SUMMARYLong QT syndrome (LQTS) is caused by functional alterations in cardiac ion channels and is associated with prolonged cardiac repolarization time and increased risk of ventricular arrhythmias. Inherited type 2 LQTS (LQT2) and drug-induced LQTS both result from altered function of the hERG channel. We investigated whether the electrophysiological characteristics of LQT2 can be recapitulated in vitro using induced pluripotent stem cell (iPSC) technology. Spontaneously beating cardiomyocytes were differentiated from two iPSC lines derived from an individual with LQT2 carrying the R176W mutation in the KCNH2 (HERG) gene. The individual had been asymptomatic except for occasional palpitations, but his sister and father had died suddenly at an early age. Electrophysiological properties of LQT2-specific cardiomyocytes were studied using microelectrode array and patch-clamp, and were compared with those of cardiomyocytes derived from control cells. The action potential duration of LQT2-specific cardiomyocytes was significantly longer than that of control cardiomyocytes, and the rapid delayed potassium channel (IKr) density of the LQT2 cardiomyocytes was significantly reduced. Additionally, LQT2-derived cardiac cells were more sensitive than controls to potentially arrhythmogenic drugs, including sotalol, and demonstrated arrhythmogenic electrical activity. Consistent with clinical observations, the LQT2 cardiomyocytes demonstrated a more pronounced inverse correlation between the beating rate and repolarization time compared with control cells. Prolonged action potential is present in LQT2-specific cardiomyocytes derived from a mutation carrier and arrhythmias can be triggered by a commonly used drug. Thus, the iPSC-derived, disease-specific cardiomyocytes could serve as an important platform to study pathophysiological mechanisms and drug sensitivity in LQT2.

The recent progress in stem cell techniques has broadened the horizon for in vitro disease modeling. For desired in vivo like phenotypes, not only correct cell type specification will be critical, the microenvironmental context will be essential to achieve relevant responses. We demonstrate how a three dimensional (3D) culture of stem cell derived neurons can induce in vivo like responses related to Alzheimer's disease, not recapitulated with conventional 2D cultures. To acquire a neural population of cells we differentiated neurons from neuroepithelial stem cells, derived from induced pluripotent stem cells. p21-activated kinase mediated sensing of Aβ oligomers was only possible in the 3D environment. Further, the 3D phenotype showed clear effects on F-actin associated proteins, connected to the disease processes. We propose that the 3D in vitro model has higher resemblance to the AD pathology than conventional 2D cultures and could be used in further studies of the disease.

BackgroundInduced pluripotent stem cells (iPSC) provide means to study the pathophysiology of genetic disorders. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a malignant inherited ion channel disorder predominantly caused by mutations in the cardiac ryanodine receptor (RyR2). In this study the cellular characteristics of CPVT are investigated and whether the electrophysiological features of this mutation can be mimicked using iPSC -derived cardiomyocytes (CM).Methodology/Principal FindingsSpontaneously beating CMs were differentiated from iPSCs derived from a CPVT patient carrying a P2328S mutation in RyR2 and from two healthy controls. Calcium (Ca2+) cycling and electrophysiological properties were studied by Ca2+ imaging and patch-clamp techniques. Monophasic action potential (MAP) recordings and 24h-ECGs of CPVT-P2328S patients were analyzed for the presence of afterdepolarizations. We found defects in Ca2+ cycling and electrophysiology in CPVT CMs, reflecting the cardiac phenotype observed in the patients. Catecholaminergic stress led to abnormal Ca2+ signaling and induced arrhythmias in CPVT CMs. CPVT CMs also displayed reduced sarcoplasmic reticulum (SR) Ca2+ content, indicating leakage of Ca2+ from the SR. Patch-clamp recordings of CPVT CMs revealed both delayed afterdepolarizations (DADs) during spontaneous beating and in response to adrenaline and also early afterdepolarizations (EADs) during spontaneous beating, recapitulating the changes seen in MAP and 24h-ECG recordings of patients carrying the same mutation.Conclusions/SignificanceThis cell model shows aberrant Ca2+ cycling characteristic of CPVT and in addition to DADs it displays EADs. This cell model for CPVT provides a platform to study basic pathology, to screen drugs, and to optimize drug therapy.

BackgroundThe growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.Methodology/Principal FindingsHere, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.Conclusion/SignificanceOur results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.