Huamin Liang

Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro

Several types of terminally differentiated somatic cells can be reprogrammed into a pluripotent state by ectopic expression of Klf4, Oct3/4, Sox2, and c‐Myc. Such induced pluripotent stem (iPS) cells have great potential to serve as an autologous source of cells for tissue repair. In the process of developing iPS‐cell‐based therapies, the major goal is to determine whether differentiated cells derived from iPS cells, such as cardiomyocytes (CMs), have the same functional properties as their physiological in vivo counterparts. Therefore, we differentiated murine iPS cells to CMs in vitro and characterized them by RTPCR, immunocytochemistry, and electrophysiology. As key markers of cardiac lineages, transcripts for Nkx2.5, αMHC, Mlc2v, and cTnT could be identified. Immunocytochemical stainings revealed the presence of organized sarcomeric actinin but the absence of mature atrial natriuretic factor. We examined characteristics and developmental changes of action potentials, as well as functional hormonal regulation and sensitivity to channel blockers. In addition, we determined expression patterns and functionality of cardiac‐specific voltage‐gated Na+, Ca2+, and K+ channels at early and late differentiation stages and compared them with CMs derived from murine embryonic stem cells (ESCs) as well as with fetal CMs. We conclude that iPS cells give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed cardiac ion channels;CMs generated from iPS cells have a ventricular phenotype;and cardiac development of iPS cells is delayed compared with maturation of native fetal CMs and of ESC‐derived CMs. This difference may reflect the incomplete reprogramming of iPS cells and should be critically considered in further studies to clarify the suitability of the iPS model for regenerative medicine of heart disorders.—Kuzmenkin, A., Liang, H., Xu, G., Pfannkuche, K., Eichhorn, H., Fatima, A., Luo, H., Saric, T., Wernig, M., Jaenisch, R., Hescheler, J. Functional characterization of cardiomyocytes derived from murine induced pluripotent stem cells in vitro. FASEB J. 23, 4168‐4180 (2009). www.fasebj.org

Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells

Cardiomyocytes generated from embryonic stem cells (ESCs) and induced pluripotent stem (iPS) cells are suggested for repopulation of destroyed myocardium. Because contractile properties are crucial for functional regeneration, we compared cardiomyocytes differentiated from ES cells (ESC‐CMs) and iPS cells (iPS‐CMs). Native myocardium served as control. Murine ESCs or iPS cells were differentiated 11 d in vitro and cocultured 5–7 d with irreversibly injured myocardial tissue slices. Vital embryonic ventricular tissue slices of similar age served for comparison. Force‐frequency relationship (FFR), effects of Ca2+, Ni2+, nifedipine, ryanodine, β‐adrenergic, and muscarinic modulation were studied during loaded contractions. FFR was negative for ESC‐CMs and iPS‐CMs. FFR was positive for embryonic tissue and turned negative after treatment with ryanodine. In all groups, force of contraction and relaxation time increased with the concentration of Ca2+ and decreased with nifedipine. Force was reduced by Ni2+. Isoproterenol (1 µM) increased the force most pronounced in embryonic tissue (207±31%, n=7;ESC‐CMs: 123±5%, n=4; iPS‐CMs: 120 ±4%, n=8). EC50 values were similar. Contractile properties of iPS‐CMs and ESC‐CMs were similar, but they were significantly different from ventricular tissue of comparable age. The results indicate immaturity of the sarcoplasmic reticulum and the β‐adrenergic response of iPS‐CMs and ESC‐CMs.—Xi, J., Khalil, M., Shishechian, N., Hannes, T., Pfannkuche, K., Liang, H., Fatima, A., Haustein, M., Suhr, F., Bloch, W., Reppel, M., Šarić, T., Wernig, M., Jaenisch, R., Brockmeier, K., Hescheler, J., Pillekamp, F. Comparison of contractile behavior of native murine ventricular tissue and cardiomyocytes derived from embryonic or induced pluripotent stem cells. FASEB J. 24, 2739–2751 (2010). www.fasebj.org

Cardiac myocytes derived from murine reprogrammed fibroblasts: intact hormonal regulation, cardiac ion channel expression and development of contractility.

