Chronic kidney disease (CKD) and its cardiac complications represent a large and growing problem in Canada. The progression of CKD is driven by the activation of several final common pathways of injury, including fibrosis and oxidative stress. If left unchecked, these inter-connected processes lead to progressive damage and subsequent organ dysfunction. Current clinical therapies, consisting of aggressive blood pressure control and blockade of the renin-angiotensin system, fail to arrest this progressive injury in a significant number of patients. Early outgrowth cells (EOCs) represent a novel bone marrow-derived cell population that have been recently described to have tissue protective activity. In this work, we examined the effects of intravascular EOC infusion in two independent models of CKD, demonstrating potent anti-fibrotic renoprotective effects in the subtotally nephrectomized (SNX) rat, a well-established model of non-diabetic progressive CKD, and anti-fibrotic and anti-oxidant effects in the db/db mouse, a commonly used model of type 2 diabetic nephropathy. In the SNX rat, which is characterized by impaired cardiac relaxation reminiscent of a common and high risk clinical CKD phenotype, EOC infusion was also associated with improved cardiac structure and function. In both cases, infused EOCs were not retained in significant numbers within the diseased kidney or heart, but rather localized to distant organs such as the liver, spleen, and bone marrow. We further demonstrated that EOCs release soluble factors with anti-oxidant and anti-fibrotic activity in vitro, and that a cell-free preparation of EOC-derived factors can mimic the reno- and cardiac protective effects of the cells themselves when infused into the SNX rat. Taken together, our results demonstrate the therapeutic potential of an EOC-based strategy for the treatment of CKD and its cardiac complications, and provide the preclinical rationale for the design of clinical trials of EOC-based therapies for this devastating disease.

Cellular therapies rely on the delivery of therapeutic cells into patients, but their safety can be compromised by the manipulation of cells ex vivo or their placement outside of their natural context in vivo. Cell Fate Control Gene Therapy (CFCGT) offers the possibility of establishing pharmacological controls over gene-modified cells (GMCs) with regards to their proliferation, differentiation, or function. In its simplest form, 'suicide' gene therapy (SGT), stable introduction of a 'suicide' gene that can activate a non-toxic prodrug establishes control over the survival of GMCs. Current SGT modalities are sub-optimal in clinical setting. To overcome the many limitation of current strategies, we have developed a next-generation CFCGT approach based on the active site-engineered variants of human deoxyCytidine Kinase (dCK), which enable robust activation of multiple Nucleoside Analogue (NA)-based prodrugs, act early in the pathway enabling rapid accumulation of activated NAs in target cells, and also provide the capabilities for the direct imaging of GMCs. Stable introduction of dCK variants into target cells by means of Lentiviral (LV) gene transfer significantly increases their sensitivity to multiple prodrugs. Our dCK variant with only two active site amino acid substitutions is expected to be non-immunogenic yet capable of specifically activating deoxythymidine- and deoxyuridine-based NAs that are not substrates for the wild-type enzyme, such as bromovinyldeoxyuridine (BVdU) and L-deoxythymidine (LdT). We show here that dCK can be used for controlling the survival of GMCs, in cell lines and primary cells in vitro and in a murine xenogeneic transplant models in vivo. To characterize dCK/prodrug-mediated killing mechanisms in GMCs, we have examined the levels of active metabolites in cells and the cellular pathways they antagonize. We describe here the experimental basis for the application of this novel CFCGT in bone marrow transplantation for management of Graft-versus-Host Disease (GvHD) and in enhancing chemotherapy in direct treatment of tumors. In summary, we have developed a novel and robust strategy for effective CFCGT that addresses the many shortcomings of existing modalities. Future studies will validate this novel system in a variety of primary cells and animal disease models, including models of hematopoietic transplantation and ES/iPS-based cell therapies.

Embryonic stem cells (ESC) constitute a promising source of cells for cardiac transplantation strategies. However, complexities associated with in vivo studies have made it difficult to develop a thorough understanding of cell integration. We have engineered an in vitro system that recapitulates the native cardiac environment using 300μm thick collagen scaffolds seeded with neonatal cardiomyocytes (CM) and electrical field stimulation. The injection of undifferentiated ESC served as a baseline to assess the validity of studying cell transplantation in this model. Yfp-ESC survived and proliferated over several days in model tissue. ESC were not observed to significantly differentiate into the cardiac lineage, and did not integrate with the cardiac cell population. While the injection of ESC improved cardiac cell number, tissue functional properties were hindered. The methods developed herein can be readily adapted to study ESC derived progenitor and differentiated cells, to elucidate the optimal cell state for ESC-mediated cell therapy.

