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Indiana University-Purdue University Indianapolis (IUPUI) / Insulin-like Growth Factor I (IGF-I) is a growth factor that stimulates both mitogenic and anabolic responses in articular chondrocytes. While it has been shown that exogenous IGF-I can regulate chondrocyte integrins, little is known regarding regulatory effects of IGF-I produced from a transiently expressed plasmid based adeno-associated virus (pAAV) vector. Because chondrocytes are using cellular machinery to overexpress IGF-I, it is of interest to see whether or not pAAV IGF-I will significantly upregulate or downregulate chondrocyte integrins. Additionally, it is of interest to know whether chondrocyte adhesion through integrins will have any regulatory effects on the production of IGF-I from the transgene. Therefore, this study will ascertain if pAAV IGF-I will have similar effects that exogenous IGF-I has on integrin regulation and if integrin silencing mechanisms will affect the production of IGF-I from the transgene. To test these hypotheses, adult articular chondrocytes were doubly transfected with the pAAV vector for IGF-I and short interference ribonucleic acid (siRNA) for integrins beta 1 and alpha V. Gene products were monitored at the transcriptional levels using quantitative real time polymerase chain reactions (qPCR) and IGF-I protein production was monitored at the translational level using enzyme linked immunoabsorbant assays (ELISAs). Adult articular chondrocytes doubly transfected were encapsulated in a three dimensional hydrogel system to simulate an in vivo environment. Samples were collected for analysis at days 2, 4, and 6 post encapsulation. Results show that IGF-I treatment with the pAAV vector does not cause significant changes in the transcriptional regulation of the beta 1 integrin in a three dimensional hydrogel system. The pAAV IGF-I vector did not cause significant regulatory changes on integrin alpha V at any time point during the experiment. Additionally, by knocking down the expression levels of integrins by using siRNA, it was shown that integrin knockdown does not have a significant regulatory effect on transcriptional or translational expression levels of IGF-I from the pAAV vector.

Induced pluripotent stem cells (iPSC) have the capacity to self renew and differentiate into a myriad of cell types making them potential candidates for cell therapy and regenerative medicine. The goal of this thesis was to determine the characteristics of equine iPSC (eiPSC) that can be harnessed for potential use in veterinary regenerative medicine. Trauma to a horse’s limb often leads to the development of a chronic non-healing wound that lacks a keratinocyte cover, vital to healing. Thus, the overall hypothesis of this thesis was that eiPSC might offer a solution for providing wound coverage for such problematic wounds. Prior to considering eiPSC for clinical applications, their immunogenicity must be studied to ensure that the transplanted cells will be accepted and integrate into host tissues. The first objective of this thesis was to determine the immune response to eiPSC. To investigate the immunogenicity of eiPSC, the expression of major histocompatibility complex (MHC) molecules by the selected lines was determined, then the cells were used in an intradermal transplantation model developed for this study. While transplantation of allogeneic, undifferentiated eiPSC elicited a moderate cellular response in experimental horses, it did not cause acute rejection. This strategy enabled the selection of weakly immunogenic eiPSC lines for subsequent differentiation into lineages of therapeutic importance. Equine iPSC offer a potential solution to deficient epithelial coverage by providing a keratinocyte graft with the ability to differentiate into other accessory structures of the epidermis. The second objective of this thesis was to develop a protocol for the differentiation of eiPSC into a keratinocyte lineage. The protocol was shown to be highly efficient at inducing the anticipated phenotype within 30 days. Indeed, the eiPSC derived vi keratinocytes (eiPSC-KC) showed both morphologic and functional characteristics of primary equine keratinocytes (PEK). Moreover, the proliferative capacity of eiPSC-KC was superior while the migratory capacity, measured as the ability to epithelialize in vitro wounds, was comparable to that of PEK, suggesting exciting potential for grafting onto in vivo wound models. In conclusion, equine iPSC-derived keratinocytes exhibit features that are promising to the development of a stem cell-based skin construct with the potential to fully regenerate lost or damaged skin in horses. However, since eiPSC do not fully escape immune surveillance despite low MHC expression, strategies to improve engraftment of iPSC derivatives must be pursued. / Les cellules souches pluripotentes induites (iPSC) ont la capacité de s'auto renouveler et de se différencier en une myriade de types cellulaires, ce qui en fait des outils