Engraftment of engineered ES cell–derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium

Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)–derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6–10-fold because of induction of proliferation on purification. Long-term engraftment (4–5 months) was observed when co-transplanting selected ES cell–derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell–derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells

Development of a molecular detection and differentiation system for ochratoxin A producing Penicillium species and its application to analyse the occurrence of Penicillium nordicum in cured meats

A PCR method for differentiation and detection of the two known ochratoxin A producing Penicillium species, Penicillium verrucosum and Penicillium nordicum has been developed. It is based upon two genes of the ochratoxin A biosynthetic pathway, namely the ochratoxin A polyketide synthase gene (otapksPN) and a non-ribosomal peptide syntethase gene (otanpsPN) from P. nordicum. Both ochratoxin A producing Penicillia differ characteristically in the PCR result, making a taxonomic differentiation possible. P. verrucosum gives consistently only a positive reaction with the primers for the otanpsPN gene, whereas P. nordicum is positive for both genes. The PCR reaction is negative with all of other food related fungal species tested. This PCR system has been used to analyse 62 Penicillium strains isolated from cured meat products or ripening rooms, the natural habitat of P. nordicum. Among the 62 analysed strains 11 (18%) were positive with all specific PCR reactions. All 11 strains were able to produce ochratoxin A. In a RAPD analysis performed in parallel all 11 strains showed a pattern characteristic of P. nordicum, indicating the congruence of all data. None of the other strains isolated from cured meat produced ochratoxin A; most of them (30 out of 62) had a RAPD pattern characteristic for Penicillium nalgiovense. Interestingly some of the P. nalgiovense strains showed weak PCR product bands with varying length after electrophoresis. This was true for both primer pairs. None of these P. nalgiovense strains however produced detectable amounts of ochratoxin A. A more detailed analysis revealed that P. nalgiovense carries similar but non-transcribed sequences to the ochratoxin A biosynthetic genes of P. nordicum

Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells

Connexins (Cx) are a large family of membrane proteins that can form intercellular connections, so-called gap junctions between adjacent cells. Cx43 is widely expressed in mammals and has a variety of different functions, such as the propagation of electrical conduction in the cardiac ventricle. Despite Cx43 knockout models, many questions regarding the biology of Cx43 in health and disease remain unanswered. Herein we report the establishment of a Cre-inducible Cx43 overexpression system in murine embryonic stem (ES) cells. This enables the investigation of the impact of Cx43 overexpression in somatic cells. We utilized a double reporter system to label Cx43-overexpressing cells via mCherry fluorescence and exogenous Cx43 via fusion with P2A peptide to visualize its distribution pattern. We proved the functionality of our systems in ES cells, HeLa cells, and 3T3-fibroblasts and demonstrated the formation of functional gap junctions based on dye diffusion and FRAP experiments. In addition, Cx43-overexpressing ES cells could be differentiated into viable cardiomyocytes, as shown by the formation of cross striation and spontaneous beating. Analysis revealed faster and more rhythmic beating of Cx43-overexpressing cell clusters. Thus, our Cx43 overexpression systems enable the investigation of Cx43 biology and function in cardiomyocytes and other somatic cells

Transgenic systems for unequivocal identification of cardiac myocyte nuclei and analysis of cardiomyocyte cell cycle status

Even though the mammalian heart has been investigated for many years, there are still uncertainties in the fields of cardiac cell biology and regeneration with regard to exact fractions of cardiomyocytes (CMs) at different developmental stages, their plasticity after cardiac lesion and also their basal turnover rate. A main shortcoming is the accurate identification of CM and the demonstration of CM division. Therefore, an in vivo model taking advantage of a live reporter-based identification of CM nuclei and their cell cycle status is needed. In this technical report, we describe the generation and characterization of embryonic stem cells and transgenic mice expressing a fusion protein of human histone 2B and the red fluorescence protein mCherry under control of the CM specific alpha MHC promoter. This fluorescence label allows unequivocal identification and quantitation of CM nuclei and nuclearity in isolated cells and native tissue slices. In ventricles of adults, we determined a fraction of <20 % CMs and binucleation of 77-90 %, while in atria a CM fraction of 30 % and a binucleation index of 14 % were found. We combined this transgenic system with the CAG-eGFP-anillin transgene, which identifies cell division and established a novel screening assay for cell cycle-modifying substances in isolated, postnatal CMs. Our transgenic live reporter-based system enables reliable identification of CM nuclei and determination of CM fractions and nuclearity in heart tissue. In combination with CAG-eGFP-anillin-mice, the cell cycle status of CMs can be monitored in detail enabling screening for proliferation-inducing substances in vitro and in vivo

