Anita Schamberger

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].

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.

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.

The nonstop decay and the RNA silencing systems operate cooperatively in plants

Abstract Translation-dependent mRNA quality control systems protect the protein homeostasis of eukaryotic cells by eliminating aberrant transcripts and stimulating the decay of their protein products. Although these systems are intensively studied in animals, little is known about the translation-dependent quality control systems in plants. Here, we characterize the mechanism of nonstop decay (NSD) system in Nicotiana benthamiana model plant. We show that plant NSD efficiently degrades nonstop mRNAs, which can be generated by premature polyadenylation, and stop codon-less transcripts, which are produced by endonucleolytic cleavage. We demonstrate that in plants, like in animals, Pelota, Hbs1 and SKI2 proteins are required for NSD, supporting that NSD is an ancient and conserved eukaryotic quality control system. Relevantly, we found that NSD and RNA silencing systems cooperate in plants. Plant silencing predominantly represses target mRNAs through endonucleolytic cleavage in the coding region. Here we show that NSD is required for the elimination of 5′ cleavage product of mi- or siRNA-guided silencing complex when the cleavage occurs in the coding region. We also show that NSD and nonsense-mediated decay (NMD) quality control systems operate independently in plants.

Generation of multidrug resistant human tissues by overexpression of the ABCG2 multidrug transporter in embryonic stem cells

The ABCG2 multidrug transporter provides resistance against various endo- and xenobiotics, and protects the stem cells against toxins and stress conditions. We have shown earlier that a GFP-tagged version of ABCG2 is fully functional and may be used to follow the expression, localization and function of this transporter in living cells. In the present work we have overexpressed GFP-ABCG2, driven by a constitutive (CAG) promoter, in HUES9 human embryonic stem cells. Stem cell clones were generated to express the wild-type and a substrate-mutant (R482G) GFP-ABCG2 variant, by using the Sleeping Beauty transposon system. We found that the stable overexpression of these transgenes did not change the pluripotency and growth properties of the stem cells, nor their differentiation capacity to hepatocytes or cardiomyocytes. ABCG2 overexpression provided increased toxin resistance in the stem cells, and protected the derived cardiomyocytes against doxorubicin toxicity. These studies document the potential of a stable ABCG2 expression for engineering toxin-resistant human pluripotent stem cells and selected stem cell derived tissues.

Correction: Reliable transgene-independent method for determining sleeping beauty transposon copy numbers

Authors’ contributions OK established the HEK clones, OK and VK optimized the real-time PCR and performed copy number measurements, AS, ZE and AA established the HUES9 clones, GV helped in FACS measurements, LM measured copy numbers in HeLa clones, ZsI and ZI gave technical help and advices with the SB transposon work, BS provided financial support and discussed the data and TIO designed the overall strategy, analyzed the data and wrote the paper. All authors read and approved the final manuscript.