intéressants pour la thérapie cellulaire et la médecine régénérative. Le but de cette thèse était de déterminer les caractéristiques des iPSC équines (eiPSC) qui peuvent être exploitées pour l'usage potentiel en médecine régénérative vétérinaire. Chez le cheval, une plaie cutanée est souvent cicatrisée par seconde intention et est sujette à de nombreuses complications lorsque située sur le membre, notamment une épithélialisation lente. Ainsi, l'hypothèse globale de cette thèse était que les eiPSC pourraient offrir une solution novatrice de couverture pour de telles blessures. Avant d'envisager l’utilisation d'eiPSC à des fins cliniques, leur immunogénicité doit être étudiée afin de s'assurer que les cellules transplantées seront acceptées et intégrées dans les tissus du receveur. Le premier objectif de cette thèse était de définir la réponse immunitaire suscitée par les eiPSC. Afin d'étudier l'immunogénicité d'eiPSC, l'expression de molécules du complexe majeur d’histocompatibilité (MHC) des lignes choisies a été déterminée, puis les cellules ont été utilisées dans un modèle de transplantation intradermique développé pour cette étude. Bien que la transplantation allogénique d'eiPSC non différenciées ait induit une réponse cellulaire modérée chez les chevaux d'expérimentation, elle n'a pas provoqué de rejet. Cette stratégie a permis la sélection de lignées d'eiPSC faiblement immunogènes pour la différenciation ultérieure en des lignées d'importance thérapeutique. Les eiPSC représentent une solution intéressante et qui, par l’entremise du développement d’une lignée de kératinocytes, pourraient servir à la création d’une greffe ayant la capacité de former non seulement l’épithélium manquant mais aussi d'autres structures accessoires de l'épiderme. Le deuxième objectif de cette thèse était donc de iv développer un protocole pour la différentiation des eiPSC en lignée de kératinocytes. Un protocole visant cette différenciation fut ainsi développé et ce dernier a démontré une grande efficacité à produire le phénotype attendu dans une période de 30 jours. En effet, les kératinocytes dérivés d'eiPSC (eiPSC-KC) ont montré des caractéristiques morphologiques et fonctionnelles des kératinocytes primaires équins (PEK). En outre, la capacité de prolifération d'eiPSC-KC est supérieure tandis que la capacité migratoire, mesurée comme l'aptitude à cicatriser les plaies in vitro, est comparable à celle du PEK. En conclusion, les eiPSC-KC ont des caractéristiques intéressantes pour le développement d'un substitut cutané à base de cellules souches, ayant le potentiel de régénérer la peau perdue lors de trauma ou de maladie, chez le cheval. Cependant, parce que les eiPSC n'échappent pas totalement à la surveillance immunitaire, malgré une faible expression du MHC, des stratégies pour améliorer la prise de greffe eiPSC-KC doivent être élaborées.

Garantir la qualité des produits sanguins est crucial. Les lysats plaquettaires (LP) riches en facteurs de croissance (FC) s’imposent comme le complément idéal pour l’expansion ex vivo des cellules souches, et comme produit thérapeutique pour la régénération cellulaire. L’intérêt est croissant pour les microparticules (MPs) extracellulaires, mais l’expression de phosphatidylsérine à leur surface peut induire des effets thrombotiques et inflammatoires. L’autre risque transfusionnel, la transmission de virus, dont le virus de l’hépatite C (VHC), est maîtrisable par traitements de réduction virale par solvant/détergent (S/D), chauffage, ou nanofiltration. Nous avons étudié des technologies de sécurisation des produits sanguins: (a) élimination des MPs par nanofiltration sur filtres de 75 nm et (b) traitements S/D, chauffage à 56°C ou nanofiltration pour inactiver ou éliminer le VHC. Les informations ont été utilisées pour développer des LP utiles en médecine régénérative. L’un d’eux destiné à la neurorégénération, a été préparé en émettant l’hypothèse qu’un lysat de culot plaquettaire (LCP) enrichi en facteurs neurotrophiques et dépourvu de protéines plasmatiques se montrerait efficace contre les maladies neurodégénératives. Nos résultats montrent que la nanofiltration sur des filtres de 75 nm préserve la composition en protéines plasmatiques, et le pouvoir hémostatique. La nanofiltration retire les MPs et évite, in vitro, la génération de thrombine. Par ailleurs le traitement S/D à 31°C pour 30 minutes élimine le pouvoir infectieux du VHC. Pris globalement les traitements de nanofiltration et S/D apparaissent donc comme des méthodes de choix pour l’amélioration de la sécurité du plasma vis à vis de risques thrombogènes et infectieux. Nous avons ensuite préparé un LCP appauvri en protéines plasmatiques (dont le fibrinogène) et enrichi en un mélange pléiotrope physiologique de FC destiné à l’administration cérébrale. Les analyses par ELISA et par protéomique ont montré qu’un chauffage de 56°C pour 30 min réduisait le contenu en protéines et modifiait favorablement la composition relative en facteurs neurotrophiques. Par ailleurs le chauffage