Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 78-84). Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 78-84). Also available online.

<p>The recent discovery that fibroblasts can be reprogrammed directly to functional neurons with lentivirus has reinvigorated the belief that autologous human cell therapies against many neurodegenerative diseases may be achievable in the near future. To increase the clinical translatability of this approach, we have developed a technique to perform this direct conversion without the use of virus. We transfected fibroblasts with plasmids condensed into non-viral nanoparticulate carriers with a bioerodible peptidomimetic polymer, pCBA-ABOL. Gene delivery with pCBA-ABOL was exceptionally effective and nontoxic, producing high transfection efficiencies and enabling serial dosing of plasmid cocktails. We delivered plasmids encoding neural lineage-instructive transcription factors to primary mouse embryonic fibroblasts (PMEFs), eliciting: drastic morphological changes, activation of endogenous neuronal transcription factor expression, neuronal promoter activity, and the appearance of neuronal proteins. Serial dosing of pCBA-ABOL complexes produced increasingly higher yields of these non-virally induced neurons (NiNs). The majority of NiNs fired action potentials under patch clamp. This is the first description of a method capable of directly inducing functional neuronal cells from fibroblasts without the use of virus.</p><p>We then moved on to further increase the yield of NiN generation, in an effort to produce a sufficient quantity of neurons for cell therapies. Informed by neurodevelopmental cues and by precedents set by the induced pluripotent stem cell (iPSC) field, we performed non-viral neuronal reprogramming in the presence of soluble microenvironmental factors that either inhibited GSK-3beta; and SMAD signaling, or induced chronic membrane depolarization. Chronic depolarization doubled the number of cells expressing neuronal markers produced with glutamatergic as well as with dopaminergic transcription factor cocktails. Inhibition of GSK-3beta; and SMAD signaling similarly doubled the yield of glutamatergic NiNs, and enabled induction of neuronal markers and morphological transformation in human fibroblasts.</p><p>In addition to soluble microenvironmental factors, the physical microenvironment can also strongly influence various cellular phenotypes. This list includes many phenotypes related to endocytosis - the transit mechanism of nanoparticulate gene carriers entering cells during non-viral transfection. As such, we set out to determine if patterned physical substrate topography could be used to increase non-viral transfection. We employed a high-throughput screen of micropitted substrate topographies, and indeed found that larger, denser micropits could support significantly higher transfection efficiencies in fibroblasts, compared to smooth substrates. The same topographies produced higher reverse transfection efficiencies, and greater siRNA-mediated knockdown of a reporter gene. The control of transfection with substrate topography is a new design parameter that could find broad application in in vitro non-viral reprogramming strategies, as well as in the intelligent design of nucleic acid-eluting scaffolds in vivo. </p><p>Encouraged by our success with direct neuronal reprogramming, and armed with a greater understanding of some microenvironmental mediators thereof, we attempted to produce non-virally-induced oligodendroglial progenitor cells (NiOPCs), which has been historically challenging for other investigators. We discovered the fibroblastic intracellular microenvironment is a significant barrier to the function of Olig2 - a master regulator of OPC phenotype - in direct reprogramming. Fibroblasts do not express Olig2 chaperones which are normally expressed in OPCs, causing Olig2 to become sequestered in the cytoplasm of transfected PMEFs. We relieved this barrier through fusion of a strong nuclear localization sequence (NLS) to Olig2, which repartitioned Olig2-NLS from the cytoplasm to the nucleus in transfected fibroblasts. The use of Olig2-NLS in iOPC reprogramming cocktails resulted in a drastic improvement in the yield of OPC-specific marker expression. The improvement associated with Olig2-NLS was insufficient to elicit significant myelin protein expression with the non-viral system, but the yield of virally-induced oligodendrocyte-like cells (iOLs) was improved dramatically. Further enhancements will be required to generate fully-reprogrammed NiOPCs, but the increased efficiency of viral iOPC generation is immediately useful for disease modeling and potentially in cell replacement therapies if human cells can be converted for the first time using this technique. During direct reprogramming, CNS-specific transcription factors are delivered to foreign intracellular contexts as a rule, which may reduce their ability to function effectively; we have shown this can be a critical yet under-appreciated determinant of the success or failure of a direct reprogramming system.</p><p>Taken together, the technological and intellectual advancements we describe herein represent significant improvements to non-viral transfection and reprogramming systems. These techniques can find broad appeal to the many researchers and clinicians deploying these systems. More specifically, we present significant steps towards realization of the dream of safe and effective autologous cell therapies against devastating and currently-intractable neurodegenerative diseases.</p> / Dissertation