Chronic kidney disease (CKD) and its cardiac complications represent a large and growing problem in Canada. The progression of CKD is driven by the activation of several final common pathways of injury, including fibrosis and oxidative stress. If left unchecked, these inter-connected processes lead to progressive damage and subsequent organ dysfunction. Current clinical therapies, consisting of aggressive blood pressure control and blockade of the renin-angiotensin system, fail to arrest this progressive injury in a significant number of patients. Early outgrowth cells (EOCs) represent a novel bone marrow-derived cell population that have been recently described to have tissue protective activity. In this work, we examined the effects of intravascular EOC infusion in two independent models of CKD, demonstrating potent anti-fibrotic renoprotective effects in the subtotally nephrectomized (SNX) rat, a well-established model of non-diabetic progressive CKD, and anti-fibrotic and anti-oxidant effects in the db/db mouse, a commonly used model of type 2 diabetic nephropathy. In the SNX rat, which is characterized by impaired cardiac relaxation reminiscent of a common and high risk clinical CKD phenotype, EOC infusion was also associated with improved cardiac structure and function. In both cases, infused EOCs were not retained in significant numbers within the diseased kidney or heart, but rather localized to distant organs such as the liver, spleen, and bone marrow. We further demonstrated that EOCs release soluble factors with anti-oxidant and anti-fibrotic activity in vitro, and that a cell-free preparation of EOC-derived factors can mimic the reno- and cardiac protective effects of the cells themselves when infused into the SNX rat. Taken together, our results demonstrate the therapeutic potential of an EOC-based strategy for the treatment of CKD and its cardiac complications, and provide the preclinical rationale for the design of clinical trials of EOC-based therapies for this devastating disease.

Growing evidence from animal and clinical studies suggests that cardiac cell therapy can restore perfusion and improve function in the ischemic/infarcted myocardium. However, cell therapy is hindered by insufficient cell numbers, inefficient cell homing and engraftment, and inadequate cellular interactions. Furthermore, the biological mechanisms and local effects of transplanted cells have not been well-elucidated. The research presented herein attempts to address some of these issues. In manuscript #1, a new subpopulation of circulating progenitor cells (CPCs), termed derived CD133+ cells, was generated from the CD133- fraction of human peripheral blood. The derived CD133+ progenitors appeared to have superior vasculogenic potential in vitro, which may prove to be beneficial in inducing vasculogenesis in ischemic tissues. Positron emission tomography (PET) with direct cell labeling and reporter gene techniques were employed to assess the fate of transplanted human CPCs in vivo at different subjects of investigation, and different stages of cell transplantation. In manuscript #2, PET imaging with 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) direct cell labeling was used to demonstrate that collagen-based matrices improve the early homing and retention of delivered CPCs in a rat ischemic hindlimb model. This mechanism conferred by the matrix may have implications on cell therapy at the early stages after transplantation. In manuscript #3, a more efficient, stable and accurate labeling method, hexadecyl-4-[18F]fluorobenzoate (18F-HFB) direct cell labeling, was developed to quantify cell distribution of transplanted CPCs in a rat myocardial infarction model. PET imaging of 18F-HFB-CPCs revealed significant cell washout from the myocardium immediately after intramyocardial injection, with only a small proportion of transplanted CPCs remaining in the target area in the first 4 hours after delivery. In manuscript #4, human CPCs transduced with lentiviral vectors showed stable expression of PET reporter genes. This reporter gene based-cell labeling technique can be developed for noninvasive tracking cells within a bioengineered matrix by PET, while preserving cell phenotype, viability and function. These studies contribute important insights into the biology and physiology of transplanted stem cells and the ability of delivery matrices to improve transplanted cell engraftment, survival, and function. I believe with further refinement, cell expansion, tissue engineering and PET imaging could facilitate the clinical applications of cell therapies in years to come.

