Rationale: Autologous bone marrow- or cardiac-derived stem cell therapy for heart disease has demonstrated safety and efficacy in clinical trials but has only offered limited functional improvements. Finding the optimal stem cell type best suited for cardiac regeneration remains a key goal toward improving clinical outcomes. Objective: To determine the mechanism by which novel bone-derived stem cells support the injured heart. Methods and Results: Cortical bone stem cells (CBSCs) and cardiac-derived stem cells (CDCs) were isolated from EGFP+ transgenic mice and were shown to express c-kit and Sca-1 as well as 8 paracrine factors involved in cardioprotection, angiogenesis and stem cell function. Wild-type C57BL/6 mice underwent sham operation (n=21) or myocardial infarction (MI) with injection of CBSCs (n=57), CDCs (n=31) or saline (n=57). Cardiac function was monitored using echocardiography with strain analysis. EGFP+ CBSCs in vivo were shown to express only 2/8 factors tested (basic fibroblast growth factor and vascular endothelial growth factor) and this expression was associated with increased neovascularization of the infarct border zone. CBSC and CDC therapy improved survival, cardiac function, attenuated adverse remodeling, and decreased infarct size relative to saline-treated MI controls. CBSC treated animals showed the most pronounced improvements in all parameters. By 6 weeks post-MI, EGFP+ cardiomyocytes, vascular smooth muscle cells and endothelial cells could be identified on histology in CBSC-treated animals but not in CDC-treated animals. EGFP+ myocytes isolated from CBSC-treated animals were smaller, more frequently mononucleated, and demonstrated fractional shortening and calcium currents indistinguishable from EGFP- myocytes from the same hearts. Conclusions: CBSCs improve survival, cardiac function, and attenuate remodeling more so than CDCs and this occurs through two mechanisms: 1) secretion of the proangiogenic factors bFGF and VEGF (which stimulates endogenous neovascularization), and 2) differentiation into functional adult myocytes and vascular cells. / Physiology

Current cartilage repair methods are ineffective in restoring the mechanical and biological functions of native hyaline cartilage. Therefore, using the paracrine actions of stem cell therapies to stimulate endogenous cartilage regeneration has gained momentum. Adipose stem cells (ASCs) are an attractive option for this endeavor because of their accessibility, chondrogenic potential, and secretion of factors that promote connective tissue repair. In order to use the factors secreted by ASCs to stimulate cartilage regeneration, the signaling pathways that affect postnatal cartilage development and morphology need to be understood. Next, approaches need to be developed to tailor the secretory profile of ASCs to promote cartilage regeneration. Finally, delivery methods that localize ASCs within a defect site while facilitating paracrine factor secretion need to be optimized. The overall objective of this thesis was to develop an ASC therapy that could be effectively delivered in cartilage defects and stimulate regeneration via its paracrine actions. The general hypothesis was that the secretory profile of ASCs can be tailored to enhance cartilage regeneration and be effectively delivered to regenerate cartilage in vivo. The overall approach used the growth plate as an initial model to study changes in postnatal cartilage morphology and the molecular mechanisms that regulate it, different media treatments and microencapsulation to tailor growth factor production, and alginate microbeads to deliver ASCs in vivo to repair cartilage focal defects.