Vectors based on lentivirus backbones have revolutionized our ability to transfer genesinto many cell types. Lentiviral vectors integrate into the chromatin of target cells and do not transfer any viral genes causing vector replication. Both of these features arecommonly used in gene therapy and have been used clinically in individuals sufferingfrom cancer, infections and genetic diseases. It has been discovered that T-cells can be genetically modified to be used as effective weapons against cancer: therefore virus mustbe produced to deliver the gene of interest into the T-cells. In this project, lentiviralvectors have been produced to transfer the gene coding for a chimeric antigen receptor(CAR) which is directed to CD19 on B-cells. The vectors will, hence, be used to generateCD19 retargeted T-cells in purpose to kill CD19 cells such as B-cell lymphoma andleukemia. We have evaluated two production protocols to determine a feasible method toculture these vectors. We have also stimulate T-cells with two different antibodies (anti-CD3 and anti-CD28) and transduced T-cells. Our results demonstrate that theconcentration of virus was higher after prolonged incubation in 4˚C, which can not beexplained. The stimulation demonstrated that bound anti-CD3 was the best stimulator,and moreover the FACS-analysis showed that addition of anti-CD28 gave a highertransduction level. In conclusion, the viral vectors may be kept in 4˚C for two days beforeconcentrating the virus, and bound anti-CD3 is a better choice than soluble anti-CD3 forstimulation of T-cells.

<p>The development of a functional tissue-engineered human skeletal muscle model in vitro would provide an excellent platform on which to study the process of myogenesis, various musculoskeletal disease states, and drugs and therapies for muscle toxicity. We developed a protocol to culture human skeletal muscle bundles in a fibrin hydrogel under static conditions capable of exerting active contractions. Additionally, we demonstrated the use of joint miR-133a and miR-696 inhibition for acceleration of muscle differentiation, elevation of active contractile force amplitudes, and increasing Type II myofiber formation in vitro. </p><p>The global hypothesis that motivated this research was that joint inhibition of miR-133a and miR-696 in isolated primary human skeletal myoblasts would lead to accelerated differentiation of tissue-engineered muscle constructs with higher proportion of Type I myofibers and that are capable of significantly increased active contractile forces when subjected to electrical stimulus. The proposed research tested the following specific hypotheses: (1) that HSkM would require different culture conditions than those optimal for C2C12 culture (8% equine serum in differentiation medium on uncoated substrates), as measured by miR expression, (2) that joint inhibition of miR-133a and miR-696 would result in 2D human skeletal muscle cultures with accelerated differentiation and increased Type I muscle fibers compared to control and individual inhibition of each miR, as measured by protein and gene expression, (3) that joint inhibition of miR-133a and miR-696 in this functional 3D human skeletal muscle model would result in active contraction significantly higher than control and individual inhibition by each miR, as measured by isometric force testing, and finally (4) that specific co-culture conditions could support a lamellar co-culture model in 3D of human cord blood-derived endothelial cells (hCB-ECs) and HSkM capable of active contraction, as measured by isometric force testing and immunofluorescence. </p><p>Major results of the dissertation are as follows. Culture conditions of 100 &#956;g/mL growth factor reduced-Matrigel-coated substrates and 2% equine serum in differentiation medium were identified to improve human skeletal myoblast culture, compared to conditions optimal for C2C12 cell culture (uncoated substrates and 8% equine serum media). Liposomal transfection of human skeletal myoblasts with anti-miR-133a and anti-miR-696 led to increased protein presence of sarcomeric alpha-actinin and PGC-1alpha when cells were cultured in 2D for 2 weeks. Presence of mitochondria and distribution of fiber type did not change with miR transfection in a 2D culture. Joint inhibition also resulted in increased PPARGC1A gene expression after 2 weeks of 2D culture. For muscle bundles in 3D, results suggest there exists a myoblast seeding density threshold for the production of functional muscle. 5 x 106 myoblasts/mL did not produce active contraction, while 10 x 106 myoblasts/mL and above were successful. Of the seeding densities studied, 15 x 106 myoblasts/mL resulted in constructs that exerted the highest twitch and tetanus forces. Engineering of human skeletal muscle from transfected cells led to significant increases in force amplitude in joint inhibition compared to negative control (transfection with scrambled miR sequence). Joint inhibition in myoblasts seeded into 3D constructs led to decreased presence of slow myosin heavy chain and increased fast myosin heavy chain. Finally, co-culture of functional human skeletal muscle with human cord blood-derived endothelial cells is possible in 3.3% FBS in DMEM culture conditions, with significant increases in force amplitudes at 48 and 96 hours of co-culture.</p> / Dissertation

