Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are widely used in studyin... more Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are widely used in studying basic mechanisms of cardiac arrhythmias that are caused by ion channelopathies. Unfortunately, the action potential profile of hiPSC-CMs-and consequently the profile of individual membrane currents active during that action potential-differs substantially from that of native human cardiomyocytes, largely due to almost negligible expression of the inward rectifier potassium current (IK1). In the present study, we attempted to "normalize" the action potential profile of our hiPSC-CMs by inserting a voltage dependent in silico IK1 into our hiPSC-CMs, using the dynamic clamp configuration of the patch clamp technique. Recordings were made from single hiPSC-CMs, using the perforated patch clamp technique at physiological temperature. We assessed three different models of IK1, with different degrees of inward rectification, and systematically varied the magnitude of the inserted IK1. Also, we modified the inserted IK1 in order to assess the effects of loss- and gain-of-function mutations in the KCNJ2 gene, which encodes the Kir2.1 protein that is primarily responsible for the IK1 channel in human ventricle. For our experiments, we selected spontaneously beating hiPSC-CMs, with negligible IK1 as demonstrated in separate voltage clamp experiments, which were paced at 1 Hz. Upon addition of in silico IK1 with a peak outward density of 4-6 pA/pF, these hiPSC-CMs showed a ventricular-like action potential morphology with a stable resting membrane potential near -80 mV and a maximum upstroke velocity >150 V/s (n = 9). Proarrhythmic action potential changes were observed upon injection of both loss-of-function and gain-of-function IK1, as associated with Andersen-Tawil syndrome type 1 and short QT syndrome type 3, respectively (n = 6). We conclude that injection of in silico IK1 makes the hiPSC-CM a more reliable model for investigating mechanisms underlying cardiac arrhythmias.
Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent c... more Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro
Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent c... more Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro
Embryonic stem (ES) cells are able to differentiate in vitro via embryo-like aggregates, so-calle... more Embryonic stem (ES) cells are able to differentiate in vitro via embryo-like aggregates, so-called "embryoid bodies", into derivatives of the endodermal, ectodermal and mesodermal lineage. We established standardised protocols for cardiogenesis, myogenesis, neurogenesis and vascular smooth muscle cell differentiation in vitro. The developmentally controlled expression of tissue-specific genes, proteins, ion channels and receptors during ES cell differentiation is the basis of several in vitro approaches: (1) "Loss of function" assays with ES cells containing homozygous mutations of specific genes, (2) "Gain of function" assays with ES cells overexpressing exogenous genes, (3) Developmental analysis of teratogenic/embryotoxic compounds in vitro, (4) Pharmacological assays and the establishment of model systems for pathological cell functions, and (5) The application of differentiation and growth factors for induction of selectively differentiated cells which, in the future, may be used as a source for tissue grafts. We propose the ES cell technology as valuable in vitro system to substitute and reduce the use of animals in basic and applied research.
During early mammalian development, primitive endoderm (PrE) is specified and segregated away fro... more During early mammalian development, primitive endoderm (PrE) is specified and segregated away from the pluripotent epiblast. At a later developmental stage, PrE forms motile parietal endoderm (PE) lying proximal to the trophectoderm, and visceral endoderm (VE) that contacts the developing epiblast and extraembryonic ectoderm. Mouse extraembryonic endoderm (XEN) cells were isolated and became widely used to study signals governing lineage specification. Rat XEN cell lines have also been derived, but were distinguished from mouse by expression of SSEA1 and Oct4. We showed here that rat XEN cells grown in the presence of a GSK3 inhibitor or overexpressing β-catenin exhibited enhanced formation of cell contacts and decreased motility. Rat XEN cells treated with BMP4 revealed similar morphological changes. Furthermore, we observed that rat XEN cells cultured with GSK3 inhibitor formed adhesion and tight junctions, and acquired bottom-top polarity, indicating the formation of VE cells. In...
Monitoring and controlling cardiac myocyte activity with optogenetic tools offers exciting possib... more Monitoring and controlling cardiac myocyte activity with optogenetic tools offers exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level. To test the hypothesis that cardiac myocyte-targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electrical activity in isolated cardiac myocytes and the whole heart as well as function and maturity in induced pluripotent stem cell (iPSC)-derived cardiac myocytes. We first generated mice with cardiac myocyte-restricted expression of VSFP2.3 and demonstrated distinct localization of VSFP2.3 at the t-tubulus/junctional sarcoplasmic reticulum microdomain without any signs for associated pathologies (assessed by echocardiography, RNA-sequencing, and patch clamping). Optically reco...
