[Show abstract][Hide abstract] ABSTRACT: 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 recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch-clamping. The utility of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiac myocytes. Optical cardiograms (OCGs) could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in iPSC-derived cardiac myocytes.
We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof-of-concept for its utility in optogenetic sensing of physiological and pathological excitation in mature and immature cardiac myocytes in vitro and in vivo.
Circulation Research 06/2015; DOI:10.1161/CIRCRESAHA.117.306143 · 11.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human induced pluripotent stem cells (hiPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, iPSC-derived grafts are at risk of giving rise to teratomas in the host, if residuals of tumorigenic cells are not rejected by the recipient. We have analyzed the susceptibility of hiPSC lines to allogeneic and autologous natural killer (NK) cells. IL-2-activated, in contrast to resting NK cells killed hiPSC lines efficiently (P=1.69x10-39). Notably, the specific lysis of the individual hiPSC lines by IL-2-activated NK cells was significantly different (P=1.72x10-6) and ranged between 46 % and 64 % in 51Cr-release assays when compared to K562 cells. The hiPSC lines were killed by both allogeneic and autologous NK cells although autologous NK cells were less efficient (P=8.63x10-6). Killing was partly dependent on the activating NK receptor DNAM-1 (P=8.22x10-7). The DNAM-1 ligands CD112 and CD155 as well as the NKG2D ligands MICA and MICB were expressed on the hiPSC lines. Low amounts of human leukocyte antigen (HLA) class I proteins, which serve as ligands for inhibitory and activating NK receptors were also detected. Thus, the susceptibility to NK cell killing appears to constitute a common feature of hiPSCs. Therefore, NK cells might reduce the risk of teratoma formation even after autologous transplantations of pluripotent stem cell-derived grafts that contain traces of pluripotent cells.
PLoS ONE 05/2015; 10(5):e0125544. DOI:10.1371/journal.pone.0125544 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose: The ability to generate patient-specific human induced pluripotent stem cells (ps-iPSCs) provides a unique opportunity for modeling heart disease. Dilated cardiomyopathy (DCM) is due to a progressive enlargement of the heart leading finally to heart failure. Mutations in many genes have been implicated in the pathogenesis of DCM including the RNA-binding motif 20 (RBM20). We aimed to generate iPSCs from DCM patients with a RBM20 mutations, and to analyze the functionality and cell biology of cardiomyocytes derived from these ps-iPSCs (ps-iPSC-CM) with regard to the cardiac DCM phenotype.
Methods and Results: Dermal fibroblasts from two patients (34y; 75y) with DCM harboring different RBM20 mutations were reprogrammed to ps-iPSCs via lentiviral infection using the STEMCCA system. The ps-iPSCs show pluripotent characteristics by expressing pluripotency markers on mRNA and protein level similar to human embryonic stem cells. Furthermore, bisulfite sequencing analysis showed that the OCT4 and NANOG promoters were significantly demethylated in ps-iPSCs compared to their parental cells. In addition, the generated RBM20-iPSCs are able to differentiate in vitro and in vivo into derivatives of all three germ layers. The cardiac in vitro differentiation efficiency into beating cardiomyocytes of the RBM20-iPSCs is similar to control iPSCs. Electrophysiological studies showed that under basal conditions ps-iPSC-CM exhibited spontaneous action potentials characteristic for pacemaker-, atrial-, ventricular- and Purkinje-like cells in a similar manner as control iPSC-CM. Using Fast Fourier Transformation the sarcomere-associated periodic signal amplitude was analyzed for α-actinin. A significant higher percentage of RBM20-iPSC-CM showed abnormal sarcomeric α-actinin distribution compared to control CM, suggesting disorganized myofilament structure. But no significant difference was observed in cell size between RBM20-iPSC-CMs and control cells. Further, we found a different isoform expression pattern of the cardiac titin in the mutant ps-iPSC-CM compared to the control cells. This demonstrates a RBM20-dependent regulation of organized myofilament structure and titin splicing in an in vitro ps-DCM-iPSC-model.
Conclusion: We demonstrate that iPSC-derived cardiomyocytes from young and old DCM patients harboring a RBM20 mutation recapitulate the abnormalities that were found in individuals with DCM caused by the same mutation. The differentiated cardiomyocytes may be used for individual drug testing and development of novel treatments for this inherited disorder.