<p>Excess cardiac myofibroblasts in fibrotic heart diseases as well as cell-based therapies involving implantation of stem cells or genetically engineered somatic cells in the heart may all lead to a situation where a cardiomyocyte becomes electrically coupled to an unexcitable cell. In these settings, electrotonic loading of cardiomyocytes by unexcitable cells can affect cardiac action potential generation, propagation, and repolarization depending on the properties of both cardiomyocytes and unexcitable cells. The objective of this dissertation was to advance our understanding of the electrical interactions between cardiomyocytes and unexcitable cells using a variety of electrophysiological, molecular, and cell culture techniques.</p><p>First, we utilized aligned cardiomyocyte monolayers covered with unexcitable cardiac fibroblasts or human embryonic kidney-293 (HEK) cells that expressed similar levels of the gap junction protein connexin-45. These cells weakly coupled to cardiomyocytes and marginally slowed cardiac conduction only at high coverage density, while producing no other measurable electrophysiological changes in cardiomyocytes. In contrast, unexcitable HEK cells genetically engineered to stably express the more conductive connexin-43 channels (Cx43 HEK) strongly coupled to cardiomyocytes, depolarized cardiac resting membrane potential, significantly slowed impulse propagation, decreased maximum capture rate, and increased action potential duration (APD) at high coverage density. None of the studied unexcitable cells significantly altered conduction velocity anisotropy ratio or the relatively low incidence of pacemaking activity of cardiac monolayers at any coverage density.</p><p>Next, we utilized individual micropatterned cell pairs consisting of a cardiomyocyte and an unexcitable Cx43 HEK cell with or without stably overexpressed inward rectifier potassium channels (Kir2.1+Cx43 HEK). By systematically varying the relative sizes of micropatterned cells, we showed that Cx43 HEK cells significantly depolarized cardiomyocytes, reduced maximum upstroke velocity and action potential amplitude, prolonged APD, and modulated beating rate as a function of HEK:CM area ratio. In contrast, in cell pairs formed between cardiomyocytes and Kir2.1+Cx43 HEK cells we observed significant reduction in cardiomyocyte action potential amplitude, duration, and maximum upstroke velocity, but no change in other measured parameters.</p><p>Finally, we utilized a hybrid dynamic clamp setting consisting of a live micropatterned cardiomyocyte coupled in real time to a virtual model of capacitive and/or ionic current components of Cx43 HEK or Kir2.1+Cx43 HEK cells. We found that coupling of cardiomyocytes to the ionic current components of Cx43 HEK or Kir2.1+Cx43 HEK cells was sufficient to reproduce the dependence of cardiomyocyte maximal diastolic potential and pacemaking behavior on HEK:CM area ratio observed in micropatterned cell pairs, but did not replicate the observed changes in action potential upstroke or duration. The pure capacitance model with no ionic current, on the other hand, significantly decreased cardiomyocyte maximum upstroke velocity and prolonged cardiomyocyte APD as function of HEK:CM area ratio without affecting maximal diastolic potential or pacemaking behavior. When the unexcitable cell model containing both capacitive and ionic currents was connected to cardiomyocytes, all changes in action potential shape observed in micropatterned cell pairs were accurately reproduced. </p><p>These studies describe how coupling of unexcitable cells to cardiomyocytes can alter cardiomyocyte electrophysiological properties dependent on the unexcitable cell connexin isoform expression, ion channel expression, and cell size. This knowledge is expected to aid in the design of safe and efficient cell and gene therapies for myocardial infarction, fibrotic heart disease, and cardiac arrhythmias.</p> / Dissertation

Bone tissue engineering is a promising approach to address the limitations of currently used bone tissue substitutes. However, an optimal cell source for the production of osteoblastic matrix proteins and mineral deposition has yet to be defined. In response to this deficiency, ex vivo gene therapy of easily accessible non-osteogenic cells, such as skeletal myoblasts, has become a prevalent strategy for inducing an osteoblastic phenotype. The majority of these approaches focus on constitutive overexpression of soluble osteogenic growth factors such as bone morphogenetic proteins (BMPs). In order to avoid aberrant effects of unregulated growth factor secretion, this work focuses on delivery of the osteoblastic transcription factor Runx2 as an autocrine osteogenic signal under the control of an inducible expression system. The overall objective of this research was to engineer an inducible cell source for bone tissue engineering that addresses the limitations of current cell-based approaches to orthopedic regeneration. Our central hypothesis was that inducible Runx2 overexpression in skeletal myoblasts would stimulate differentiation into a regulated osteoblastic phenotype. We have demonstrated that Runx2 overexpression stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype. Furthermore, we have established Runx2-engineered skeletal myoblasts as a potent cell source for bone tissue engineering applications in vitro and in vivo, similar to BMP-2-overexpressing controls. Finally, we exogenously regulated osteoblastic differentiation by myoblasts engineered to express a tetracycline-inducible Runx2 transgene. This conversion into an osteoblastic phenotype was inducible, repressible, recoverable after suppression, and dose-dependent with tetracycline concentration. This work is significant because it addresses cell sourcing limitations of bone tissue engineering, develops controlled and effective gene therapy methods for orthopedic regeneration, and establishes a novel strategy for regulating the magnitude and kinetics of osteoblastic differentiation.