Embryonic stem cells (ESCs) are being considered as a cell source for cardiac regeneration because of their potency and availability. We studied the electrophysiological implications using co-cultures of ESCs and neonatal rat ventricular myocytes (NRVM) grown on a multi-electrode array (MEA). To mimic expected engraftment rates 5% mouse ESCs were co-cultured with NRVMs. Comparing cultures without and with 5% ESCs at 4 days, the mean bipolar field potential duration (FPD) of NRVMs increased from 26.3 ± 2.2 ms (n=10) to 44.3 ± 6.2 ms (n=9; p < 0.05), the interspike interval (ISI) increased from 358.3 ± 62.8 ms (n=10) to 947.8 ± 214.6 ms (n=7; p < 0.01), and conduction velocity (CV) decreased from 14.2 ± 1.3 cm/s (n=8) to 4.6 ± 1.2 cm/s (n=5; p < 0.01). To evaluate whether ESC were having direct or paracrine effects on NRVMs, media conditioned by 3x106 ESCs for 24 hr was diluted 1:1 with fresh media and then introduced to NRVM cultures on the day of plating. Conditioned media was changed daily and altered mean FPD, ISI, and CV to 46.1 ± 7.8 ms, ISI to 682.0 ± 128.5 ms, and 4.2 ± 0.4 cm/s (n=8; p < 0.01 for each measure), respectively at 4 days. However, changes were not seen in media that was incubated for 24hrs and diluted 1:1 with fresh media and introduced to NRVM cultures in a similar fashion (n=7; p > 0.05). Slowed CV is associated with increased arrhythmic risk and reports demonstrate an inverse relationship between CV and nonphosphorylated Cx43(NP-Cx43). Western blots for total Cx43 expression revealed a decrease in ratio of P-Cx43/NP-Cx43 in the 5% mouse ESCs and ESC conditioned media cultures as compared to controls (n=8; p < 0.01 for each). There was not significant increase in the total Cx43 expression (n=6; p > 0.05). Culturing ESCs with NRVMs resulted in a decreased ISI, prolonged FPD, and slowed CV of the co-cultures as compared to controls leading to pro-arrhythmic conditions. Similar effects on NRVMs were observed when applying media conditioned by ESCs, suggesting that the electrophysiological changes were mediated by soluble factors. The increase in NP-Cx43 leads to gap junction uncoupling being a potential mechanism for these arrhythmogenic substrates. Further research into preventing NP-Cx43 in cultures is currently underway.

In open angle glaucoma, dysfunction of the trabecular meshwork (TM) results in impaired aqueous humour outflow leading to an elevated intraocular pressure (IOP) that underlies optic nerve damage and irreversible blindness. Currently, no curative treatment is available for the disease. Indeed, most pharmacological and surgical interventions usually provide only temporary relief from elevated IOP while little progress has been made in targeting the root cause of this disease: correcting the dysfunctional TM. In this context, we hypothesized that regeneration/refunctionalization of the TM may represent an effective therapeutic option to halt disease progression or even reverse the pathologic process. We previously demonstrated in a rat model of glaucoma that the injection of mesenchymal stromal cells (MSCs) cultured under hypoxic conditions or their conditioned media (MSC-CM) into laser-damaged TM area results in tissue regeneration. Injection of MSC or conditioned media in our glaucoma model led to activation and proliferation of ocular progenitor cells culminating in TM regeneration and a decrease in IOP. However, the mechanistic basis for this regenerative process remained elusive. Thus, the aim of this thesis is to elucidate the mechanistic basis of MSC secretome-mediated TM regeneration and the subsequent decrease in IOP. We now demonstrate that injection of hypoxic MSC-CM into laser-induced glaucomatous eyes resulted in massive immune cell recruitment. We also demonstrate that these hypoxic MSC-CM conditioned cells produced pro-regenerative factors in vitro and in vivo. Next, employing a proteomic approach, we identified and verified the pro-regenerative effect of several factors secreted by hypoxic MSC-CM recruited cells, which in turn induced the activation/proliferation of ocular progenitor cells leading to TM regeneration and decreased IOP. Upon individual injection of the purified factors into glaucomatous rat eyes, we observed a partial and delayed but significant decrease in IOP that correlated with an increase in the activation and proliferation of neuronal progenitor cells in the TM area. The co-injection of these factors resulted in a significant decrease in IOP compared with individual factor injection. Importantly, this drop in IOP was associated with restoration of retinal functionality, thus demonstrating the importance of these factors in the TM regeneration process and