Zsuzsanna Polgar

Liver transplantation has been established as a curative therapy for acute and chronic liver failure, as well as liver-based inherited metabolic diseases. Because of the complexity of organ transplantation and the worldwide shortage of donor organs, hepatocyte transplantation is being developed as a bridging therapy until donor organs become available, or for amelioration of inherited liver-based diseases. The Gunn rat is a molecular and metabolic model of Crigler-Najjar syndrome type 1, which is characterized by lifelong unconjugated hyperbilirubinemia due to the lack of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-mediated bilirubin glucuronidation. Gunn rats are convenient for evaluating the effect of hepatocyte transplantation or gene therapy, because the extent of UGT1A1 replacement can be assessed by serial determination of serum bilirubin levels, and excretion of bilirubin glucuronides in bile provide definitive evidence of the function of the transplanted hepatocytes or the effect of gene therapy. The core techniques involved in hepatocyte transplantation in Gunn rats are discussed in this chapter.

ABSTRACT Induced pluripotent stem (iPS) cell technology involves reprogramming somatic cells to a pluripotent state. The original technology used to produce these cells requires viral gene transduction and results in the permanent integration of exogenous genes into the genome. This can lead to the development of abnormalities in the derived iPS cells. Here, we report that non-viral transfection of a Sleeping Beauty (SB) transposon containing the coding sequences Oct3/4 (Pouf1), Sox-2, Klf-4 and c-Myc (OSKM) linked with 2A peptides, can reprogram mouse fibroblasts. We have established reprogrammed mouse cell lines from three different genetic backgrounds: (1) ICR-outbred, (2) C57BL/6-inbred and (3) F1-hybrid (C57BL/6 x DBA/2J), with parallel robust expression of all exogenous (Oct3/4, Sox-2, Klf-4, and c-Myc) and endogenous (e.g. Oct3/4 and Nanog) pluripotency genes. The iPS cell lines exhibited characteristics typical for undifferentiated embryonic stem (ES) cell lines: ES cell-like morphology, alkaline phosphatase (ALP) positivity and gene expression pattern (shown by reverse transcription PCR, and immunofluorescence of ES cell markers-e.g. Oct3/4, SSEA1, Nanog). Furthermore, cells were able to form embryoid bodies (EBs), to beat rhythmically, and express cardiac (assayed by immunofluorescence, e.g. cardiac Troponin T, desmin) and neuronal (assayed by immunofluorescence e.g. nestin, Tuj1) markers. The in vitro differentiation potential was found to be the highest in the ICR-derived iPS lines (ICR-iPS). Interestingly, the ICR-iPS lines had even higher differentiation potential than the ICR-ES cell lines: the rate of EBs forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. In vivo, the ICR and F1 hybrid iPS cells formed chimeras and one of the iPS cells from the F1 hybrid background transmitted to the germline. Our results suggest that iPS technology may be useful for generating pluripotent stem cells from genetic backgrounds of which good quality ES cell generation is difficult. These studies provide insights into viral-free iPS technology and may contribute towards defining future cell-based therapies, drug-screening methods and production of transgenic animals using genetically modified iPS cells.

Osteosarcoma is a frequent and extremely aggressive type of pediatric cancer. New therapeutic approaches are needed to improve the overall survival of osteosarcoma patients. Our previous results suggest that NMNAT1, a key enzyme in nuclear NAD+ synthesis, facilitates the survival of cisplatin-treated osteosarcoma cells. A high-throughput cytotoxicity screening was performed to identify novel pathways or compounds linked to the cancer-promoting role of NMNAT1. Nine compounds caused higher toxicity in the NMNAT1 KO U2OS cells compared to their wild type counterparts, and actinomycin D (ActD) was the most potent. ActD-treatment of NMNAT1 KO cells increased caspase activity and secondary necrosis. The reduced NAD+ content in NMNAT1 KO cells was further decreased by ActD, which partially inhibited NAD+-dependent enzymes, including the DNA nick sensor enzyme PARP1 and the NAD+-dependent deacetylase SIRT1. Impaired PARP1 activity increased DNA damage in ActD-treated NMNAT1 knockout cells, ...