Lentiviral Vector Mediated Thymidine Kinase Expression in Pluripotent Stem Cells Enables Removal of Tumorigenic Cells

<div><p>Embryonic stem cells (ES) and induced pluripotent stem (iPS) cells represent promising tools for cell-based therapies and regenerative medicine. Nevertheless, implantation of ES cell derived differentiated cells holds the risk of teratoma formation due to residual undifferentiated cells. In order to tackle this problem, we used pluripotent stem cells consisting of ES and iPS cells of mouse genetically modified by lentiviral vectors (LVs) carrying herpes simplex virus thymidine kinase (HSV-TK) under the control of different promoters of pluripotency genes. Cells expressing TK in turn are eliminated upon administration of the prodrug ganciclovir (GCV). Our aim was to study the conditions required for a safe mechanism to clear residual undifferentiated cells but using low MOIs of lentiviruses to reduce the risk of insertional mutagenesis. Our <i>in vitro</i> data demonstrated that TK expression in pluripotent stem cells upon treatment with GCV led to elimination of undifferentiated cells. However, introduction of hygromycin resistance in the LV transduced ES cells followed by pre-selection with hygromycin and GCV treatment was required to abolish undifferentiated cells. Most importantly, transplantation of pre-selected ES cells that had been transduced with low MOI LV in mice resulted in no teratoma development after GCV treatment <i>in vivo</i>. Taken together, our data show that pre-selection of ES cells prior to <i>in vivo</i> application is necessary if vector integration events are minimized. The study presented here gives rise to safer use of pluripotent stem cells as promising cell sources in regenerative medicine in the future.</p> </div

Analysis of ES cells transduced with LVs carrying a hygromycin resistance gene.

<p>(<b>A</b>) Construct of LV carrying additional hygromycin resistance gene driven by PGK promoter and cDNA of TK under control of EOS-S4-promoter (STPH). (<b>B</b>) ES cells were transduced with STPH (1.5 copy numbers per genome in average) or not transduced (WT) and treated with (+) or without (-) 20 µM GCV for 72 hours after pre-selection with (+Hygro) or without hygromycin (-Hygro). Representative brightfield images are shown (n=3). (<b>C</b>) (<b>D</b>) and (<b>E</b>) Relative cell survival of untransduced ES cells (WT) (C), STPH-transduced ES cells (1.5 copy numbers per genome in average) without pre-selection (D) and STPH-transduced ES cells (1.5 copy numbers per genome in average) with pre-selection (E) with (+) or without (-) 20 µM GCV treatment (n=3, Mean±SEM; ***P<0.001 compared to without GCV treatment, respectively, Student’s <i>t</i>-test). Data is based on images representatively shown in (B); undifferentiated cells were manually counted using three different fields of view that were counted twice. (<b>F</b>) The untreated ES cell populations shown in (B) were differentiated as EBs with (+) or without (-) 20 µM GCV. After 14 days of differentiation dissociated EBs were immunostained with Oct-3/4 (red) and Hoechst (blue) indicating Oct-3/4-positive cells (ES cells) and nuclei, respectively. Representative images are shown (n=3).</p

Analysis of mixed ES cell populations transduced with lentiviral NT or OT with low copy number.

<p>(<b>A</b>) ES cells were transduced with different amounts of lentiviral NT or OT or were not transduced (WT). The copy number of LVs in mixed ES cell populations was analyzed by qPCR. Shown are the results of mixed ES cell populations carrying ^≈1.5 copy numbers per genome in average (n=3, Mean±SEM; N.S., not significant, ANOVA). As control, two transgenic mice previously analyzed by Southern Blot to have one or two integrants (data not shown), were also analyzed by qPCR. (<b>B</b>) ES cells were transduced with NT or OT (1.5 copy numbers per genome in average) or not transduced (WT) and treated with (+) or without (-) 20 µM GCV. Representative brightfield images are shown (n=3). (<b>C</b>) The untreated ES cell populations shown in (B) were differentiated as EBs with (+) or without (-) 20 µM GCV treatment. After 14 days of differentiation dissociated EBs were immunostained with Oct-3/4 (red) and Hoechst (blue) indicating Oct-3/4-positive cells (ES cells) and nucleus, respectively. Representative images are shown (n=3). (<b>D</b>) Percentage of Oct-3/4-positive cells on day 14 of differentiation with and without GCV treatment analyzed by manual counting of Oct-3/4-positive cells on images representatively shown in (C) using three different fields of view that were counted five times (Mean±SEM, **P<0.01, ANOVA).</p

