Transgenic approaches for the investigation of putative airway stem cells as potential targets for gene correction therapy

Since the discovery of the CFTR gene over a decade ago, Cystic Fibrosis (CF) has been regarded as amenable to intervention by gene therapy. The ultimate aim of gene therapy must be the correction, within cells capable of repopulating the tissue, of the genetic defect in its chromosomal context. Towards that end, a mouse model designed to evaluate the efficiency of gene correction was created, and a transgenic approach was taken to the investigation of a putative progenitor cell population in the adult murine respiratory tract. Before gene correction systems can be considered as valid therapeutic agents, their utility in the cells and tissues of living animals must be demonstrated. Thus, an in vivo system permitting the simple quantification of correction frequency in a wide range of tissues would be a valuable resource for the gene correction community. The generation and analysis of a transgenic mouse carrying an inactivated, but potentially correctable, reporter transgene is described. The full potential of a gene correction strategy to provide a single-dose, permanent solution to a genetically-diseased tissue will only be realised once the therapy is able to target resident stem cells. For CF lung disease, this will require the prior identification of stem cells in the respiratory epithelium. Previous work has indicated that potential stem cells are spatially coincident with small groups of cells expressing high levels of keratin 5 (K5) protein in the proximal murine trachea. In order to investigate lineage arising from this putative stem cell niche, transgenic mice have been generated which express an inducible form of Cre recombinase from the K5 promoter. Preliminary experiments demonstrate recombination of a conditional reporter gene after induction of Cre activity in K5-expressing tissue. Comparison of the inducible system with a constitutive K5 promoter-driven Cre line validated the choice of the former, as the clarity of data obtained from the conventional system was undermined as a result of K5 promoter activity causing reporter gene activation prior to the onset ofthe experiment. In the course of these studies it became evident that the conventional, constitutive Cre line gave rise to segregating patterns of reporter gene activation. While some mice displayed the expected K5-derived expression profile, other animals demonstrated ubiquitous expression. Universal activation of the conditional reporter was detected only in animals derived from females carrying the Cre transgene, and was found to be the result of unanticipated production of Cre protein in the maternal germline. This transgenic line is unusual and valuable in offering a choice of tissuespecific and generalised recombination of floxed alleles

Intravenously delivered mesenchymal stem cell-derived exosomes target M2-type macrophages in the injured spinal cord

In a previous report we showed that intravenous infusion of bone marrow-derived mesenchymal stem cells (MSCs) improved functional recovery after contusive spinal cord injury (SCI) in the non-immunosuppressed rat, although the MSCs themselves were not detected at the spinal cord injury (SCI) site [1]. Rather, the MSCs lodged transiently in the lungs for about two days post-infusion. Preliminary studies and a recent report [2] suggest that the effects of intravenous (IV) infusion of MSCs could be mimicked by IV infusion of exosomes isolated from conditioned media of MSC cultures (MSCexos). In this study, we assessed the possible mechanism of MSCexos action on SCI by investigating the tissue distribution and cellular targeting of DiR fluorescent labeled MSCexos at 3 hours and 24 hours after IV infusion in rats with SCI. The IV delivered MSCexos were detected in contused regions of the spinal cord, but not in the noninjured region of the spinal cord, and were also detected in the spleen, which was notably reduced in weight in the SCI rat, compared to control animals. DiR "hotspots" were specifically associated with CD206-expressing M2 macrophages in the spinal cord and this was confirmed by co-localization with anti-CD63 antibodies labeling a tetraspanin characteristically expressed on exosomes. Our findings that MSCexos specifically target M2-type macrophages at the site of SCI, support the idea that extracellular vesicles, released by MSCs, may mediate at least some of the therapeutic effects of IV MSC administration

Embryonic Stem Cells

Embryonic stem cells are one of the key building blocks of the emerging multidisciplinary field of regenerative medicine, and discoveries and new technology related to embryonic stem cells are being made at an ever increasing rate. This book provides a snapshot of some of the research occurring across a wide range of areas related to embryonic stem cells, including new methods, tools and technologies; new understandings about the molecular biology and pluripotency of these cells; as well as new uses for and sources of embryonic stem cells. The book will serve as a valuable resource for engineers, scientists, and clinicians as well as students in a wide range of disciplines