Cardiovascular Research 07/2014; 103(suppl 1):S48. DOI:10.1093/cvr/cvu083.2 · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract 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, the hepatic progenitors were capable of differentiating further into mature hepatic cells, demonstrated by the expression of liver-specific markers Aat, Alb, Tdo2, Krt18, Krt8, Krt19, Cps1, Sek, Cyp7a1, Otc, and Pah. A high percentage of maGSC-derived hepatic progenitors (51% AFP- and 61% DLK1-positive) and mature hepatic-like cells (26% ALB-positive) were achieved using this OP9 co-culture system. These generated hepatic cells successfully demonstrated in vitro functions associated with mature hepatocytes, including albumin and urea secretion, glycogen storage, and uptake of low-density lipoprotein. The established co-culture system for maGSCs into functional hepatic cells might serve as a suitable model to delineate the differentiation process for the generation of high numbers of mature hepatocytes in humans without genetic manipulations and make germ line-derived stem cells a potential autologous and alternative cell source for hepatic transplants in metabolic liver disorders.
[Show abstract][Hide abstract] ABSTRACT: Barth syndrome (BTHS) patients carrying mutations in tafazzin (TAZ1), which is involved in the final maturation of cardiolipin, present with dilated cardiomyopathy, skeletal myopathy, growth retardation and neutropenia. To study how mitochondrial function is impaired in BTHS patients, we generated induced pluripotent stem cells (iPSCs) to develop a novel and relevant human model system for BTHS. BTHS-iPSCs generated from dermal fibroblasts of three patients with different mutations in TAZ1 expressed pluripotency markers, and were able to differentiate into cells derived from all three germ layers both in vitro and in vivo. We used these cells to study the impact of tafazzin deficiency on mitochondrial oxidative phosphorylation. We found an impaired remodeling of cardiolipin, a dramatic decrease in basal oxygen consumption rate and in the maximal respiratory capacity in BTHS-iPSCs. Simultaneous measurement of extra-cellular acidification rate allowed us a thorough assessment of the metabolic deficiency in BTHS patients. Blue native gel analyses revealed that decreased respiration coincided with dramatic structural changes in respiratory chain supercomplexes leading to a massive increase in generation of reactive oxygen species. Our data demonstrate that BTHS-iPSCs are capable of modeling BTHS by recapitulating the disease phenotype and thus are important tools for studying the disease mechanism.
Stem Cell Research 05/2013; 11(2):806-819. DOI:10.1016/j.scr.2013.05.005 · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AimsInduced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes.Methods and resultsWe generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca(2+) transients and Ca(2+) sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated.Conclusions
Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell origins are similar. However, MSC-derived hiPSCs revealed a higher cardiac differentiation efficiency than keratinocyte- and fibroblast-derived hiPSCs.
European Heart Journal 07/2012; 34(33). DOI:10.1093/eurheartj/ehs203 · 14.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The basic zipper Gcn4 protein activates transcription in the yeast Saccharomyces cerevisiae in response to amino acid starvation. This includes numerous metabolic genes of amino acid or purine biosynthesis and the developmental cell-surface flocculin gene FLO11, which is required for diploid pseudohyphae formation and for adhesion upon nutrient starvation. We separated the metabolic from the developmental response by screening for GCN4 alleles that allow growth during amino acid starvation but are impaired in adhesion and are unable to form pseudohyphae. The identified Gcn4(L267S) variant carries an amino acid substitution in the third of the four conserved leucines of the zipper dimerization domain. This mutation abolished FLO11 expression and results in reduced but sufficient transcriptional activity for amino acid biosynthetic genes. The Leu267Ser substitution impairs Gcn4 homodimer formation and is a significantly more stable protein than the wild-type protein. A helix-breaker substitution in Leu253 results in a transcriptionally inactive but highly stable protein variant. This is due to a feedback circuit between transcriptional activity of Gcn4 and its own stability, which depends on the Gcn4-controlled cyclin PCL5. Gcn4(L253G) reduces the expression of Pcl5 and therefore reduces its own degradation. This self-controlled buffer system to restrict transcriptional activity results in a reciprocal correlation between Gcn4 transcriptional activity and protein stability.