disease control. The findings presented in this thesis provide a novel acellular therapeutic approach for glaucoma treatment via in situ TM regeneration. Moreover, the knowledge gained here could have a lasting impact on how we induce tissue regeneration in other degenerative diseases and lead to novel therapeutic advances in regenerative medicine. / Dans le glaucome à angle ouvert, le dysfonctionnement du trabéculum (TM), un tissu nécessaire à la filtration de l'humeur aqueuse, entraîne une élévation de la pression intraoculaire (PIO). Ceci cause des lésions au niveau du nerf optique et une cécité irréversible. Présentement, aucun traitement curatif n'a été développé pour cette maladie. Nous émettons l'hypothèse que la régénération et re-fonctionnalisation du trabéculum peut représenter une option thérapeutique efficace pour arrêter ou inverser la progression de la maladie dans de nombreux cas de glaucome. Nous avons précédemment démontré les effets régénérateurs des cellules mésenchymateuses (MSCs) et de leurs milieux conditionnés par l'hypoxie (MSC-CM) dans la régénération du TM suite à un dommage par laser. Ce processus a conduit à l'activation et à la prolifération des cellules progénitrices oculaires résultant en une diminution de la PIO dans un modèle de glaucome induit par laser chez le rat. Cependant, la base mécanistique de ce processus de régénération reste encore inconnue. Ainsi, le but de cette thèse de recherche est d'élucider cette base mécanistique de la régénération du TM médiée par le sécrétome des MSC et la diminution subséquente de la PIO. À cette fin, l'injection de MSC-CM hypoxique dans les yeux glaucomateux induits par laser a entraîné un important recrutement de cellules immunitaires. Sous l’action du MSC-CM, ces cellules produisent des facteurs pro-régénératifs in vitro et in vivo. Ensuite, nous avons utilisé une approche protéomique et vérifié l'effet pro-régénératif des facteurs sécrétés par ces cellules exposées au MSC-CM hypoxique, sur l'activation et la prolifération des cellules progénitrices oculaires et la PIO. Lors de l'injection de ces facteurs chez le rat glaucomateux, nous avons observé une augmentation significative de l'activation et de la prolifération des cellules progénitrices neuronales présentes dans la zone du TM, résultant en une diminution de la PIO. De plus, l’injection combinée de ces facteurs résulte en une diminution synergique importante de la PIO. Cette baisse de la PIO était associée à une restauration de la fonction rétinienne, démontrant ainsi l'importance de ces facteurs dans le processus de régénération du TM et de contrôle de la maladie. Les résultats présentés dans cette thèse pourraient amener à une nouvelle approche thérapeutique acellulaire pour le traitement du glaucome via la régénération du TM. De plus, les connaissances acquises au cours de cette thèse pourraient avoir un impact durable sur la manière d’aborder la régénération tissulaire dans d'autres maladies dégénératives et amener des avancées thérapeutiques nouvelles en médecine régénératrice

Les cellules souches ont été présentées comme la clé d’une médecine régénératrice, où la réparation d’un organe, la guérison de maladies dégénératives et la création de nouveaux tissus seraient des objectifs réalisables à court ou moyen terme. Les cellules souches isolées chez un adulte ont un certain degré de spécialisation limitant leur potentiel à leur organe d’origine. Mais l’une d’elles, la cellule souche mésenchymateuse (MSC), possède une versatilité qui en fait une candidate idéale pour plusieurs traitements. Il est communément accepté que les MSC exercent leur effet grâce à la production de facteurs solubles. Toutefois, leur mécanisme d’action reste jusqu’à maintenant imprécis. Ces facteurs ayant le potentiel d’agir dans plusieurs maladies dégénératives, nous avons voulu évaluer leur effet régénératif dans l’une des maladies oculaires les plus répandues, le glaucome à angle ouvert. Cette maladie est caractérisée par une destruction des cellules ganglionnaires de la rétine suite à une élévation de la pression intraoculaire. Cette hausse de pression est souvent engendrée par une dysfonction du trabéculum, le tissu régulant la sortie de l’humeur aqueuse de l’œil. Actuellement, la progression du glaucome peut être contrôlée, mais la maladie ne peut pas être guérie. Or la régénération du trabéculum pourrait arrêter la progression de la maladie et même renverser le processus. Ainsi, l’objectif de cette thèse était de vérifier si les MSC ont le potentiel de favoriser la régénération oculaire dans des cas de glaucome et de comprendre les mécanismes sous-jacents. Pour ce faire, nous avons injecté des MSC ou leurs facteurs