Melanin-binding colorants: updating molecular modeling, staining and labeling mechanisms, and biomedical perspectives

Melanin and melanoma tumors are two fields of increasing interest in biomedical research. Melanins are ubiquitous biopigments with adaptive value and multiple functions, and occur in the malignant melanoma. Although several chemical structures have been proposed for eumelanin, molecular modeling and orbitals indicate that a planar or spiral benzoquinone-porphycene polymer would be the model that better explains the broad-band light and ultrasound absorption, electric conductivity, and graphite-like organization shown by X-ray crystallography and electron microscopy. Lysosomes and melanosomes are selectively labeled by vital probes, and melanin also binds to metal cations, colorants, and drugs, with important consequences in pharmacology, pathology, and melanoma therapy. In addition to traditional and recent oncologic treatments, photodynamic, photothermal, and ultrasound protocols represent novel modalities for melanoma therapy. Since eumelanin is practically the ideal photothermal and ultrasound sensitizer, the vibrational decay from photo-excited electrons after NIR irradiation, or the electrochemical production of ROS and radicals after ultrasound absorption, induce an efficient heating or oxidative response, resulting in the damage and death of tumor cells. This allows repetitive treatments due to the remaining melanin contained in tumoral melanophages. Given that evolution and prognosis of the advanced melanoma is still a concern, new biophysical procedures based on melanin properties can now be developed and applie

Impedance-Based Real-Time Monitoring of Mammalian Cells upon Introduction of Xenobiotics into the Cytoplasm by In Situ Electroporation

Whole-cell biosensors are irreplaceable tool for studies of cellular mechanisms and behavior of the cell as a smallest living unit. Their development have progressed rapidly over past decades and nowadays we have powerful tools to study cell-based assays and to examine behavior of the cells exposed to different kinds of stimuli and challenges. Limited and selective permeability of the plasma membrane prevents the introduction of hydrophilic xenomolecules into the cytoplasm of mammalian cells. However, it is essential for many fields of cell biology, biomedicine or biotechnology to allow transport of such molecules (e.g. nucleic acids, antibodies, peptides or drugs) across the cell membrane. An ultimate goal of this thesis was to establish proof-of-principle assays for delivery of various bioactive molecules into adherent cells by in situ electroporation, and to monitor how these compounds influence cellular behavior, once they are internalized within the cell cytosol. Studies of in situ electroporation (ISE) were conducted using different types of mammalian cells (BAEC, CHO-K1/CHO-GFP, HaCaT, NRK and NIH-3T3) grown to confluence on small planar gold film electrodes. For every cell line individually, electric pulse parameters were optimized to achieve maximal loading efficiency, while keeping the invasiveness of the operation as low as possible. Impedance monitoring of in situ electroporation conducted with high time resolution showed biphasic changes of impedance signal after pulse application, indicating (i) fast recovery of the cell membrane integrity and (ii) relatively slow process of cell recovery after changes induced by membrane permeabilization. For the first time, release of intracellular material from the cells by ISE was studied using ECIS setup. Direct time-resolved imaging of NRK cells showed measurable efflux of fluorescence-labeled probes upon multiply applied electric pulses. In situ electroporation allowed transfer of second messenger (8-OH-)cAMP in the cell monolayers. Subsequent changes in impedance signal were in agreement with those observed after stimulation of the cells with membrane-permeable compounds CPT-cAMP and forskolin, as a consequence of triggering of the corresponding signaling cascades. Transport of nucleic acids into cytoplasm and nuclei of NRK and CHO-GFP cells was conducted in a highly-efficient manner. Besides fluorescent DNA aptamers, various types of siRNA molecules were successfully delivered into cells by in situ electroporation and their long-term sequence-specific silencing effect on cells was demonstrated and quantified by using microscopy and/ or impedance analysis. Transfection performance of ISE was compared with conventional and widespread delivery transfection method. In conclusion, this thesis demonstrated that in situ electroporation allows for highly efficient delivery of emerging types of molecules into monolayers of various types of cells

Tumourigenic and tumour suppresive properties of KSHV cyclin in vitro and in vivo