Stress impacts upon all organisms and a robust stress response is required for adaptive interactions of the organism with the environment. In most higher organisms, an individual’s response to stress is mediated by the hypothalamic pituitary adrenal (HPA) axis. Inappropriate regulation of this axis can cause debilitating mental health disorders including depression and anxiety. These disorders can affect an individual’s ability to interact and respond appropriately as different situations arise. An important component of this axis is the vasopressin V1b receptor (V1bR), which mediates adrenocorticotropin (ACTH) secretion from the anterior pituitary in response to stimulation by arginine vasopressin (AVP). AVP also potentiates the ACTH secretion mediated by corticotropin-releasing hormone type 1 receptor (CRH-R1) in response to corticotropin- releasing hormone (CRH) stimulation. Both the V1bR and CRH-R1 are G protein coupled receptors (GPCRs). A common feature of GPCR signalling is desensitisation of the response following prolonged or repeated exposure to an agonist. Phosphorylation of the receptor is one of the mechanisms of desensitisation. This directly, or indirectly, results in rapid and reversible uncoupling of the receptor from its heterotrimeric guanine nucleotide binding protein (G-protein). Previous research has shown that G protein coupled receptor kinases (GRKs) are key phosphorylators involved in the molecular mechanism of GPCR desensitisation. One of the mains goals of the research carried out in the Mason laboratory is to examine the molecular mechanisms of V1bR desensitisation. The current short term aim is to examine the potential role for GRKs in this mechanism. It is difficult to study a single receptor type and the molecular mechanisms involved in its regulation in a system larger than a cell based assay. As the proposed method of assessing the involvement of GRKs in desensitisation of the V1bR is to use RNA interference (RNAi) to knock down the expression of the GRKs, primary cell cultures of pituitary corticotrophs are an inappropriate choice. This is due to a number of factors, including the difficulty involved in transfecting primary cells, and the difficulty involved in interpreting the results from primary cell culture experiments as these cultures are composed heterogenous population of cells. Therefore, the main aim of this research was to develop a model cell system from an immortalised cell line, stably-transfected with the V1bR, in which the involvement of GRKs in the molecular mechanism of V1bR desensitisation could be studied. Development of stably-transfected cell lines requires substantial preliminary work and planning in order to produce a successful outcome. Once developed, characterisation of the clonal cell lines is required. The preliminary work involved determining the cell proliferation rate of the parental cell line, plasmid sub-cloning and production of a large quantity of plasmid DNA, optimisation of the antibiotic selection conditions, and optimisation of the transfection protocol, as well as modification of the inositol phosphate (IP) assay protocol. The V1bR activates the phospholipase Cβ (PLCβ) second messenger signalling pathway in response to stimulation with AVP. This results in the production of IPs and therefore, measurement of IPs in response to AVP stimulation of cells labelled with myo-[³H]inositol can be used as an indicator of functional V1bR expression. In this research a total of nine clonal cell lines resistant to the antibiotic G418 were generated. Initial testing of these lines indicated that four probably expressed the V1bR and these were selected for characterisation in greater detail. All of these four lines showed significantly increased IP production in response to AVP stimulation (P<0.05; t-test). A significant decrease in IP production in response to AVP stimulation following an AVP pre-treatment was also seen with all four lines (P<0.05; t-test). Current evidence therefore suggests that the V1bR in these clonal cell lines signals and desensitises in the normal way. Although further characterisation of the clonal cell line is desirable, the data to date indicate that these lines should be considered to provide an appropriate model system for examining the molecular mechanisms involved in the regulation of the V1bR. It appears that there are some minor differences in signalling between the clonal cell lines and therefore this should be a consideration when deciding which line is most appropriate to use for investigating a particular question.

