporter hESC line in conditions that promote pluripotency, we tested the ability of combinations of 15 genes to induce cardiac specification. Screening identified GATA4 plus TBX5 as the minimum requirement to activate the cardiac gene regulatory network and produce mRFP(+) cells, while a combination of GATA4, TBX5, NKX2.5, and BAF60c (GTNB) was necessary to generate beating cardiomyocytes positive for cTnI and α-actinin. Including the chemotherapeutic agent, Ara-C, from day 10 of induced differentiation enriched for cTnI/α-actinin double positive cells to 45%. Transient expression of GTNB for 5-7 days was necessary to activate the cardiogenesis through progenitor intermediates in a manner consistent with normal heart development. This system provides a route to test the effect of different factors on human cardiac differentiation and will be useful in understanding the network failures that underlie disease phenotypes.

Exon Skipping and Gene Transfer Restore Dystrophin Expression in Human Induced Pluripotent Stem Cells-Cardiomyocytes Harboring DMD Mutations

With an incidence of ∼1:3,500 to 5,000 in male children, Duchenne muscular dystrophy (DMD) is an X-linked disorder in which progressive muscle degeneration occurs and affected boys usually die in their twenties or thirties. Cardiac involvement occurs in 90% of patients and heart failure accounts for up to 40% of deaths. To enable new therapeutics such as gene therapy and exon skipping to be tested in human cardiomyocytes, we produced human induced pluripotent stem cells (hiPSC) from seven patients harboring mutations across the DMD gene. Mutations were retained during differentiation and analysis indicated the cardiomyocytes showed a dystrophic gene expression profile. Antisense oligonucleotide-mediated skipping of exon 51 restored dystrophin expression to ∼30% of normal levels in hiPSC-cardiomyocytes carrying exon 47-50 or 48-50 deletions. Alternatively, delivery of a dystrophin minigene to cardiomyocytes with a deletion in exon 35 or a point mutation in exon 70 allowed expression levels similar to those seen in healthy cells. This demonstrates that DMD hiPSC-cardiomyocytes provide a novel tool to evaluate whether new therapeutics can restore dystrophin expression in the heart.

Faster generation of hiPSCs by coupling high-titer lentivirus and column-based positive selection

The protocols described here address methods used in two crucial stages in the retroviral reprogramming of somatic cells to produce human induced pluripotent stem cell (hiPSC) lines. The first is an optimized method for producing lentivirus at an efficiency 600-fold greater than previously published, and it includes conjugation of the lentivirus to streptavidin superparamagnetic particles; this process takes 8 d. The second method enables the isolation of true hiPSCs immediately after somatic cell reprogramming and involves column-based positive selection of cells expressing the pluripotency marker TRA-1-81. This process takes 2 h and, as it is directly compatible with feeder-free culture, the time burden of manually identifying and mechanically propagating hiPSC colonies is reduced drastically. Taken together, these methods accelerate the production of hiPSCs and enable lines to be isolated, expanded to ∼107 cells and cryopreserved within 6–8 weeks.

Exon skipping and gene transfer restore dystrophin expression in hiPSC-cardiomyocytes harbouring DMD mutations.

With an incidence of ~1:3,500 to 5,000 in male children, Duchenne muscular dystrophy (DMD) is an X-linked disorder in which progressive muscle degeneration occurs and affected boys usually die in their twenties or thirties. Cardiac involvement occurs in 90% of patients and heart failure accounts for up to 40% of deaths. To enable new therapeutics such as gene therapy and exon skipping to be tested in human cardiomyocytes, we produced human induced pluripotent stem cells (hiPSC) from seven patients harbouring mutations across the DMD gene. Mutations were retained during differentiation and analysis indicated the cardiomyocytes showed a dystrophic gene expression profile. Antisense oligonucleotide-mediated skipping of exon 51 restored dystrophin expression to ~30% of normal levels in hiPSC-cardiomyocytes carrying exon 47-50 or 48-50 deletions. Alternatively, delivery of a dystrophin minigene to cardiomyocytes with a deletion in exon 35 or a point mutation in exon 70 allowed expression levels similar to those seen in healthy cells. This demonstrates that DMD hiPSC-cardiomyocytes provide a novel tool to evaluate whether new therapeutics can restore dystrophin expression in the heart.

19 Drug evaluation in cardiomyocytes derived from human induced pluripotent stem cells carrying a Long QT Syndrome type 2 mutation

Background Congenital Long QT Syndromes (LQTS) are associated with prolonged ventricular repolarisation and sudden cardiac death. Limitations to existing clinical therapeutic management strategies prompted us to develop a novel human in vitro drug-evaluation system for LQT2 that will complement existing in vitro and in vivo models. Methods and Results Skin fibroblasts from a patient with a KCNH2 G1681A mutation (encodes IKr potassium ion channel) were reprogrammed to human induced pluripotent stem cells (hiPSCs), which were subsequently differentiated to functional cardiomyocytes. Relative to controls (including the patients mother), multi-electrode array and patch-clamp electrophysiology of LQT2-hiPSC cardiomyocytes showed prolonged field/action potential duration. When LQT2-hiPSC cardiomyocytes were exposed to E4031 (an IKr blocker), arrhythmias developed and these presented as early after depolarisations (EADs) in the action potentials. In contrast to control cardiomyocytes, LQT2-hiPSC cardiomyocytes also developed EADs when challenged with the clinically-used stressor, isoprenaline. This effect was reversed by ß-blockers, propranolol and nadolol, the latter being used for the patients therapy. Treatment of cardiomyocytes with experimental potassium channel enhancers, nicorandil and PD118057, caused action potential shortening. Notably, combined treatment with isoprenaline (enhancers/isoprenaline) caused EADs, but this effect was reversed by nadolol. Conclusions Findings from this paper demonstrate that patient LQT2-hiPSC cardiomyocytes respond appropriately to clinically-relevant pharmacology and will be a valuable human in vitro model for testing experimental drug combinations.