Osteosarcoma (OS) is the most common bone tumor in children and adolescents. Modern OS treatment, based on the combination of neoadjuvant chemotherapy (cisplatin + doxorubicin + methotrexate) with subsequent surgical removal of the primary tumor and metastases, has dramatically improved overall survival of OS patients. However, further research is needed to identify new therapeutic targets. Here we report that expression level of the nuclear NAD synthesis enzyme, nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1), increases in U-2OS cells upon exposure to DNA damaging agents, suggesting the involvement of the enzyme in the DNA damage response. Moreover, genetic inactivation of NMNAT1 sensitizes U-2OS osteosarcoma cells to cisplatin, doxorubicin, or a combination of these two treatments. Increased cisplatin-induced cell death of NMNAT1−/− cells showed features of both apoptosis and necroptosis, as indicated by the protective effect of the caspase-3 inhibitor z-DEVD-FMK and th...

Abstract Landmark discoveries during the last decade have shown that the so-called “terminally differentiated” somatic cells can transition to more primitive progenitor cells. On the other hand, directed differentiation of embryonic stem cells, as well as pluripotent stem cells obtained by reprogramming of somatic cells (induced pluripotent stem cells, iPSCs) has enabled the generation of differentiated cells belonging to each of the three germ layers. These studies continue to provide novel insights into the basic mechanisms of cell plasticity underlying the transition of cells to various cell types. Furthermore, the ability to differentiate iPSCs to various cell types, including hepatocyte-like cells (iHep) has initiated the translational application of such cells in disease modeling, pharmacological testing, and regenerative medicine. Despite these developments, currently available methods do not yield cells that fully resemble adult primary hepatocytes in the qualitative and quantitative gene expressions. Generating hepatocytes from iPSCs remains a work in progress. In addition to describing these exciting developments, this review will discuss some emerging new approaches to generate iPSCs, improving their differentiation to iHeps and maintaining them in culture for longer duration and with improved function.

Stages with different letters are significantly (P ≤ 0.05) different for the levels of the gene.<b>Copyright information:</b>Taken from "Expression profiles of the pluripotency marker gene and validation of reference genes in rabbit oocytes and preimplantation stage embryos"http://www.biomedcentral.com/1471-2199/9/67BMC Molecular Biology 2008;9():67-67.Published online 28 Jul 2008PMCID:PMC2507718.

<b>Copyright information:</b>Taken from "Expression profiles of the pluripotency marker gene and validation of reference genes in rabbit oocytes and preimplantation stage embryos"http://www.biomedcentral.com/1471-2199/9/67BMC Molecular Biology 2008;9():67-67.Published online 28 Jul 2008PMCID:PMC2507718.

Stages with different letters are significantly (P ≤ 0.05) different for the levels of the gene. An asterisk (*) indicates significant differences {(* = P ≤ 0.05) and (** = P ≤ 0.01)} with the levels of the same stage.<b>Copyright information:</b>Taken from "Expression profiles of the pluripotency marker gene and validation of reference genes in rabbit oocytes and preimplantation stage embryos"http://www.biomedcentral.com/1471-2199/9/67BMC Molecular Biology 2008;9():67-67.Published online 28 Jul 2008PMCID:PMC2507718.

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with...

The effects of cryopreservation on membrane of rabbit spermatozoa were studied by Trypan blue-Giemsa staining. Semen samples of 11 White New Zealand bucks (N=31) were evaluated before freezing and after freezing-thawing. The applied cryopreservation method based on two cryoprotectants (DMSO, glycerol). The integrity of the head and tail membrane and the acrosome status were evaluated on smears stained by trypan blue and Giemsa stains according to KOVACS, FOOTE (1992). The freezing-thawing process increased the number of dead cells from 18% to 46%. At the same time live cell heads were also observed and significant damage of acrosomal and tail membrane integrity (p<0.001) was detected. The most destroying effect was shown on sperm tail, which was found as the doubled number of stained tail spermatozoa. The freezingthawing effects were not influenced by collection date but some differences were detected between semen of different bucks in term of tolerance to freezing.