Constructs of LVs carrying TK expression cassette and analysis of NT- or OT-transduced ES and iPS cells <i>in vitro</i>.

<p>(<b>A</b>) Constructs of LVs carrying thymidine kinase (TK) cDNA from herpes simplex virus driven by promoters of pluripotency genes Nanog (NT) or Oct-3/4 (OT). (<b>B</b>) ES cells were transduced with LVs (NT or OT, 300 ng of reverse transcriptase) or not transduced (WT) and further treated with 0, 10, 20, 40, 60, 80 or 100 µM GCV for 72 hours. Representative brightfield images are shown (n=3). (<b>C</b>) and (<b>D</b>) iPS cells (C) or iPS-Oct-GFP cells (D) were transduced with LVs (NT or OT, 300 ng of reverse transcriptase) or not transduced (WT) and further treated with (+) or without (-) 20 µM GCV for 72 hours. Representative brightfield images (C, D) and fluorescence images (D) are shown (n=3).</p

Analysis of ES cells transduced with LVs using different promoters of pluripotency genes.

<p>(<b>A</b>) In addition to NT and OT (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070543#pone-0070543-g001" target="_blank">Figure 1A</a>) LVs carrying TK cDNA under the control of the EOS-C3 (CT) and EOS-S4 (ST) promoters were constructed. (<b>B</b>) The copy number of LVs in mixed ES cell populations transduced with NT, OT, CT and ST or not transduced (WT) was analyzed by qPCR (n=3, Mean±SEM, N.S., not significant, ANOVA). As control, DNAs of two transgenic mice were used, that were previously analyzed by Southern Blot to have one or two integrants. (<b>C</b>) TK expression on mRNA level of NT, OT, CT and ST transduced ES cells (1.5 copy numbers per genome in average) or not transduced (WT) was analyzed by qPCR and normalized to GAPDH (n=3, Mean±SEM, *P<0.05, **P<0.01, ***P<0.001 compared to ST, ANOVA). (<b>D</b>) Brightfield images of NT, OT, CT, ST (1.5 copy numbers per genome in average) or not transduced (WT) ES cells after treatment with (+) or without (-) 20 µM GCV for 72 hours. Representative images are shown (n=4). (<b>E</b>) ES cells from (D) with GCV treatment were analyzed with LDH assay as described in experimental methods section. Shown are the relative numbers of NT, OT, CT or ST transduced ES cells that survived GCV treatment as compared to untransduced (WT) ES cells (n=3, Mean±SEM; ***P<0.001, compared to WT; N.S., not significant, ANOVA).</p

Analysis of single LV-integrant NT- or OT-transduced ES cell clones <i>in vitro</i>.

<p>(<b>A</b>) ES cells were transduced with NT or OT and picked ES cell clones carrying one integrant (NT #8, NT #11, OT #4, OT#11) or not transduced ES cells (WT) were treated with (+) or without (-) 20 µM GCV for 72 hours. Representative brightfield images are shown (n=3). (<b>B</b>) The untreated ES cell clones shown in (A) were differentiated as EBs with (+) or without (-) 20µM GCV. After 14 days of differentiation dissociated EBs were immunostained with Oct-3/4 (red) and Hoechst (blue) indicating Oct-3/4-positive cells (ES cells) and nuclei, respectively. Representative images are shown (n=3). (<b>C</b>) Percentage of Oct-3/4-positive cells of dissociated EBs on day 14 after differentiation with and without GCV treatment analyzed by manual cell counting of images shown in (B) by using one field of view that was counted five times (Mean±SEM; ***P<0.001 compared to without GCV, respectively, ANOVA)..</p