améliore l’action neuroprotectrice et, associé aux traitements S/D et de nanofiltration, contribue à l’inactivation du VHC. Ce LCP exerce une neuroprotection élevée dans des modèles de la maladie de Parkinson (MP) tout à la fois (a) in vitro (cellules LUHMES différentiées en neurones dopaminergiques et exposées au MPP+) et (b) in vivo (souris intoxiquées par MPTP). L’expression de la tyrosine hydroxylase (TH) dans la Substantia nigra pars compacta montre que l’administration intracérébroventriculaire (ICV) ou intranasale (i.n.) apparait comme une option thérapeutique possible des maladies neuro-dégénératives. Les études cellulaires In vitro sur LUHMES et NSC34 ont montré que l’inhibition spécifique des voies signalétiques relayées par AkT et ERK altère l’activité neuroprotectrice du LC. Des événements neuro-inflammatoires pouvant aggraver l’évolution des maladies neurodégénératives, nous avons vérifié que le LCP n’induit pas de marqueurs inflammatoires (COX-2, iNOS) chez des cellules microgliales BV2, et pouvait même diminuer celle de COX-2 après exposition à des lipopolysaccharides. De plus, nous avons identifié que le LCP contenait 1.7 x 1012 MP/mL d’une taille moyenne de 160 nm. Isolées, ces MPs pourraient exercer un rôle neuroprotecteur des cellules LUHMES exposées à des agents neurotoxiques. En conclusion, nos résultats montrent la faisabilité technique à préparer des lysats plaquettaires viro-inactivés pour des usages dans le domaine de la médecine régénérative, y compris comme agent neuroprotecteur du système nerveux central. / Ensuring quality and safety of blood products is crucial. Platelet lysates (PL) rich in growth factors (GFs) have emerged as ideal clinical-grade supplement for ex vivo expansion of mesenchymal stromal cells, and as therapeutic product promote cellular regeneration. Interest for platelet extracellular microparticles (MPs) is growing but expression of phosphatidylserine on their surface may cause thrombotic and inflammatory side effects. Another transfusional risk, transmission of viruses, including hepatitis C virus (HCV), can be fully controlled by dedicated viral reduction methods as solvent/detergent (S/D) or heat treatments, or nanofiltration. We have evaluated technologies to secure therapeutic blood products: (a) removal of MPs by 75nm-nanofiltration and (b) inactivation/removal of HCV by S/D or 56°C heat treatments, or nanofiltration. Data have been used to develop LP of interest for regenerative medicine. In particular, one, targeting neuroregenerative applications, has been prepared based on the hypothesis that a platelet pellet lysate (PPL) enriched in multiple neurotrophic growth factors and depleted of plasma proteins could exert potent neuroprotective actions in neurodegenerative disease models. Our data show that 75 nm-plasma nanofiltration preserved plasma protein biochemical profile, and hemostatic power. Nanofiltration removes MPs and avoids in vitro the generation of thrombin. In addition, the S/D treatment at 31°C for 30 minutes fully inactivates HCV infectivity. Therefore, altogether, nanofiltration and S/D emerge as choice procedures to improve the safety of plasma for thrombogenic and infectious risks. We have then prepared a PPL depleted of plasma proteins (in particular fibrinogen), and rich in a physiological pleiotropic mixture of neurotrophins for brain administration. ELISA and proteomics studies revealed that the heat-treatment at 56°C for 30 min decreased the protein content and favorably modified the relative composition in neurotrophic factors. Heat-treatment improved the neuroprotective activity and, together with S/D and nanofiltration contributed to HCV inactivation. This PPL exerted strong neuroprotective effects in Parkinson’s disease (PD) models (a) in vitro, using LUHMES cells exposed to MPP+ neurotoxin, and (b) in vivo, in mice intoxicated by MPTP neurotoxin. Expression of tyrosine hydroxylase (TH) in the Substantia nigra pars compacta indicated that brain delivery by intracerebroventricular (ICV) or intranasal (i.n.) administration may be a therapeutic option for disease-modifying strategies of neurodegenerative diseases. In vitro studies in LUHMES and NSC34 cells showed that specific inhibition of signal transduction pathways through AkT and ERK influenced PPL neuroprotective function. Since neuro-inflammation detrimentally affects neurodegenerative disorders, we verified that the PPL did not stimulate the release of inflammatory markers (e.g. COX-2, iNOS) by BV2 microglial cells in culture, and could even restrict COX-2 expression when cells were exposed to LPS. In addition, the PPL was found to contain 1.7 x 1012 MP/mL with a mean size of 160 nm. These MPs may exert neuroprotective activity on LUHMES cells exposed to neurotoxins. Altogether, our data demonstrate the technical feasibility of developing virally-safe customized platelet lysate preparations with specific applications for cell therapy and regenerative medicine, in particular as neuroprotective agents of the central nervous system.