In cell therapy the pathway from bench to bedside has shown a quick adoption into clinical trials. Thus preclinical and clinical studies progress along parallel trajectories. A great effort is being undertaken to find out universal tools and strategies, which can be applied to pluripotent stem cells differentiated to distinct cell types. The major unresolved problem is obtaining the sufficient number of autologous pluripotent cells and the inherited association of pluripotency with the risk of teratoma formation. The second obstacle, easier to be solved, is a massive death of progenitor cells after transplantation. It has been convincingly shown that the cell survival can be improved by overexpression of cytoprotective genes. One of the candidates is heme oxygenase-1 (HO-1), the antioxidative, anti-apoptotic and anti-inflammatory enzyme. We have demonstrated that overexpression of HO-1 significantly improved the survival of murine proangiogenic progenitors (PPC, a population enriche...

During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, 'naïve' or 'primed', depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells. We document conserved and divergent traits in gene expression, and identify predictor genes shared across the species that are associated with pluripotent states and Amongst these are the pluripotency-linked genes and The novel genes discovered include naïve- ( and ) and primed-associated ( and ) genes as well as naïve to primed transition genes ( and ). Both and play a role in pluripotency since their knockdown results in differentiation and downregulation of key pluripotency genes. Our interspecies comparison revealed new insights of pluripotency, pluripotent stem cell identity and...

Redox regulation has been proposed to control various aspects of carcinogenesis, cancer cell growth, metabolism, migration, invasion, metastasis and cancer vascularization. As cancer has many faces, the role of redox control in different cancers and in the numerous cancer-related processes often point in different directions. In this review, we focus on the redox control mechanisms of tumor cell destruction. The review covers the tumor-intrinsic role of oxidants derived from the reduction of oxygen and nitrogen in the control of tumor cell proliferation as well as the roles of oxidants and antioxidant systems in cancer cell death caused by traditional anticancer weapons (chemotherapeutic agents, radiotherapy, photodynamic therapy). Emphasis is also put on the role of oxidants and redox status in the outcome following interactions between cancer cells, cytotoxic lymphocytes and tumor infiltrating macrophages.

Liver transplantation has been established as a curative therapy for acute and chronic liver failure, as well as liver-based inherited metabolic diseases. Because of the complexity of organ transplantation and the worldwide shortage of donor organs, hepatocyte transplantation is being developed as a bridging therapy until donor organs become available, or for amelioration of inherited liver-based diseases. The Gunn rat is a molecular and metabolic model of Crigler-Najjar syndrome type 1, which is characterized by lifelong unconjugated hyperbilirubinemia due to the lack of uridinediphosphoglucuronate glucuronosyltransferase-1 (UGT1A1)-mediated bilirubin glucuronidation. Gunn rats are convenient for evaluating the effect of hepatocyte transplantation or gene therapy, because the extent of UGT1A1 replacement can be assessed by serial determination of serum bilirubin levels, and excretion of bilirubin glucuronides in bile provide definitive evidence of the function of the transplanted h...

Although several types of somatic cells have been reprogrammed to induced pluripotent stem cells (iPSC) and then differentiated to hepatocyte-like cells (iHep), the method for generating such cells from renal tubular epithelial cells shed in human urine and transplanting them in animal livers has not been described systematically. We report reprogramming of human urinary epithelial cells to iPSCs and subsequent hepatic differentiation, followed by a detailed characterization of the newly generated iHeps. The epithelial cells were reprogrammed into iPSCs by delivering the pluripotency factors OCT3/4, SOX2, KLF4 and MYC using methods that do not involve transgene integration, such as nucleofection of episomal (oriP/EBNA-1) plasmids or infection with recombinant Sendai viruses. After characterization of stable iPS cell lines, a 3-step differentiation toward hepatocytes was performed. The iHeps expressed a large number of hepatocyte-preferred genes, including nuclear receptors that regu...

