Kornélia Szebényi

Applying a “Double-Feature” Promoter to Identify Cardiomyocytes Differentiated from Human Embryonic Stem Cells Following Transposon-Based Gene Delivery†‡

Human embryonic stem (HuES) cells represent a new potential tool for cell‐therapy and gene‐therapy applications. However, these approaches require the development of efficient, stable gene delivery, and proper progenitor cell and tissue separation methods. In HuES cell lines, we have generated stable, enhanced green fluorescent protein (EGFP)‐expressing clones using a transposon‐based (Sleeping Beauty) system. This method yielded high percentage of transgene integration and expression. Similarly to a lentiviral expression system, both the undifferentiated state and the differentiation pattern of the HuES cells were preserved. By using the CAG promoter, in contrast to several other constitutive promoter sequences (such as CMV, elongation factor 1α, or phosphoglycerate kinase), an exceptionally high EGFP expression was observed in differentiated cardiomyocytes. This phenomenon was independent of the transgene sequence, methods of gene delivery, copy number, and the integration sites. This “double‐feature” promoter behavior, that is providing a selectable marker for transgene expressing undifferentiated stem cells, and also specifically labeling differentiated cardiomyocytes, was assessed by transcriptional profiling. We found a positive correlation between CAG promoter‐driven EGFP transcription and expression of cardiomyocyte‐specific genes. Our experiments indicate an efficient applicability of transposon‐based gene delivery into HuES cells and provide a novel approach to identify differentiated tissues by exploiting a nontypical behavior of a constitutively active promoter, thereby avoiding invasive drug selection methods. Stem Cells 2009;27:1077–1087

Calcium signaling in pluripotent stem cells

Pluripotent stem cells represent a new source of biological material allowing the exploration of signaling phenomena during normal cell development and differentiation. Still, the calcium signaling pathways and intracellular calcium responses to various ligands or stress conditions have not been sufficiently explored as yet in embryonic or induced pluripotent stem cells and in their differentiated offspring. This is partly due to the special culturing conditions of these cell types, the rapid morphological and functional changes in heterogeneous cell populations during early differentiation, and methodological problems in cellular calcium measurements. In this paper, we review the currently available data in the literature on calcium signaling in pluripotent stem cells and discuss the potential shortcomings of these studies. Various assay methods are surveyed for obtaining reliable data both in undifferentiated embryonic stem cells and in specific, stem cell-derived human tissues. In this paper, we present the modulation of calcium signaling in human embryonic stem cells (hESC) and in their derivates; mesenchymal stem cell like (MSCl) cells and cardiac tissues using the fluorescent calcium indicator Fluo-4 and confocal microscopy. LPA, trypsin and angiotensin II were effective in inducing calcium signals both in HUES9 and MSCl cells. Histamine and thrombin induced calcium signal exclusively in the MSCl cells, while ATP was effective only in HUES9 cells. There was no calcium signal evoked by GABA, even at relatively high concentrations. In stem cell-derived cardiomyocytes a rapid increase in the beating rate and an increase of the calcium signal peaks could be observed after the addition of adrenaline, while verapamil led to a strong decrease in cellular calcium and stopped spontaneous contractions in a relaxed state.

Evaluation of ABCG2 expression in human embryonic stem cells: Crossing the same river twice?

