Role of oxygen exposure on the differentiation of human induced pluripotent stem cells in 2D and 3D cardiac organoids

Introduction Human induced pluripotent stem cells (hiPSC) have the ability to differentiate theoritically into any cell type. The development of organoid systems exhibiting the essential features of human organ such as liver and heart is of high interest. Optimizing the culture conditions to obtain the highest cardiac organoids efficacy is crucial. In fact, cardiac differentiation protocols have been established by essentially focusing on specific growth factors on hiPSC differentiation efficiency. However, the optimal environmental factors such as the optimal oxygen exposure to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Yet, the effect of low oxygen exposure on the molecular and functional maturity of the hiPSC-derived cardiomyocytes remains unexplored. Aims We aimed here at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hiPSCs in 2D monolayer and 3D organoids protocols. Methods hiPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed using qRT-PCR and immunofluorescence. The mitochondrial localization and metabolic properties were evaluated by high-resolution respirometry and mitochondrial staining. The intracellular Ca2+ handling and contractile properties were also monitored using confocal fluorescent microscopy and atomic force microscopy. Results Our results indicated that the 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac organoids containing hiPSC-CMs in LOE exhibited higher cardiac markers expression such as troponin T (TnTc), RyR2, Serca2a, alpha and beta heavy myosin chains. Moreover, we found enhanced contractile force, hypertrophy and steadier SR Ca2+ release reflected by a more regular spontaneous Ca2+ transients associated with a higher maximal amplitude and lower spontaneous Ca2+ events revealing a better SR Ca2+ handling in LOE. Similar beat rate, preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Conclusions Our results brought evidences that LOE is moderately beneficial for the 3D cardiac organoids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.Introduction Human induced pluripotent stem cells (hiPSC) have the ability to differentiate theoritically into any cell type. The development of organoid systems exhibiting the essential features of human organ such as liver and heart is of high interest. Optimizing the culture conditions to obtain the highest cardiac organoids efficacy is crucial. In fact, cardiac differentiation protocols have been established by essentially focusing on specific growth factors on hiPSC differentiation efficiency. However, the optimal environmental factors such as the optimal oxygen exposure to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Yet, the effect of low oxygen exposure on the molecular and functional maturity of the hiPSC-derived cardiomyocytes remains unexplored. Aims We aimed here at comparing the molecular and functional consequences of low (5% O2 or LOE) and high oxygen exposure (21% O2 or HOE) on cardiac differentiation of hiPSCs in 2D monolayer and 3D organoids protocols. Methods hiPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed using qRT-PCR and immunofluorescence. The mitochondrial localization and metabolic properties were evaluated by high-resolution respirometry and mitochondrial staining. The intracellular Ca2+ handling and contractile properties were also monitored using confocal fluorescent microscopy and atomic force microscopy. Results Our results indicated that the 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac organoids containing hiPSC-CMs in LOE exhibited higher cardiac markers expression such as troponin T (TnTc), RyR2, Serca2a, alpha and beta heavy myosin chains. Moreover, we found enhanced contractile force, hypertrophy and steadier SR Ca2+ release reflected by a more regular spontaneous Ca2+ transients associated with a higher maximal amplitude and lower spontaneous Ca2+ events revealing a better SR Ca2+ handling in LOE. Similar beat rate, preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Conclusions Our results brought evidences that LOE is moderately beneficial for the 3D cardiac organoids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE

Boronic acids for sensing and other applications - a mini-review of papers published in 2013

Boronic acids are increasingly utilised in diverse areas of research. Including the interactions of boronic acids with diols and strong Lewis bases as fluoride or cyanide anions, which leads to their utility in various sensing applications. The sensing applications can be homogeneous assays or heterogeneous detection. Detection can be at the interface of the sensing material or within the bulk sample. Furthermore, the key interaction of boronic acids with diols allows utilisation in various areas ranging from biological labelling, protein manipulation and modification, separation and the development of therapeutics. All the above uses and applications are covered by this mini-review of papers published during 2013

Post-Translational Modifications and Diastolic Calcium Leak Associated to the Novel RyR2-D3638A Mutation Lead to CPVT in Patient-Specific hiPSC-Derived Cardiomyocytes.