Human Herpes Simplex viruses and Simian Herpes Simplex viruses share a high degree of genome homology, but despite this, important differences arise when the viruses are compared at the level of gene expression and virulence in non-host primates. In Human Herpes viruses (HSV-1 and HSV-2); 5 genes (RL02, US01, RS01, UL54 and US12) are expressed with an immediate early kinetics, i.e. their transcriptional activation does not require de novo synthesis of host or viral factors. The five immediate early (IE) genes regulate the cascade of expression of the other early and late HSV genes. Literature indicates that in HSV-1 infections, ICP4, ICP27 and to a lesser extent, ICP0, are mandatory for the full expression of the early and late gene classes. In contrast, our data on the Simian simplexviruses SA8, HVP-2 and B virus indicate that ICP0 (RL2) is the only gene with true IE kinetics. It is possible that in Simian Herpes viruses, ICP0 is necessary for the expression of all other viral genes, and to test this hypothesis I have cloned and expressed in Vero cells the ICP0 protein for the simian simplexvirus SA8 and studied its effect on the SA8 genes that are homologous to the immediate early genes in HSV. Results demonstrate that ICP0 does not appear to be sufficient to activate the transcription of the other IE genes but it is likely that ICP0 functionality is a necessary component in the activation process.

Human Herpes Simplex viruses and Simian Herpes Simplex viruses share a high degree of genome homology, but despite this, important differences arise when the viruses are compared at the level of gene expression and virulence in non-host primates. In Human Herpes viruses (HSV-1 and HSV-2); 5 genes (RL02, US01, RS01, UL54 and US12) are expressed with an immediate early kinetics, i.e. their transcriptional activation does not require de novo synthesis of host or viral factors. The five immediate early (IE) genes regulate the cascade of expression of the other early and late HSV genes. Literature indicates that in HSV-1 infections, ICP4, ICP27 and to a lesser extent, ICP0, are mandatory for the full expression of the early and late gene classes. In contrast, our data on the Simian simplexviruses SA8, HVP-2 and B virus indicate that ICP0 (RL2) is the only gene with true IE kinetics. It is possible that in Simian Herpes viruses, ICP0 is necessary for the expression of all other viral genes, and to test this hypothesis I have cloned and expressed in Vero cells the ICP0 protein for the simian simplexvirus SA8 and studied its effect on the SA8 genes that are homologous to the immediate early genes in HSV. Results demonstrate that ICP0 does not appear to be sufficient to activate the transcription of the other IE genes but it is likely that ICP0 functionality is a necessary component in the activation process.

Current treatment methods for non-union bone defects present problems. The objective of this study was to genetically engineer primary and immortalized cell types to express osteogenic molecules BMP2, RUNX2, OSX, or VEGF in a doxycycline dose-dependent manner for tissue regeneration. Coding cDNA sequences for all four factors were sub-cloned into the pRTS-1 expression plasmid and transfected into HUCPVCs, RBMCs, ROS cells. Electroporation was the most effective method of transfection for all cells but stably transfected cells could only be established for RBMCs and ROS cells. Cells achieved maximum expression within 72hours of induction and returned to basal levels after 18 days. Enhanced osteogenic bioactivity was only observed upon activation of BMP-2. The tight regulation of the pRTS-1 system allowed for a controlled gene expression. Future transplantation experiments using these engineered RBMC and ROS cells in vivo will evaluate the usefulness of the dox-inducible gene expression system in bone defects.

Current treatment methods for non-union bone defects present problems. The objective of this study was to genetically engineer primary and immortalized cell types to express osteogenic molecules BMP2, RUNX2, OSX, or VEGF in a doxycycline dose-dependent manner for tissue regeneration. Coding cDNA sequences for all four factors were sub-cloned into the pRTS-1 expression plasmid and transfected into HUCPVCs, RBMCs, ROS cells. Electroporation was the most effective method of transfection for all cells but stably transfected cells could only be established for RBMCs and ROS cells. Cells achieved maximum expression within 72hours of induction and returned to basal levels after 18 days. Enhanced osteogenic bioactivity was only observed upon activation of BMP-2. The tight regulation of the pRTS-1 system allowed for a controlled gene expression. Future transplantation experiments using these engineered RBMC and ROS cells in vivo will evaluate the usefulness of the dox-inducible gene expression system in bone defects.