The pharmacologic treatment of painful neuropathy continues to pose problems and challenges in clinical practice. This is largely due to a limited understanding of the underlying etiologies of such neuropathic pain and insufficient knowledge of the optimal effective doses that would cause only minimal systemic side effects. The use of molecular methods, such as gene deletion from knockout mice and the development of cellular mini-pumps for the delivery of biologic antinociceptive molecules have led to a better understanding of the underlying mechanisms involved in the induction of intractable neuropathic pain. It is now known that the initiation of an excitatory cascade after injury or disease leads to the induction of various second messenger systems, and the loss or down-regulation of the endogenous inhibitory spinal system and central sensitization, both of which cause such pain. Currently, there are novel approaches that use genetic therapy in the management of neuropathic pain. Two such approaches which have been determined to be safe are proposed to be investigated in this study using animal models of pain. The first approach involves cell-mediated delivery of antinociceptive molecules to the cerebrospinal fluid using cultivated spinal progenitor cells transplanted into the subarachnoid space. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the hind paw of rats. 1x106 spinal progenitor cells (SPCs) were implanted intrathecally on the third day after the CCI surgery. The behavioral response to thermal hyperalgesia was observed and recorded during the 14 days post surgery. Various techniques were utilized to trace the progenitor cells, confirm the differentiation, and identify the neurotransmitters involved. Glutamic acid decarboxylase (GAD) immunoreactivity was revealed for 65% of the cultivated SPCs in our study. We also determined that transplanted cells could survive more than four weeks post intrathecal implantation. Significant reductions were demonstrated for responses to thermal stimuli for the CCI rats that had received intrathecal SPC transplantation. A novel intrathecal delivery with SPCs reduced CCI-induced neuropathic pain. The second approach involves the use of a newly developed intrathecal electroporation probe in the delivery of antinociceptive peptides to reduce expression of endogenous nociceptive molecules in the spinal cord. To investigate the feasibility of delivering exogenous genes into spinal cord using direct in vivo electrotransfection, pE-GFP C1 vector was used to achieve the goal. Gene transfer to the spinal cord was accomplished via direct intrathecal injection of, followed by 5 electric pulses for 50 ms at 200 V delivered intrathecally. The spinal cords were retrieved and analyzed with fluorescence microscopy, reverse transcription polymerase chain reaction (RT-PCR), and western blotting. At day 1, 3 or 7 following electroporation a clear green fluorescence protein (GFP) expression in spinal cord tissue was detected. The most prominent transfection occurred in the meningeal cells and superficial layer of the spinal cord. Successful transfection was also confirmed with RT-PCR and western blotting. The expression of GFP protein was peaked between 3-7 days after electroporation and significantly decreased at 14 days. No behavioral or spinal neurodegenerative changes were detected at any time point. This study demonstrates that direct in vivo electrotransfection represents an effective and simple method for spinal gene delivery. Furthermore, the optimal pulse characteristics (voltage, pulse duration, number of shocks) were investigated for in vivo electroporation for gene transfer into the spinal cord. The expression of pre-opiomelanocortin (POMC) gene from electroporated plasmid DNA was then evaluated in this study using RT-PCR and western blot. We conclude that the optimal conditions for electroporation are a pulse voltage of 200 V, 75-ms duration, 925-ms interval, for five iterations. Also, electroporation treatment for neuropathic pain was attempted for CCI rats using plasmid DNA that expresses the POMC gene. Intrathecal administrations of the POMC plasmid elevated spinal beta-endorphin levels, as manifested in significantly elevated pain threshold for the CCI limbs. We also tested whether intrathecal electric stimulation would reduce the tolerance of chronic morphine usage and the severity of precipitated morphine withdrawal symptoms. Rats received intrathecal electrode catheter implantation and a continuous intrathecal infusion of morphine (2 nmol/hr) or saline for seven days. Intrathecal electric stimulations (0, 20V, 200V) were performed once daily during the same period. Daily tail flick and intrathecal morphine challenge tests were performed to assess the effect of intrathecal electric stimulation on antinociception and tolerance of morphine. Naloxone withdrawal (2mg/kg) was performed to assess morphine dependence, and changes in spinal neurotransmitters were monitored by microdialysis. The antinoceptive effect of intrathecal morphine was increased by 200V electric stimulation. The magnitude of tolerance was decreased in the rats receiving 2 nmol/hr infusion with daily intrathecal electric stimulation. The severity of naloxone-induced withdrawal symptom was lower in the rats receiving 200V stimulation. Intrathecal stimulation thus enhances analgesia and attenuates naloxone-induced withdrawal symptoms in rats receiving chronic intrathecal morphine infusion. Increases in spinal glycine release may be the underlying mechanisms. The promise is that, both approaches attenuate or reverse persistent nociceptive states; they could be exploited for use in the development of gene therapy for the management of pain.