A recent publication in Stem Cells states that human embryonicstem (ES) cells do not express ABCG2 and ‘‘…absence ofABCG2 isa novelfeatureof humanpluripotentstem cells,whichdistinguishes them from many other stem cells including mouseES cells’’ [1]. This is in sharp contrast to our observations [2]and the report of several other investigators who detectedABCG2 mRNA in various human ES cells [3–6]. The presenceof multidrug resistance ABC (MDR-ABC) transporters may sig-nificantly contribute to stem cell defense mechanisms; thus, thisis an important question that should be addressed properly.Our interest in ABC transporters dates back to the discov-ery of their role in cancer drug resistance over two decadesago. Since then, we have had ample opportunity to experiencehow insufficient methodology and a simplifying approachmay obscure the assessment of the impact of MDR-ABCtransporters on cancer patient survival. Measuring the func-tional expression of ABC transporters proved challengingbecause of the heterogeneity of tumors, the varying levels ofexpression, and the unreliability of the assay systems usedthroughout the trials. As a result, most reports were consid-ered controversial, and the true contribution of MDR-ABCtransporters to therapy failure could only be established onceassay conditions were standardized [7]. The key teaching ofthese extensive studies have immediate relevance to exploringtransporter expression in stem cells. First, MDR-ABC trans-porters are active extrusion pumps that may significantly mod-ify cellular homeostasis or endobiotic and xenobiotic resist-ance even at low levels. Therefore, the assays measuring theirimpact should be sensitive, quantitative, and should preferablytarget the function of the MDR-ABC transporters. Second,samples are often heterogeneous for MDR-ABC expression,as these proteins are rapidly regulated by numerous mecha-nisms, both at the transcriptional and processing levels. How-ever, this initial heterogeneity may be relevant in circumstan-ces of stress, survival, or proliferation. Third, in many cases,the cell type, the mechanism of cell transformation, or differ-entiation does not determine the expression or function ofMDR-ABC transporters. Rather, ABC transporters are modu-lated by numerous environmental conditions [7, 8].In the case of the paper by Zeng et al. [1], the appre-ciation of these features is not possible as there are manyexperimental flaws that are reminiscent of the limitationsthat our field had to overcome to evaluate the MDR ofcancer. First, the reverse transcription polymerase chainreaction (RT-PCR) results are not quantitated, and there isno effort to perform quantitative PCR studies for thedetection of the relevant messages. Second, the Hoechstdye efflux studies lack the essential negative control. Third,instead of using a highly specific ABCG2 inhibitor, theauthors make their case on the basis of the effect of vera-pamil, which is a weak and nonspecific inhibitor ofABCG2. Fourth, the immunostaining studies are not con-vincing, the antibody used requires cell permeabilization,and the membrane localization of ABCG2 is not examined.Fifth, detection of subpopulations is contradictory and isnot evaluated in the context of co-expression of stem cellmarkers. Therefore, this study does not allow conclusionsto be drawn regarding the presence or up- and downregu-lation of ABCG2 in human ES cells.In contrast, we emphasize again that with appropriateexperimental tools, the functional although heterogeneousexpression of membrane ABCG2 is detectable in undifferenti-ated human stem cells. Detailed documentation is not possi-ble here, but the key features of ABCG2 expression in fourdifferent ES cell lines are depicted in Figure 1 and in thesupporting information video. Here we used properly quanti-tated real-time PCR measurements, flow cytometry, and con-focal microscopy with costaining of relevant surface markers.Furthermore, we compare ES cells grown on MEF or Matri-gel, and we also evaluate the expression pattern of a mesen-chymal-like cell line (Figure 1C (F2)). We also document amicroscopic measurement of Hoechst dye uptake in undiffer-entiated stem cells, which is modulated by a specific ABCG2inhibitor. All these measurements suggest that ABCG2 ispresent at relatively high levels in the undifferentiatedhuman ES cells, highlighting its role in the protection of thisvaluable sanctuary against the damage by toxins, drugs, orhypoxia [8, 9].

Design, synthesis and biological evaluation of thiosemicarbazones, hydrazinobenzothiazoles and arylhydrazones as anticancer agents with a potential to overcome multidrug resistance.

There is a constant need for new therapies against multidrug resistant (MDR) cancer. An attractive strategy is to develop chelators that display significant antitumor activity in multidrug resistant cancer cell lines overexpressing the drug efflux pump P-glycoprotein. In this study we used a panel of sensitive and MDR cancer cell lines to evaluate the toxicity of picolinylidene and salicylidene thiosemicarbazone, arylhydrazone, as well as picolinylidene and salicylidene hydrazino-benzothiazole derivatives. Our results confirm the collateral sensitivity of MDR cells to isatin-β-thiosemicarbazones, and identify several chelator scaffolds with a potential to overcome multidrug resistance. Analysis of structure-activity-relationships within the investigated compound library indicates that NNS and NNN donor chelators show superior toxicity as compared to ONS derivatives regardless of the resistance status of the cells.

Characterization of calcium signals in human embryonic stem cells and in their differentiated offspring by a stably integrated calcium indicator protein

Intracellular calcium signaling pathways play a major role in cellular responses such as proliferation, differentiation and apoptosis. Human embryonic stem cells (hESC) provide new possibilities to explore the development and differentiation of various cell types of the human body. Intracellular calcium responses to various ligands and the calcium signaling pathways, however, have not been thoroughly studied in embryonic stem cells and in their differentiated progenies. In our previous work we demonstrated that the use of the fluorescent calcium indicator Fluo-4 with confocal microscopy allows sensitive and reliable measurements of calcium modulation in human embryonic stem cells and stem-cell derived cardiomyocytes. Here we developed a human embryonic stem cell line stably expressing a genetically encoded Ca(2+) indicator (GCaMP2) using a transposon-based gene delivery system. We found that the differentiation properties were fully preserved in the GCaMP2-expressing hESC lines and Ca imaging could be performed without the need of toxic dye-loading of the cells. In undifferentiated hES cells the calcium signals induced by various ligands, ATP, LPA, trypsin or angiotensin II were comparable to those in Fluo-4 loaded cells. In accordance with previous findings, no calcium signal was evoked by thrombin, histamine or GABA. Cardiomyocyte colonies differentiated from hES-GCaMP2 cells could be recognized by spontaneous contractions and Ca(2+) oscillations. GCaMP2-expressing neural cells were identified based on their morphological and immuno-staining properties and Ca signals were characterized on those cells. Characteristics of both the spontaneous and ligand-induced Ca(2+) signals, as well as their pharmacological modification could be successfully examined in these model cells by fluorescence imaging.