Aims: Induced pluripotent stem (iPS) cells have a developmental potential similar to that of blastocyst-derived embryonic stem (ES) cells and may serve as an autologous source of cells for tissue repair, in vitro disease modelling and toxicity assays. Here we aimed at generating iPS cell-derived cardiomyocytes (CMs) and comparing their molecular and functional characteristics with CMs derived from native murine ES cells. Methods and Results: Beating cardiomyocytes were generated using a mass culture system from murine N10 and O9 iPS cells as well as R1 and D3 ES cells. Transcripts of the mesoderm specification factor T-brachyury and non-atrial cardiac specific genes were expressed in differentiating iPS EBs. Using immunocytochemistry to determine the expression and intracellular organisation of cardiac specific structural proteins we demonstrate strong similarity between iPS-CMs and ES-CMs. In line with a previous study electrophysiological analyses showed that hormonal response to β-adrenergic and muscarinic receptor stimulation was intact. Action potential (AP) recordings suggested that most iPS-CMs measured up to day 23 of differentiation are of ventricular-like type. Application of lidocaine, Cs+, SEA0400 and verapamil+ nifedipine to plated iPS-EBs during multi-electrode array (MEA) measurements of extracellular field potentials and intracellular sharp electrode recordings of APs revealed the presence of INa, If, INCX, and ICaL, respectively, and suggested their involvement in cardiac pacemaking, with ICaL being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to β-adrenergic stimulation. Conclusions: Our data demonstrate that the cardiogenic potential of iPS cells is comparable to that of ES cells and that iPS-CMs possess all fundamental functional elements of a typical cardiac cell, including spontaneous beating, hormonal regulation, cardiac ion channel expression and contractility. Therefore, iPS-CMs can be regarded as a potentially valuable source of cells for in vitro studies and cellular cardiomyoplasty.

Global transcriptional profiles of beating clusters derived from human induced pluripotent stem cells and embryonic stem cells are highly similar

BackgroundFunctional and molecular integrity of cardiomyocytes (CMs) derived from induced pluripotent stem (iPS) cells is essential for their use in tissue repair, disease modelling and drug screening. In this study we compared global transcriptomes of beating clusters (BCs) microdissected from differentiating human iPS cells and embryonic stem (ES) cells.ResultsHierarchical clustering and principal component analysis revealed that iPS-BCs and ES-BCs cluster together, are similarly enriched for cardiospecific genes and differ in expression of only 1.9% of present transcripts. Similarly, sarcomeric organization, electrophysiological properties and calcium handling of iPS-CMs were indistinguishable from those of ES-CMs. Gene ontology analysis revealed that among 204 genes that were upregulated in iPS-BCs vs ES-BCs the processes related to extracellular matrix, cell adhesion and tissue development were overrepresented. Interestingly, 47 of 106 genes that were upregulated in undifferentiated iPS vs ES cells remained enriched in iPS-BCs vs ES-BCs. Most of these genes were found to be highly expressed in fibroblasts used for reprogramming and 34% overlapped with the recently reported iPS cell-enriched genes.ConclusionsThese data suggest that iPS-BCs are transcriptionally highly similar to ES-BCs. However, iPS-BCs appear to share some somatic cell signature with undifferentiated iPS cells. Thus, iPS-BCs may not be perfectly identical to ES-BCs. These minor differences in the expression profiles may occur due to differential cellular composition of iPS-BCs and ES-BCs, due to retention of some genetic profile of somatic cells in differentiated iPS cell-derivatives, or both.

Global transcriptome analysis of murine embryonic stem cell-derived cardiomyocytes