Sarcoplasmic reticulum Ca2+ leak and post-translational modifications under stress have been implicated in catecholaminergic polymorphic ventricular tachycardia (CPVT), a highly lethal inherited arrhythmogenic disorder. Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. The aims were to obtain functional hiPSC-derived cardiomyocytes from a CPVT patient harboring a novel ryanodine receptor (RyR2) mutation and model the syndrome, drug responses and investigate the molecular mechanisms associated to the CPVT syndrome. Patient-specific cardiomyocytes were generated from a young athletic female diagnosed with CPVT. The contractile, intracellular Ca2+ handling and electrophysiological properties as well as the RyR2 macromolecular remodeling were studied. Exercise stress electrocardiography revealed polymorphic ventricular tachycardia when treated with metoprolol and marked improvement with flecainide alone. We found abnormal stress-induced contractile and electrophysiological properties associated with sarcoplasmic reticulum Ca2+ leak in CPVT hiPSC-derived cardiomyocytes. We found inadequate response to metoprolol and a potent response of flecainide. Stabilizing RyR2 with a Rycal compound prevents those abnormalities specifically in CPVT hiPSC-derived cardiomyocytes. The RyR2-D3638A mutation is located in the conformational change inducing-central core domain and leads to RyR2 macromolecular remodeling including depletion of PP2A and Calstabin2. We identified a novel RyR2-D3638A mutation causing 3D conformational defects and aberrant biophysical properties associated to RyR2 macromolecular complex post-translational remodeling. The molecular remodeling is for the first time revealed using patient-specific hiPSC-derived cardiomyocytes which may explain the CPVT proband's resistance. Our study promotes hiPSC-derived cardiomyocytes as a suitable model for disease modeling, testing new therapeutic compounds, personalized medicine and deciphering underlying molecular mechanisms

Post-Translational Modifications and Diastolic Calcium Leak Associated to the Novel RyR2-D3638A Mutation Lead to CPVT in Patient-Specific hiPSC-Derived Cardiomyocytes

Background: Sarcoplasmic reticulum Ca2+ leak and post-translational modifications under stress have been implicated in catecholaminergic polymorphic ventricular tachycardia (CPVT), a highly lethal inherited arrhythmogenic disorder. Human induced pluripotent stem cells (hiPSCs) offer a unique opportunity for disease modeling. Objective: The aims were to obtain functional hiPSC-derived cardiomyocytes from a CPVT patient harboring a novel ryanodine receptor (RyR2) mutation and model the syndrome, drug responses and investigate the molecular mechanisms associated to the CPVT syndrome. Methods: Patient-specific cardiomyocytes were generated from a young athletic female diagnosed with CPVT. The contractile, intracellular Ca2+ handling and electrophysiological properties as well as the RyR2 macromolecular remodeling were studied. Results: Exercise stress electrocardiography revealed polymorphic ventricular tachycardia when treated with metoprolol and marked improvement with flecainide alone. We found abnormal stress-induced contractile and electrophysiological properties associated with sarcoplasmic reticulum Ca2+ leak in CPVT hiPSC-derived cardiomyocytes. We found inadequate response to metoprolol and a potent response of flecainide. Stabilizing RyR2 with a Rycal compound prevents those abnormalities specifically in CPVT hiPSC-derived cardiomyocytes. The RyR2-D3638A mutation is located in the conformational change inducing-central core domain and leads to RyR2 macromolecular remodeling including depletion of PP2A and Calstabin2. Conclusion: We identified a novel RyR2-D3638A mutation causing 3D conformational defects and aberrant biophysical properties associated to RyR2 macromolecular complex post-translational remodeling. The molecular remodeling is for the first time revealed using patient-specific hiPSC-derived cardiomyocytes which may explain the CPVT proband’s resistance. Our study promotes hiPSC-derived cardiomyocytes as a suitable model for disease modeling, testing new therapeutic compounds, personalized medicine and deciphering underlying molecular mechanisms

Voltammetric characterisation of diferrocenylborinic acid in organic solution and in aqueous media when immobilised into a titanate nanosheet film

Diferrocenylborinic acid (Fc2BOH, 1) has been synthesized in good yield via an improved synthetic path. Characterisation by nuclear magnetic resonance (NMR), mass spectrometry (HRMS), infrared spectroscopy (FTIR), X-ray crystallography, and by electrochemical methods reveal two one-electron oxidation processes for the two electronically coupled ferrocenyl moieties. The oxidation of 1 dissolved in organic media is contrasted to the oxidation of 1 in aqueous environments (by incorporation of 1 into a lamellar film of 2D titanate nanosheets on a glassy carbon electrode). Data from cyclic voltammetry and from square wave voltammetry suggest that the bridging boron can bind to nucleophiles (hydroxide, fluoride) upon oxidation of the ferrocenyl groups. A multi-pathway ECE reaction scheme is proposed. Potential applications in sensing are discussed.</p