Abstract Landmark discoveries during the last decade have shown that the so-called “terminally differentiated” somatic cells can transition to more primitive progenitor cells. On the other hand, directed differentiation of embryonic stem cells, as well as pluripotent stem cells obtained by reprogramming of somatic cells (induced pluripotent stem cells, iPSCs) has enabled the generation of differentiated cells belonging to each of the three germ layers. These studies continue to provide novel insights into the basic mechanisms of cell plasticity underlying the transition of cells to various cell types. Furthermore, the ability to differentiate iPSCs to various cell types, including hepatocyte-like cells (iHep) has initiated the translational application of such cells in disease modeling, pharmacological testing, and regenerative medicine. Despite these developments, currently available methods do not yield cells that fully resemble adult primary hepatocytes in the qualitative and quantitative gene expressions. Generating hepatocytes from iPSCs remains a work in progress. In addition to describing these exciting developments, this review will discuss some emerging new approaches to generate iPSCs, improving their differentiation to iHeps and maintaining them in culture for longer duration and with improved function.

Sublethal high hydrostatic pressure (HHP) treatment of cells was reported to enhance stress tolerance and to increase post-thawing survival after cryopreservation in mouse, swine and cattle. The goal of this study was to define if HHP stress tolerance depends on the embryos' stage of development and culture conditions, to describe long term in vivo effects and transcriptional alterations of selected stress related genes. Studies showed that impacts greater than 60 MPa caused blastomere and membrane injuries to the two-cell stage embryos, while even 80 MPa was well tolerated by blastocysts. HHP treatment caused significant upregulation of Azin1, Sod2 and Gadd45g genes, detected by RT-qPCR. The transfer of HHP treated blastocysts revealed normal in vivo development and reproductive function in a two generation study. The cell type and the embryos' development stage shall be taken into account when optimizing sublethal HHP stress treatment protocol of different cells.

ABSTRACT Nuclear transfer using somatic cells offers new opportu¬nities for genetic engineering, genome preservation, and tissue regeneration. As described in detail in the corre¬sponding chapters of this book, nuclear transfer of somatic cells has succeeded in various species, including sheep, mice, pigs, gaurs, cats, mules, horses, dogs, ferrets, camels, and others. Although the efficiency of the overall cloning process has been low, rapid progress has been made in the improvement and application of this technology. The rabbit was one of the first species in which blasto¬mere nuclear transfer succeeded; as a model species, rab¬bits played a central role in developing micromanipulation technologies in embryos. There are several advantages of using rabbits as an experimental model.

During early mammalian development, transient pools of pluripotent
cells emerge that can be immortalised upon stem cell derivation. The
pluripotent state, ‘naïve’ or ‘primed’, depends on the embryonic stage
and derivation conditions used. Here we analyse the temporal gene
expression patterns of mouse, cattle and porcine embryos at stages
that harbour different types of pluripotent cells. We document
conserved and divergent traits in gene expression, and identify
predictor genes shared across the species that are associated with
pluripotent states in vivo and in vitro. Amongst these are the
pluripotency-linked genes Klf4 and Lin28b. The novel genes
discovered include naïve- (Spic, Scpep1 and Gjb5) and primedassociated
(Sema6a and Jakmip2) genes as well as naïve to primed
transition genes (Dusp6 and Trip6). Both Gjb5 and Dusp6 play a role
in pluripotency since their knockdown results in differentiation and
downregulation of key pluripotency genes. Our interspecies comparison
revealed new insights of pluripotency, pluripotent stem cell identity
and a new molecular criterion for distinguishing between pluripotent
states in various species, including human.

Le transfert de noyau de cellule somatique et l’utilisation des cellules souches embryonnaires offrent de nouvelles possibilités dans de nombreux champs de la recherche en biotechnologie. Les étapes techniques du clonage sont complexes, et l’efficacité encore faible, même si de nombreux efforts sont menés.

The improvement of in vitro maturation (IVM) system for rabbit oocytes could play a positive role in rabbit biotechnology. Our goal was to develop an IVM method to have an efficient source of matured rabbit oocytes for further studies on nuclear transfer and other techniques. The present study was carried out to evaluate the effects of FCS, growth factor and hormone supplementation on maturation, activation and embryo development rates. Rabbit ovaries were collected in a local slaughterhouse and were transferred within 2 h to the laboratory in PBS. Oocytes were collected by aspiration and subjected to IVM in three types of media based on M199. In one group growth factors (50 ng/ml IGF-I, 10 ng/ml EGF) and hormones (51 IU/ml hCG and 51 IU/ml PMSG) were added to the IVM medium (IVM+), in the other groups IVM medium was supplemented either with 10% (IVM+10%FCS) or 20% FCS (IVM+20%FCS). Groups of 70-100 oocytes in four replicates were compared. Maturation of oocytes was assessed by extr...

