Tamás I. Orbán

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

Reliable transgene-independent method for determining Sleeping Beauty transposon copy numbers.

BackgroundThe transposon-based gene delivery technique is emerging as a method of choice for gene therapy. The Sleeping Beauty (SB) system has become one of the most favored methods, because of its efficiency and its random integration profile. Copy-number determination of the delivered transgene is a crucial task, but a universal method for measuring this is lacking. In this paper, we show that a real-time quantitative PCR-based, transgene-independent (qPCR-TI) method is able to determine SB transposon copy numbers regardless of the genetic cargo.ResultsWe designed a specific PCR assay to amplify the left inverted repeat-direct repeat region of SB, and used it together with the single-copy control gene RPPH1 and a reference genomic DNA of known copy number. The qPCR-TI method allowed rapid and accurate determination of SB transposon copy numbers in various cell types, including human embryonic stem cells. We also found that this sensitive, rapid, highly reproducible and non-radioactive method is just as accurate and reliable as the widely used blotting techniques or the transposon display method. Because the assay is specific for the inverted repeat region of the transposon, it could be used in any system where the SB transposon is the genetic vehicle.ConclusionsWe have developed a transgene-independent method to determine copy numbers of transgenes delivered by the SB transposon system. The technique is based on a quantitative real-time PCR detection method, offering a sensitive, non-radioactive, rapid and accurate approach, which has a potential to be used for gene therapy.

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].

Type and location of isocitrate dehydrogenase mutations influence clinical characteristics and disease outcome of acute myeloid leukemia

Abstract Mutations of isocitrate dehydrogenase 1 and 2 (IDH1/2) are genetic alterations in acute myeloid leukemia (AML). The aim of our study was to investigate the frequency and prognostic effect of IDH1/2 mutations together followed by an individual analysis of each substitution in a Hungarian cohort consisting of 376 patients with AML. IDH1mut and IDH2mut were mutually exclusive, detected in 8.5% and 7.5% of cases, respectively. IDH1/2mut was associated with: older age (p = 0.001), higher average platelet count (p = 0.001), intermediate karyotype (p < 0.0001), NPM1mut (p = 0.022) and lower mRNA expression level of ABCG2 gene (p = 0.006). Overall survival (OS), remission and relapse rates were not different in IDH1mut or IDH2mut vs. IDHneg. IDH1mut and IDH2mut were associated differently with NPM1mut; co-occurrence was observed in 14.3% of IDH1 R132C vs. 70% of R132H carriers (p = 0.02) and in 47.4% of IDH2 R140Q vs. 0% of R172K carriers (p = 0.02). IDH1 R132H negatively influenced OS compared to IDHneg (p = 0.02) or R132C (p = 0.019). Particular amino acid changes affecting the same IDH1 codon influence the clinical characteristics and treatment outcome in AML.

3' IsomiR species and DNA contamination influence reliable quantification of microRNAs by stem-loop quantitative PCR.

MicroRNAs (miRNAs) are ∼20–24 nucleotide-long regulatory RNAs that have been proven to play important roles in many cellular processes. Since their discovery, a number of different techniques have been developed to detect and accurately quantify them. For individual mature miRNA measurements, quantitative stem-loop real-time PCR represents a widely used method. Although there are some data on optimization of this technique, there are still many factors that have not been investigated yet. In this study, we have thoroughly optimized this technique and pointed out several important factors that influence reliable quantification. First, we found that total RNA input can affect the measurements. Second, our data showed that carryover DNA contamination could also mislead the detection in a sequence-specific manner. Additionally, we provided evidence that different 3′ isomiR species of a particular miRNA can be reverse transcribed and cross-detected even by specifically targeted assays. Besides these, we have investigated the measurement of reaction efficiencies from total RNA samples and the accuracy of simultaneous reverse transcription reactions for increasing reliability and cost effectiveness without the loss of sensitivity and specificity. In summary, we provide a detailed, refined protocol for reliable detection of microRNA species by quantitative stem-loop PCR.

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.

Combined localization and real-time functional studies using a GFP-tagged ABCG2 multidrug transporter

ABCG2 is a half-transporter which causes multidrug resistance when overexpressed in tumor cells. Availability of combined localization and functional assays would greatly improve cell biology and drug modulation studies for this transporter. Here we demonstrate that an N-terminally GFP-tagged version of the protein (GFP-G2) can be used to directly monitor ABCG2 expression, dimerization, localization and function in living cells. GFP-G2 is fully functional when tested for drug-stimulated ATPase activity, vesicular transport assay, subcellular localization or cell surface epitope conformational changes. By measuring both GFP and Hoechst 33342 dye fluorescence in HEK-293 cells, we provide evidence that a real-time transport assay can be reliably applied to identify ABCG2 substrates, transport modulators, as well as to monitor the cellular functions of this multidrug transporter protein. This approach also avoids the need of cloning, drug selection or other further separation or characterization of the transgene-expressing cells.

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.

Human mirtrons can express functional microRNAs simultaneously from both arms in a flanking exon-independent manner

Mirtrons are short intronic microRNA (miRNA) precursors representing an alternative, Drosha/DGCR8-independent miRNA biogenesis pathway. In this study we characterized three predicted human mirtrons. Their expression was proven to be context-independent, since functional mirtrons could be derived either from their endogenous or from a heterologous coding environment. Systematic testing revealed that both 5′- and 3′-arms of mir-877 are capable of producing functional miRNA simultaneously in the various cell types examined. On the other hand, experimental validations revealed that the predicted mir-1233 is not a bona fide mirtron. For functional mirtrons, we were able to detect mature mirtron-derived miRNAs for the first time by qRT-PCR or northern blot analysis, when silencing activity was proven by functional assays. Our results emphasize the need for functional testing of both arms of miRNAs and the importance of experimentally validating human mirtrons since, in spite of being localized in a short intron, predicted species could mature via other miRNA processing pathways.

PI3-kinase and mTOR inhibitors differently modulate the function of the ABCG2 multidrug transporter.

The ATP-binding cassette (ABC) transporter ABCG2 plays an important role in tissue detoxification and confers multidrug resistance to cancer cells. Identification of expressional and functional cellular regulators of this multidrug transporter is therefore intensively pursued. The PI3-kinase/Akt signaling axis has been implicated as a key element in regulating various cellular functions, including the expression and plasma membrane localization of ABCG2. Here we demonstrate that besides inhibiting their respective target kinases, the pharmacological PI3-kinase inhibitor LY294002 and the downstream mTOR kinase inhibitor rapamycin also directly inhibit ABCG2 function. In contrast, wortmannin, another commonly used pharmacological inhibitor of PI3-kinase does not interact with the transporter. We suggest that direct functional modulation of ABCG2 should be taken into consideration when pharmacological agents are applied to dissect the specific role of PI3-kinase/Akt/mTOR signaling in cellular functions.