BackgroundCharacterization of gene expression signatures for cardiomyocytes derived from embryonic stem cells will help to define their early biologic processes.ResultsA transgenic α-myosin heavy chain (MHC) embryonic stem cell lineage was generated, exhibiting puromycin resistance and expressing enhanced green fluorescent protein (EGFP) under the control of the α-MHC promoter. A puromycin-resistant, EGFP-positive, α-MHC-positive cardiomyocyte population was isolated with over 92% purity. RNA was isolated after electrophysiological characterization of the cardiomyocytes. Comprehensive transcriptome analysis of α-MHC-positive cardiomyocytes in comparison with undifferentiated α-MHC embryonic stem cells and the control population from 15-day-old embryoid bodies led to identification of 884 upregulated probe sets and 951 downregulated probe sets in α-MHC-positive cardiomyocytes. A subset of upregulated genes encodes cytoskeletal and voltage-dependent channel proteins, and proteins that participate in aerobic energy metabolism. Interestingly, mitosis, apoptosis, and Wnt signaling-associated genes were downregulated in the cardiomyocytes. In contrast, annotations for genes upregulated in the α-MHC-positive cardiomyocytes are enriched for the following Gene Ontology (GO) categories: enzyme-linked receptor protein signaling pathway (GO:0007167), protein kinase activity (GO:0004672), negative regulation of Wnt receptor signaling pathway (GO:0030178), and regulation of cell size (O:0008361). They were also enriched for the Biocarta p38 mitogen-activated protein kinase signaling pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway.ConclusionThe specific pattern of gene expression in the cardiomyocytes derived from embryonic stem cells reflects the biologic, physiologic, and functional processes that take place in mature cardiomyocytes. Identification of cardiomyocyte-specific gene expression patterns and signaling pathways will contribute toward elucidating their roles in intact cardiac function.

Human and murine embryonic stem cell-derived cardiomyocytes serve together as a valuable model for drug safety screening.

Aims: Screening of drug safety is typically performed in diverse non-human healthy species with an intact repolarization reserve. Nevertheless, these drugs are later applied in diseased humans with a reduced repolarization reserve. It would be optimal to set up a preclinical screening tool to estimate the proarrhythmic potential of drugs in human cardiac tissue with a reduced repolarization reserve in vitro. Methods and Results: In our study spontaneously beating human embryonic stem cell-derived cardiomyocytes clusters (hESCM) and murine ES cell-derived cardiomyocytes (mESCMs) were plated onto micro-electrode arrays (MEAs) to record the extracelluar field potentials (FPs) as well as effects of several antiarrhythmic drugs. In line with clinical observations the class III antiarrhythmic drugs (±)-sotalol, E4031 and class I antiarrhythmic drug quinidine led to a prolongation of the cardiac repolarization phase (FP duration, FPdur) and a decrease of the FP frequency. Verapamil (a class IV antiarrhythmic drug) decreased the FP frequency and shortened FPdur. Both, quinidine and verapamil, but not (±)-sotalol or E4031 decreased conduction velocities in hESCM clusters. Moreover, (±)-sotalol exerted stronger effects on FPdur in early developmental stages of hESCMs, as proof for a reduced repolarization reserve. The EC50 of the (±)-sotalol-induced prolongation of the FPdur was higher in mESCMs than in hESCMs implying species-dependent differences in cardiac repolarization. Likewise, the incidence of drug-induced early recurrent depolarization (ERDs) was higher in mESCMs than hESCMs. Conclusion: The combined measurement of drug effects on FP parameters in hESCMs and mESCMs serves as a reliable in vitro model for preclinical studies of drug safety.

Functional characterization and transcriptome analysis of embryonic stem cell-derived contractile smooth muscle cells.

Complete transcriptome profiling of contractile smooth muscle cells (SMCs) differentiated from embryonic stem cells is crucial for the characterization of smooth muscle gene expression signatures and will contribute to defining biological and physiological processes in these cells. We have generated a transgenic embryonic stem cell line expressing both the puromycin acetyl transferase and enhanced green fluorescent protein cassettes under the control of the Acta2 promoter. Applying a specific monolayer culture protocol using retinoic acid, a puromycin-resistant and enhanced green fluorescent protein–positive Acta2+ SMC population of 95% purity was isolated. Acta2+ SMCs were characterized by semiquantitative and quantitative RT-PCR profiling of SMC markers and by microarray expression profiling, as well as by immunostaining for SMC-specific cytoskeletal proteins. Patch-clamp electrophysiological characterization of these cells identified SMC-specific channels such as the ATP-sensitive potassium channel and the Ca2+-activated potassium channel. Culturing of Acta2+ SMCs in serum-containing medium resulted in a significant number of hypertrophic and binucleated cells failing to complete cell division. Functional characterization of the cells has been proved by stimulation of the cells with vasoactive agents, such as angiotensin II and endothelin. We concluded that our embryonic stem cell–derived SMC population possesses the contractile and hypertrophic phenotype of SMCs incapable of proliferation. This is the first study describing the complete transcriptome of ES-derived SMCs allowing identification of specific biological and physiological processes in the contractile phenotype SMCs and will contribute to the understanding of these processes in early SMCs derived from embryonic stem cells.