Rabbit somatic cell nuclear transfer (SCNT) offers new opportunities for basic research, biomedical and agricultural applications and genome preservation. However, the technology is still in a very early development stage and it is relatively inefficient proving many early expectations premature. The technical steps of SCNT are extremely complex. They are also very sensitive to small changes and fluctuations in technical parameters. Furthermore, the biological background of the reprogramming process is not fully understood. It makes it virtually impossible to optimize a protocol providing “ideal” recipient oocytes and donor cells for the process. In this chapter we will intend to summarize the current status of nuclear transfer technology in rabbits and to give the readers a brief insight of its future use.

Rabbit somatic cell nuclear transfer (SCNT) offers new opportunities for basic research, biomedical and agricultural applications and genome preservation. However, the technology is still in a very early development stage and it is relatively inefficient proving many early expectations premature. The technical steps of SCNT are extremely complex. They are also very sensitive to small changes and fluctuations in technical parameters. Furthermore, the biological background of the reprogramming process is not fully understood. It makes it virtually impossible to optimize a protocol providing “ideal” recipient oocytes and donor cells for the process. In this chapter we will intend to summarize the current status of nuclear transfer technology in rabbits and to give the readers a brief insight of its future use.

ABSTRACT Major genome activation is a key event in early embryonic development occurring at the late 2-cell stage in the mouse. Concomitantly occurring molecular and ultrastructural changes in the nucleolus, where the ribosomal RNA genes are transcribed and their transcripts processed, enable the use of this organelle as a sensitive marker of genome activation in embryos produced by different techniques. The aim of this study was to evaluate and compare the genome activation in mouse embryos of different origin using the nucleolus as a marker. Early and late 2-cell- and late 4-cell-stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and somatic cell nuclear transfer of mouse embryonic fibroblast (MEF), and mouse HM1 embryonic stem cells (HM1) were processed for autoradiography following 3H-uridine incubation and transmission electron microscopy (5 embryos per group) and for immunofluorescence for detection of nucleolar proteins involved in rRNA synthesis (upstream binding factor; UBF) and processing (nucleophosmin; B23) (10–21 embryos per group). Early 2-cell embryos in all groups showed transcriptional activity in the nucleoplasm, but not over nucleolar precursor bodies (NPBs). UBF was localized diffusely in the cytoplasm. B23 was, likewise, localized in the cytoplasm and, in 30% of embryos, in the nucleoplasm. Late 2-cell IVF and PG embryos displayed transcriptional labelling over nucleoplasm and NPBs, which, ultrastructurally, were in the process of transformation into fibrillo-granular nucleoli presenting fibrillar centers, a dense fibrillar component, and a granular component. MEF and HM1 embryos displayed transcriptional labelling over nucleoplasm, but not over NPBs, and the transformation into functional nucleoli was never observed at this stage of development. UBF and B23 were in all groups localized in the nucleoplasm and, in 40–50% of cases, distinctly in the developing nucleoli. At the late 4-cell stage, all embryos presented transcriptional labelling over nucleoplasm and NPBs, which were at different levels of transformation into fibrillo-granular nucleoli. UBF and B23 were distinctly localized in these developing nucleoli. However, whereas fully transformed reticulated fibrillo-granular nucleoli without remnants of NPBs were found in IVF and PG embryos, despite the distinct localization of nucleolar proteins, the nucleoli in MEF and HM1 embryos were not reticulated and still displayed remnants of NPBs. Conclusively, embryos reconstructed by nuclear transfer, independent of cell origin, displayed well-timed extranucleolar genomic activation, but delayed transformation of NPBs into reticulated fibrillo-granular nucleoli. Moreover, the proper nucleolar activation noted in PG embryos activated in the same manner as MEF and HM1 embryos demonstrate that somatic and embryonic stem cell factors exert an influence on nucleolar activation and may cause reduced embryo viability. This work was supported by the Specific Targeted Project (MED-RAT; contract LSHG-CT-2006-518240) and Marie Curie ResearchTraining Networks (CLONET; contract 035468-2).