Expression of the Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) cyclin D homologue, K cyclin, may contribute to viral oncogenesis. This thesis shows that ectopic expression of K cyclin also triggers an apoptotic response. Apoptosis is caspase-dependent and is partially inhibited by expression of either a dominant negative (dn) caspase 9 or the KSHV Bcl-2 homologue, indicating involvement of the mitochondrial apoptotic pathway. Furthermore, even when K cyclin expression does not kill cells directly, it sensitises to induction of apoptosis by various stress signals. In primary fibroblasts, K cyclin expression induces accumulation of transcriptionally active p53 with consequent apoptosis and growth arrest in wild type but not in p53-deficient cells. In contrast, apoptosis is not accompanied by induction of p19ARF protein and requires neither E2F1 nor p19ARF. Interestingly, K cyclin-expressing wt and p53-deficient cells become multinucleated and polyploid. Centrosome analysis of such cells shows that K cyclin expression is associated with centrosome amplification and aneuploidy, which is further exacerbated by p53 loss. I hypothesise that K cyclin expression leads to abortive cytokinesis and polyploidy, ultimately triggering p53-dependent apoptosis and growth arrest. The in vitro data suggest that K cyclin expression is tumourigenic in a p53 null background. To corroborate this, I developed a Eμ K cyclin transgenic mouse model which expresses K cyclin protein in B and T cells. These mice develop lymphomas at a low incidence (10%) and long latency (5-9 months). Tumours often contain mutations in the p53 pathway. Eμ K cyclin mice were crossed with p53 null mice. All K cyclin transgenic p53-/- mice developed B and/or T cell lymphoblastic lymphomas extremely rapidly (around 85 days). These data show that p53 acts as a potent suppressor of K cyclin-induced tumours in vivo

Magnetische Nanopartikel-basierter Gentransfer: Mechanismen und Klinische Perspektiven

The development of safe and efficient gene delivery systems is the key to the overall success of gene therapy. This work introduced a MNP-based nonviral gene delivery approach for transfection in clinically relevant cells. Moreover, the in vitro mechanism studies could explained the beneficial properties of the proposed carrier design. This work demonstrated, that proposed system could efficiently and safely transfect human bone marrow derived mesenchymal stem cells. Additionaly, the clinical perspectives of MNP-based gene therapy were discussed and evaluated in this dissertation

The role of Fbw7 and its substrates Notch and c-Jun in neural stem cells, the brain and development

Activation of the oncogenic transcription factor c-Jun by the Jun N-terminal kinase (JNK) has been implicated in diverse biological effects, for example promoting intestinal proliferation or inducing neural apoptosis. Apart from differentiation, apoptosis plays an important role in the specification of neuronal networks in the developing brain. However, the molecular mechanisms governing differentiation and apoptosis during brain development are incompletely understood. In my PhD studies, I have shown for the first time that the E3 ubiquitin ligase substrate recognition component Fbw7 (F-box and WD repeat domain containing-7), a negative regulator of phosphorylated c-Jun and other oncoproteins such as Notch, is a key factor of differentiation and survival in the developing brain. Fbw7-deficiency caused Notchdependent accumulation of radial glia stem cells and c-Jun-dependent loss of progenitors and differentiated cells. Thus, Fbw7 acts as a key molecular switch to allow neural stem cells to differentiate and neural progenitor cells to survive by antagonising Notch and JNK/c-Jun signalling respectively. Whilst sustained JNK/c-Jun signalling contributes to abnormal brain development in conditional Fbw7-knockout mice, c-Jun activation by JNK has been suggested to be dispensable for mouse development but necessary for c-Jun oncogenic function. By mutating the four main JNK-phosphorylation sites in the Jun gene (Jun4A), I could show that Jun4A/4A mice are viable, do not exhibit histological abnormalities and are able to recover from intestinal or neural pathology. Furthermore, moderate activation of JNK/c-Jun signalling in the nervous system of ROSA26-LSL-JNKK2-JNK1ΔN/+ mice did not impair brain histology but led to slightly improved nerve regeneration. In vitro, Jun4A/4A mouse embryonic fibroblasts underwent premature senescence independent of oxidative stress and p53 levels. These findings may prove important for targeting JNK/c-Jun signalling in order to promote nerve regeneration and to inhibit tumour growth in a p53-independent manner with the potential of limited side effects