Functional Characterization of Inward Rectifier Potassium Ion Channel in Murine Fetal Ventricular Cardiomyocytes

Aims: Previous studies have shown the dramatic changes in electrical properties of murine fetal cardiomyocytes, while details on inward rectifier potassium current (IK1) are still seldom discussed. Thus we aimed to characterize the functional expression and functional role of IK1 in murine fetal ventricular cardiomyocytes. Methods: Whole cell patch clamp was applied to investigate the electrophysiological properties of IK1. Quantitative real-time PCR, western blotting and double-label immunofluorescence were further utilized to find out the molecular basis of IK1. Results: Compared to early developmental stage (EDS), IK1 at late developmental stage (LDS) displayed higher current density, stronger rectifier property and faster activation kinetics. It was paralleled with the downregulation of Kir2.3 and the upregulation of Kir2.1/Kir2.2. IK1 contributed to maintain the maximum diastolic potential (MDP), late repolarization phase (LRP) as well as the action potential duration (APD). However, the contribution to MDP and velocity of LRP did not change significantly with maturation. Conclusions: During fetal development, the switch of IK1 subtypes from Kir2.1/Kir2.3 to Kir2.1 resulted in the dramatic changes in IK1 electrophysiological properties.

The L-type Ca2+ Channels Blocker Nifedipine Represses Mesodermal Fate Determination in Murine Embryonic Stem Cells

Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca2+ channels of different cell types. Moreover, influx of Ca2+ through L-type Ca2+ channels (LTCCs) activates Ca2+ signaling pathways, which in turn contribute to numerous cellular processes. Although LTCCs are expressed in undifferentiated cells, very little is known about its contributions to the transcriptional regulation of mesodermal and cardiac genes. This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro. The murine embryonic stem (ES, cell line D3) and induced pluripotent stem (iPS, cell clone 09) cells were differentiated into enhanced green fluorescence protein (EGFP) expressing spontaneously beating cardiomyocytes (CMs). Early treatment of differentiating cells with 10 µM nifedipine led to a significant inhibition of the cardiac mesoderm formation and cardiac lineage commitment as revealed by gene regulation analysis. This was accompanied by the inhibition of spontaneously occurring Ca2+ transient and reduction of LTCCs current density (I CaL) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca2+ channel α1C (CaV1.2) subunits. Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function. Our data indicate that nifedipine blocks the determined path of pluripotent stem cells to cardiomyogenesis by inhibition of mesodermal commitment at early stages of differentiation, thus the proper upkeep Ca2+ concentration and pathways are essentially required for cardiac gene expression, differentiation and function.

Isolation and Functional Characterization of α-Smooth Muscle Actin Expressing Cardiomyocytes from Embryonic Stem Cells

Early mammalian heart development is characterized by transient expression of Α-smooth muscle actin (Acta2). To date, cardiomyocytes expressing Acta2 in the early stages of in vivo development have not been characterized. To functionally characterize Acta2-expressing cardiomyocytes, we used a transgenic ES cell line expressing both the puromycin acetyl transferase (Pac) and enhanced green fluorescent protein (EGFP) cassettes under the control of the Acta2 promoter. The onset of Acta2 expression occurred in parallel with the appearance of beating areas, indicating the formation of cardiomyocytes. Antibiotic selection resulted in a high yield of cardiomyocytes and smooth muscle cells. The green fluorescent beating areas stained positively for multiple cardiomyocyte markers. Comparative electrophysiological analysis including fetal and Α-MHC-expressing ES cell-derived cardiomyocyte controls showed that Acta2-positive cardiomyocytes contained pacemaker-, atrial- and ventricular-like phenotypes. Interestingly, the proportion of ventricular-like cells was much higher in the Acta2-positive cardiomyocytes population than in control Α-MHC-expressing cardiomyocytes (75 % and 12 %, respectively). The findings of the present study provide a novel approach for the identification and enrichment of Acta2-positive cardiomyocytes, especially of the ventricular phenotype under in vitro conditions.