sécrétés dans un modèle de glaucome induit par trabéculoplastie laser. Nous avons démontré que les MSC peuvent régénérer le trabéculum endommagé et abaisser la pression oculaire en réactivant des cellules progénitrices dans le corps ciliaire. Ces cellules prolifèrent et s’implantent dans le trabéculum une semaine après le traitement. Nous avons aussi démontré que les facteurs produits par les MSC cultivées en conditions hypoxiques induisent l’activation des cellules progénitrices. Par contre, les MSC cultivées dans un environnement normoxique n’induisent pas cet effet. Nous avons aussi observé que les facteurs produits dans des conditions hypoxiques sont incapables de réactiver les cellules progénitrices ex vivo. Nous avons donc voulu vérifier si d’autres facteurs régénératifs sont engendrés in situ suite à l’injection des MSC. Nous avons ainsi découvert qu’un facteur produit par les macrophages migrant dans la zone endommagée permet de réguler l’activation des cellules progénitrices. Cette production requiert cependant une exposition des macrophages aux facteurs paracrines des MSC. Nous avons aussi observé que l’effet régénératif des MSC était inhibé suite à l’élimination des macrophages de l’organisme. Ceci positionne les macrophages comme un intermédiaire essentiel de l’effet régénératif exercé par les MSC. Les résultats présentés dans cette thèse constituent une avancée importante pour l’utilisation des MSC en médecine régénératrice. Ils ont permis d’établir les bases d’un traitement potentiel pour le glaucome à angle ouvert et d’ajouter une pièce importante à la compréhension des mécanismes régénératifs des MSC. Ces connaissances devraient avoir un impact significatif sur la régénération du tissu oculaire et de plusieurs autres organes. / Stem cells have been presented as the key to regenerative medicine, where organ repair, cures for degenerative diseases and even the creation of new tissue could be achieved. Adult stem cells already possess a certain level of differentiation, which limits their potential effect to the organ where they were isolated. But one subtype, mesenchymal stem cells (MSC), possess a versatility which makes them ideal candidates for many treatments. It is commonly accepted that MSCs exert their effect through paracrine factors, but their precise mechanism of action remains uncertain. As such factors could have a positive effect in many degenerative diseases, we wanted to study their regenerative potential in one of the most widespread ocular disease, open angle glaucoma. This disease is characterised by the destruction of retinal ganglion cells following a rise in intraocular pressure. This pressure rise is mostly caused by a dysfunction of the trabecular meshwork, the tissue regulating the outflow of aqueous humor from the eye. Glaucoma progression can currently be controlled, but the disease cannot be cured. Meanwhile, trabecular regeneration could halt and even reverse disease progression. Thus, the objective of this thesis is to evaluate MSC potential in the regeneration of the trabecular meshwork and to understand any underlying mechanisms. To this end, we injected MSC or their secreted factors in a model of trabeculoplasty induced glaucoma. We demonstrated that MSC can regenerate the damaged trabeculum and lower ocular pressure by reactivating progenitor cells located in the ciliary body. These cells proliferate and restore the trabeculum within one week after the initial treatment. We also demonstrated that MSC factors produced under hypoxic conditions induce progenitor cell activation. Such activation does not occur with MSC factors produced under normoxic conditions. We have also observed that factors produced under hypoxic conditions are incapable of reactivating progenitor cells in an ex vivo setting. Thus, we wanted to verify if any other regenerative factors are produced in vivo after MSC injection. We have discovered that macrophages produce one such factor after their migration in the area of damage. Production of the macrophage factor requires contact with MSC paracrine factors. We have also observed that MSC regeneration was completely blocked following macrophage elimination from the organism. This positions macrophages as an essential intermediate in MSC-mediated tissue regeneration. The results presented in this thesis represent an important step forward for the use of MSC in regenerative medicine. They have established a basis for an open angle glaucoma treatment and added an important element for the comprehension of MSC regenerative mechanisms. This knowledge should have a significant impact for the treatment of ocular diseases and other illnesses in many different organs.