Human mesenchymal stem cells (hMSCs) are a promising candidate for cell-based therapies given their therapeutic potential and propensity to grow in vitro. However, to generate the cell numbers required for such applications, robust, reproducible and scalable manufacturing methods need to be developed. To address this challenge, the expansion of hMSCs in a microcarrier-based bioreactor system was investigated. Initial studies performed in T-flask monolayer cultures investigated the effect of key bioprocess parameters such as dissolved oxygen concentration (dO2), the level of medium exchange and the use of serum-free media. 20 % dO2 adversely impacted cell proliferation in comparison to 100 % dO2, whilst FBS-supplemented DMEM was found to be the most consistent and cost-effective cell culture medium despite the advances in serum-free cell culture media. Several microcarriers were screened in 100 mL agitated spinner flasks where Plastic P102-L was selected as the optimal microcarrier for hMSC expansion given the high cell yields obtained, its xeno-free composition and effective harvest capacity. The findings from the initial small-scale studies culminated in the successful expansion of hMSCs on Plastic P102-L microcarriers in a fully equipped 5 L stirred-tank bioreactor (2.5 L working volume), the largest reported volume for hMSC microcarrier culture to date. A maximum cell density of 1.68 x 105 cells/mL was obtained after 9 days in culture; further growth was limited by the low glucose concentration and lack of available surface area. A novel, scalable harvesting method was also developed, allowing for the successful recovery of hMSCs. Importantly, harvested hMSCs retained their immunophenotype, multipotency and ability to proliferate on tissue culture plastic.

Eine Vielzahl präklinischer Schlaganfallstudien zeigte die neuroprotektive und neuroregenerative Wirkung des hämatopoetischen Wachstumsfaktors G-CSF (Granulozyten-Kolonie stimulierender Faktor). Ein Wirkungsmechanismus des G-CSF ist die Mobilisation von protektiven Knochen-markzellen in die ischämische Läsion, wobei diese zeitverzögert nach G-CSF-Gabe stattfindet. Eine zusätzliche frühzeitige Transplantation mononukleärer Knochenmarkzellen (BM MNC) könnte diese therapeutische Lücke füllen. Ziel der vorliegenden Studie war es, die Wirksamkeit dieser Kombinations-therapie in einem Schlaganfallmodell der spontan hypertensiven Ratte (SHR) zu testen. Syngene BM MNC wurden aus dem Knochenmark von SHRs durch immunmagnetische Depletion der Granulozyten isoliert. Nach Verschluss der Arteria cerebri media wurde den Tieren über insgesamt 5 Tage G-CSF verabreicht und zusätzlich zu einem frühen (6h nach Schlaganfall) oder späteren (48h nach Schlaganfall) Zeitpunkt BM MNC intravenös appliziert. Unbehandelte Schlaganfalltiere sowie Tiere mit alleiniger G-CSF-Therapie dienten als Kontrolle. Das Infarktvolumen wurde weder durch die alleinige G-CSF-Gabe noch durch die zusätzliche Zelltherapie verändert. Dennoch wiesen Tiere mit G-CSF-Einzeltherapie eine anhaltende funktionelle Verbesserung des sensomotorischen Defizites auf. Während die zusätzliche frühzeitige Zelltransplantation (6h) keinen weiteren Therapieeffekt zeigte, führte die Zelltransplantation nach 48h zu einer Aufhebung des protektiven G-CSF Effektes. Die G-CSF-Therapie bewirkte erwartungsgemäß einen deutlichen Anstieg der zirkulierenden Leukozyten. Interessanterweise wurde der Granulozytengehalt im Blut und in der Milz durch die einmalige Zelltherapie nach 48h signifikant erhöht. Ein Großteil der transplantierten BM MNC (48h) konnte in der Milz nachgewiesen werden und führte dort vermutlich zu einer kompetitiven Hemmung des Granulozytenabbaus. Dies hatte sowohl den Anstieg der zirkulierenden Granulozyten als auch deren vermehrte Infiltration in das ischämische Hirngewebe zur Folge und könnte schließlich den negativen Einfluss auf die funktionelle Verbesserung erklären. Die beobachteten Interaktionsmechanismen werfen ein interessantes Licht auf die mögliche Wirkungsweise von Zelltherapien und unterstreichen die entscheidende Rolle des Immunsystems in der Pathophysiologie des Schlaganfalls.