Nóra Varga

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

Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth.

Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

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.

Resolution of a nontrivial diophantine equation without reduction methods

In this note a non-separable Runge-type diophantine equation related to the equal values of certain combinatorial numbers is solved. The novelty of our approach is avoiding the use of reduction methods, although as a first step we have a huge bound (≈ 10) for the solutions.

Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications

There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the ΔΔCt formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies.

On a generalization of a problem of Erdos and Graham

Abstract. In this paper we provide bounds for the size of the solutions of the Diophantine equation x(x+1)(x+2)(x+3) (x+a)(x+b) = y2, where a, b ∈ Z, a 6= b are parameters. We also determine all integral solutions for a, b ∈ {−4,−3,−2,−1, 4, 5, 6, 7}.

On some polynomial values of repdigit numbers

Abstract We study the equal values of repdigit numbers and the k dimensional polygonal numbers. We state some effective finiteness theorems, and for small parameter values we completely solve the corresponding equations.