[Show abstract][Hide abstract] ABSTRACT: Multipotent adult germ-line stem cells (maGSCs) and induced pluripotent stem cells (iPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, effects of the immune system on these cells have not been investigated. We have compared the susceptibility of maGSC lines to IL-2-activated natural killer (NK) cells with embryonic stem cell (ESC) lines, iPSCs, and F9 teratocarcinoma cells. The killing of pluripotent cell lines by syngeneic, allogeneic, and xenogeneic killer cells ranged between 48 and 265% in chromium release assays when compared to YAC-1 cells, which served as highly susceptible reference cells. With the exception of 2 maGSC lines, they expressed ligands for the activating NK receptor NKG2D that belong to the RAE-1 family, and killing could be inhibited by soluble NKG2D, demonstrating a functional role of these molecules. Furthermore, ligands of the activating receptor DNAM-1 were frequently expressed. The susceptibility to NK cells might constitute a common feature of pluripotent cells. It could result in rejection after transplantation, as suggested by a reduced teratoma growth after NK cell activation in vivo, but it might also offer a strategy to deplete contaminating pluripotent cells before grafting of differentiated cells.
The FASEB Journal 02/2010; 24(7):2164-77. DOI:10.1096/fj.09-134957 · 5.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently, we reported the successful establishment of multipotent adult germ-line stem cells (maGSCs) from cultured adult mouse spermatogonial stem cells. Similar to embryonic stem cells, maGSCs are able to self-renew and differentiate into derivatives of all three germ layers. These properties make maGSCs a potential cell source for the treatment of neural degenerative diseases. In this study, we describe the generation of maGSC-derived proliferating neural precursor cells using growth factor-mediated neural lineage induction. The neural precursors were positive for nestin and Sox1 and could be continuously expanded. Upon further differentiation, they formed functional neurons and glial cells, as demonstrated by expression of lineage-restricted genes and proteins and by electrophysiological properties. Characterization of maGSC-derived neurons revealed the generation of specific subtypes, including GABAergic, glutamatergic, serotonergic, and dopaminergic neurons. Electrophysiological analysis revealed passive and active membrane properties and postsynaptic currents, indicating their functional maturation. Functional networks formed at later stages of differentiation, as evidenced by synaptic transmission of spontaneous neuronal activity. In conclusion, our data demonstrate that maGSCs may be used as a new stem cell source for basic research and biomedical applications.
Stem Cell Research 04/2009; 2(2):139-54. DOI:10.1016/j.scr.2008.09.001 · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pcl5 is a Saccharomyces cerevisiae cyclin that directs the phosphorylation of the general amino acid control transcriptional activator Gcn4 by the cyclin-dependent kinase (CDK) Pho85. Phosphorylation of Gcn4 by Pho85/Pcl5 initiates its degradation via the ubiquitin/proteasome system and is regulated by the availability of amino acids. In this study, we show that Pcl5 is a nuclear protein and that artificial dislocation of Pcl5 into the cytoplasm prevents the degradation of Gcn4. Nuclear localization of Pcl5 depends on the beta-importin Kap95 and does not require Pho85, Gcn4, or the CDK inhibitor Pho81. Pcl5 nuclear import is independent on the availability of amino acids and is mediated by sequences in its C-terminal domain. The nuclear localization signal is distinct from other functional domains of Pcl5. This is corroborated by a C-terminally truncated Pcl5 variant, which carries the N-terminal nuclear domain of Pho80. This hybrid is still able to fulfill Pcl5 function, whereas Pho80, which is another Pho85 interacting cyclin, does not mediate Gcn4 degradation.
[Show abstract][Hide abstract] ABSTRACT: Nutrient starvation results in the interaction of Saccharomyces cerevisiae cells with each other and with surfaces. Adhesive growth requires the expression of the FLO11 gene regulated by the Ras/cAMP/cAMP-dependent protein kinase, the Kss1p/MAPK, and the Gcn4p/general amino acid control pathway, respectively. Proteomics two-dimensional DIGE experiments revealed post-transcriptionally regulated proteins in response to amino acid starvation including the ribosomal protein Cpc2p/Asc1p. This putative translational regulator is highly conserved throughout the eukaryotic kingdom and orthologous to mammalian RACK1. Deletion of CPC2/ASC1 abolished amino acid starvation-induced adhesive growth and impaired basal expression of FLO11 and its activation upon starvation in haploid cells. In addition, the diploid Flo11p-dependent pseudohyphal growth during nitrogen limitation was CPC2/ASC1-dependent. A more detailed analysis revealed that a CPC2/ASC1 deletion caused increased sensitivity to cell wall drugs suggesting that the gene is required for general cell wall integrity. Phosphoproteome and Western hybridization data indicate that Cpc2p/Asc1p affected the phosphorylation of the translational initiation factors eIF2 alpha and eIF4A and the ribosome-associated complex RAC. A crucial role of Cpc2p/Asc1p at the ribosomal interface coordinating signal transduction, translation initiation, and transcription factor formation was corroborated.