<p>Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage damage and loss in the joints that affects approximately 27 million adults in the US. Tissue that is damaged by OA is a major health concern since cartilage tissue has a limited ability to self-repair due to the lack of vasculature in cartilage and low cell content. Tissue engineering efforts aim towards the development of cartilage repair strategies that mimic articular cartilage and are able to halt the progression of the disease as well as restore cartilage to its normal function.</p><p>This study harnesses the biological activity of collagen type II, present in articular cartilage, and the superior mechanical properties of collagen type I by characterizing gels made of collagen type I and II blends (1:0, 3:1, 1:1, 1:3, and 0:1). The collagen blend hydrogels were able to incorporate both types of collagen and retain chondroitin sulfate (CS) and hyaluronic acid (HA). Cryoscanning electron microscopy images showed that the 3:1 ratio of collagen type I to type II gels had a lower void space percentage (36.4%) than the 1:1 gels (46.5%) and the complex modulus was larger for the 3:1 gels (G*=5.0 Pa) compared to the 1:1 gels (G*=1.2 Pa). The 3:1 blend consistently formed gels with superior mechanical properties compared to the other blends and has the potential to be implemented as a scaffold for articular cartilage engineering.</p> <p>Following the work done to characterize the collagen scaffolds, we studied whether an aggrecan mimic, CS-GAHb, composed of CS and HA binding peptides, GAH, and not its separate components, is able to prevent glycosaminoglycan (GAG) and collagen release when incorporated into chondrocyte-embedded collagen gels. Bovine chondrocytes were cultured and embedded in collagen type I scaffolds with CS, GAH, CS and GAH, or CS-GAHb molecules. Gels composed of 3:1 collagen type I and II with CS or CS-GAHb were also studied. The results obtained showed CS-GAHb is able to decrease GAG and collagen release and increase GAG retention in the gels. CS-GAHb also stimulated cytokine production during the initial days of scaffold culture. However, the addition of CS-GAHb into the chondrocyte-embedded collagen scaffolds did not affect ECM protein expression in the gels. The incorporation of collagen type II into the collagen type I scaffolds did not significantly affect GAG and cytokine production and ECM protein synthesis, but did increase collagen release. The results suggest the complex interaction between CS-GAHb, the chondrocytes, and the gel matrix make these scaffolds promising constructs for articular cartilage repair.</p> <p>Finally, we used Dunkin Hartley guinea pigs, a commonly used animal model of osteoarthritis, to determine if high frequency ultrasound can ensure intra-articular injections of the aggrecan mimic are accurately positioned in the knee joint. A high-resolution small animal ultrasound system with a 40 MHz transducer was used for image-guided injections. We assessed our ability to visualize important anatomical landmarks, the needle, and anatomical changes due to the injection. From the ultrasound images, we were able to visualize clearly the movement of anatomical landmarks in 75% of the injections. The majority of these showed separation of the fat pad (67.1%), suggesting the injections were correctly delivered in the joint space. The results demonstrate this image-guided technique can be used to visualize the location of an intra-articular injection in the joints of guinea pigs and we are able to effectively inject the aggrecan mimic into knee joints.</p><p>All of the work presented here suggests that the addition of the aggrecan mimic to collagen I and collagen I and II scaffolds has shown that this type of construct could be useful for treating cartilage damage in the future.</p>