On the basis of their self-renewal capacity and their ability to differentiate into derivatives o... more On the basis of their self-renewal capacity and their ability to differentiate into derivatives of all three germ layers, germ line-derived multipotent adult stem cells (maGSCs) from mouse testis might serve as one of preferable sources for pluripotent stem cells in regenerative medicine. In our study, we aimed for an efficient hepatic differentiation protocol that is applicable for both maGSCs and embryonic stem cells (ESCs). We attempted to accomplish this goal by using a new established co-culture system with OP9 stroma cells for direct differentiation of maGSCs and ESCs into hepatic cells. We found that the hepatic differentiation of maGSCs was induced by the OP9 co-culture system in comparison to the gelatin culture. Furthermore, we showed that the combination of OP9 co-culture with activin A resulted in the increased expression of endodermal and early hepatic markers Gata4, Sox17, Foxa2, Hnf4, Afp, and Ttr compared to differentiated cells on gelatin or on OP9 alone. Moreover, ...
Cellular interactions with substrata of the microenvironment are one of the major mechanisms for ... more Cellular interactions with substrata of the microenvironment are one of the major mechanisms for differentiation and morphogenesis. Many of these interactions are mediated via the beta 1 integrin subfamily of cell surface receptors, which are believed to transduce signals upon cell adhesion. We have used beta 1 integrin-deficient embryonic stem cells to test their ability to differentiate into cardiac muscle cells. We show here by several approaches that beta 1 integrin is important for normal cardiogenesis. First, the in vitro differentiation of beta 1 integrin-deficient embryonic stem cells into cardiac muscle cells is retarded. This is demonstrated by the delayed expression of cardiac muscle-specific genes and action potentials. Second, the specification of cardiac precursor cells into pacemaker-, atrial- and ventricular-like cells is significantly impaired in beta 1 integrin-deficient cells. The occurrence of atrial- and ventricular-like cells is reduced and transient. Only cell...
Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are widely used in studyin... more Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are widely used in studying basic mechanisms of cardiac arrhythmias that are caused by ion channelopathies. Unfortunately, the action potential profile of hiPSC-CMs-and consequently the profile of individual membrane currents active during that action potential-differs substantially from that of native human cardiomyocytes, largely due to almost negligible expression of the inward rectifier potassium current (IK1). In the present study, we attempted to "normalize" the action potential profile of our hiPSC-CMs by inserting a voltage dependent in silico IK1 into our hiPSC-CMs, using the dynamic clamp configuration of the patch clamp technique. Recordings were made from single hiPSC-CMs, using the perforated patch clamp technique at physiological temperature. We assessed three different models of IK1, with different degrees of inward rectification, and systematically varied the magnitude of the inserted IK1. Also, we modified the inserted IK1 in order to assess the effects of loss- and gain-of-function mutations in the KCNJ2 gene, which encodes the Kir2.1 protein that is primarily responsible for the IK1 channel in human ventricle. For our experiments, we selected spontaneously beating hiPSC-CMs, with negligible IK1 as demonstrated in separate voltage clamp experiments, which were paced at 1 Hz. Upon addition of in silico IK1 with a peak outward density of 4-6 pA/pF, these hiPSC-CMs showed a ventricular-like action potential morphology with a stable resting membrane potential near -80 mV and a maximum upstroke velocity >150 V/s (n = 9). Proarrhythmic action potential changes were observed upon injection of both loss-of-function and gain-of-function IK1, as associated with Andersen-Tawil syndrome type 1 and short QT syndrome type 3, respectively (n = 6). We conclude that injection of in silico IK1 makes the hiPSC-CM a more reliable model for investigating mechanisms underlying cardiac arrhythmias.
Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent c... more Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro
Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent c... more Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro
Embryonic stem (ES) cells are able to differentiate in vitro via embryo-like aggregates, so-calle... more Embryonic stem (ES) cells are able to differentiate in vitro via embryo-like aggregates, so-called "embryoid bodies", into derivatives of the endodermal, ectodermal and mesodermal lineage. We established standardised protocols for cardiogenesis, myogenesis, neurogenesis and vascular smooth muscle cell differentiation in vitro. The developmentally controlled expression of tissue-specific genes, proteins, ion channels and receptors during ES cell differentiation is the basis of several in vitro approaches: (1) "Loss of function" assays with ES cells containing homozygous mutations of specific genes, (2) "Gain of function" assays with ES cells overexpressing exogenous genes, (3) Developmental analysis of teratogenic/embryotoxic compounds in vitro, (4) Pharmacological assays and the establishment of model systems for pathological cell functions, and (5) The application of differentiation and growth factors for induction of selectively differentiated cells which, in the future, may be used as a source for tissue grafts. We propose the ES cell technology as valuable in vitro system to substitute and reduce the use of animals in basic and applied research.
During early mammalian development, primitive endoderm (PrE) is specified and segregated away fro... more During early mammalian development, primitive endoderm (PrE) is specified and segregated away from the pluripotent epiblast. At a later developmental stage, PrE forms motile parietal endoderm (PE) lying proximal to the trophectoderm, and visceral endoderm (VE) that contacts the developing epiblast and extraembryonic ectoderm. Mouse extraembryonic endoderm (XEN) cells were isolated and became widely used to study signals governing lineage specification. Rat XEN cell lines have also been derived, but were distinguished from mouse by expression of SSEA1 and Oct4. We showed here that rat XEN cells grown in the presence of a GSK3 inhibitor or overexpressing β-catenin exhibited enhanced formation of cell contacts and decreased motility. Rat XEN cells treated with BMP4 revealed similar morphological changes. Furthermore, we observed that rat XEN cells cultured with GSK3 inhibitor formed adhesion and tight junctions, and acquired bottom-top polarity, indicating the formation of VE cells. In...
Monitoring and controlling cardiac myocyte activity with optogenetic tools offers exciting possib... more Monitoring and controlling cardiac myocyte activity with optogenetic tools offers exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level. To test the hypothesis that cardiac myocyte-targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electrical activity in isolated cardiac myocytes and the whole heart as well as function and maturity in induced pluripotent stem cell (iPSC)-derived cardiac myocytes. We first generated mice with cardiac myocyte-restricted expression of VSFP2.3 and demonstrated distinct localization of VSFP2.3 at the t-tubulus/junctional sarcoplasmic reticulum microdomain without any signs for associated pathologies (assessed by echocardiography, RNA-sequencing, and patch clamping). Optically reco...
On the basis of their self-renewal capacity and their ability to differentiate into derivatives o... more On the basis of their self-renewal capacity and their ability to differentiate into derivatives of all three germ layers, germ line-derived multipotent adult stem cells (maGSCs) from mouse testis might serve as one of preferable sources for pluripotent stem cells in regenerative medicine. In our study, we aimed for an efficient hepatic differentiation protocol that is applicable for both maGSCs and embryonic stem cells (ESCs). We attempted to accomplish this goal by using a new established co-culture system with OP9 stroma cells for direct differentiation of maGSCs and ESCs into hepatic cells. We found that the hepatic differentiation of maGSCs was induced by the OP9 co-culture system in comparison to the gelatin culture. Furthermore, we showed that the combination of OP9 co-culture with activin A resulted in the increased expression of endodermal and early hepatic markers Gata4, Sox17, Foxa2, Hnf4, Afp, and Ttr compared to differentiated cells on gelatin or on OP9 alone. Moreover, ...
Cellular interactions with substrata of the microenvironment are one of the major mechanisms for ... more Cellular interactions with substrata of the microenvironment are one of the major mechanisms for differentiation and morphogenesis. Many of these interactions are mediated via the beta 1 integrin subfamily of cell surface receptors, which are believed to transduce signals upon cell adhesion. We have used beta 1 integrin-deficient embryonic stem cells to test their ability to differentiate into cardiac muscle cells. We show here by several approaches that beta 1 integrin is important for normal cardiogenesis. First, the in vitro differentiation of beta 1 integrin-deficient embryonic stem cells into cardiac muscle cells is retarded. This is demonstrated by the delayed expression of cardiac muscle-specific genes and action potentials. Second, the specification of cardiac precursor cells into pacemaker-, atrial- and ventricular-like cells is significantly impaired in beta 1 integrin-deficient cells. The occurrence of atrial- and ventricular-like cells is reduced and transient. Only cell...
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