Visualization of Calcium Dynamics in Kidney Proximal Tubules

Intrarenal changes in cytoplasmic calcium levels have a key role in determining pathologic and pharmacologic responses in major kidney diseases. However, cell-specific delivery of calcium-sensitive probes in vivo remains problematic. We generated a transgenic rat stably expressing the green fluorescent protein-calmodulin-based genetically encoded calcium indicator (GCaMP2) predominantly in the kidney proximal tubules. The transposon-based method used allowed the generation of homozygous transgenic rats containing one copy of the transgene per allele with a defined insertion pattern, without genetic or phenotypic alterations. We applied in vitro confocal and in vivo two-photon microscopy to examine basal calcium levels and ligand- and drug-induced alterations in these levels in proximal tubular epithelial cells. Notably, renal ischemia induced a transient increase in cellular calcium, and reperfusion resulted in a secondary calcium load, which was significantly decreased by systemic administration of specific blockers of the angiotensin receptor and the Na-Ca exchanger. The parallel examination of in vivo cellular calcium dynamics and renal circulation by fluorescent probes opens new possibilities for physiologic and pharmacologic investigations.

Expression pattern of the human ABC transporters in pluripotent embryonic stem cells and in their derivatives

ATP‐binding cassette (ABC) transporters have key roles in various physiological functions as well as providing chemical defense and stress tolerance in human tissues. In this study, we have examined the expression pattern of all ABC proteins in pluripotent human embryonic stem cells (hESCs) and in their differentiated progenies. We paid special attention to the cellular expression and localization of multidrug transporter ABC proteins.

Generation of a Homozygous Transgenic Rat Strain Stably Expressing a Calcium Sensor Protein for Direct Examination of Calcium Signaling

In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na+/Ca2+ exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies.

Extensive astrocyte synchronization advances neuronal coupling in slow wave activity in vivo

Slow wave activity (SWA) is a characteristic brain oscillation in sleep and quiet wakefulness. Although the cell types contributing to SWA genesis are not yet identified, the principal role of neurons in the emergence of this essential cognitive mechanism has not been questioned. To address the possibility of astrocytic involvement in SWA, we used a transgenic rat line expressing a calcium sensitive fluorescent protein in both astrocytes and interneurons and simultaneously imaged astrocytic and neuronal activity in vivo. Here we demonstrate, for the first time, that the astrocyte network display synchronized recurrent activity in vivo coupled to UP states measured by field recording and neuronal calcium imaging. Furthermore, we present evidence that extensive synchronization of the astrocytic network precedes the spatial build-up of neuronal synchronization. The earlier extensive recruitment of astrocytes in the synchronized activity is reinforced by the observation that neurons surrounded by active astrocytes are more likely to join SWA, suggesting causality. Further supporting this notion, we demonstrate that blockade of astrocytic gap junctional communication or inhibition of astrocytic Ca2+ transients reduces the ratio of both astrocytes and neurons involved in SWA. These in vivo findings conclusively suggest a causal role of the astrocytic syncytium in SWA generation.

Pegylated liposomal formulation of doxorubicin overcomes drug resistance in a genetically engineered mouse model of breast cancer

Abstract Success of cancer treatment is often hampered by the emergence of multidrug resistance (MDR) mediated by P‐glycoprotein (ABCB1/Pgp). Doxorubicin (DOX) is recognized by Pgp and therefore it can induce therapy resistance in breast cancer patients. In this study our aim was to evaluate the susceptibility of the pegylated liposomal formulation of doxorubicin (PLD/Doxil®/Caelyx®) to MDR. We show that cells selected to be resistant to DOX are cross‐resistant to PLD and PLD is also ineffective in an allograft model of doxorubicin‐resistant mouse B‐cell leukemia. In contrast, PLD was far more efficient than DOX as reflected by a significant increase of both relapse‐free and overall survival of Brca1−/−;p53−/− mammary tumor bearing mice. Increased survival could be explained by the delayed onset of drug resistance. Consistent with the higher Pgp levels needed to confer resistance, PLD administration was able to overcome doxorubicin insensitivity of the mouse mammary tumors. Our results indicate that the favorable pharmacokinetics achieved with PLD can effectively overcome Pgp‐mediated resistance, suggesting that PLD therapy could be a promising strategy for the treatment of therapy‐resistant breast cancer patients. Graphical abstract Figure. No Caption available.