Mesenchymal stem cell therapy for the treatment of myocardial infarction

Mesenchymal stem cells (MSCs) are currently under investigation as repair agents in the preservation of cardiac function following myocardial infarction (MI). However concerns have emerged regarding the safety of acute intracoronary (IC) MSC delivery specifically related to mortality, micro-infarction and microvascular flow restriction post cell therapy in animal models. This thesis aimed to firstly identify an optimal dose of MSC that could be tolerated when delivered via the coronary artery in a porcine model of acute MI (AMI). Initial dosing studies identified 25x106 MSC to be a safe MSC cell dose, however, angiographic observations from these studies recognised that on delivery of MSC there was a significant adverse decrease in distal blood flow within the artery. This observation along with additional supportive data in the literature (published during the course of this thesis) suggested MSC may be contributing to such adverse events through the propagation of thrombosis. Therefore further studies aimed to investigate the innate prothrombotic activity of MSC. Expression of the initiator of the coagulation cascade initiator tissue factor (TF) on MSC was detected in high levels on the surface of these cells. MSC-derived TF antigen was catalytically active, capable of supporting thrombin generation in vitro and enhancing platelet-driven thrombus deposition on collagen under flow. Infusion of MSC via IC route was associated with a decreased coronary flow reserve when delivered but not when coadministered with an antithrombin agent heparin. Heparin also reduced MSC-associated in situ thrombosis incorporating platelets and VWF in the microvasculature. Heparin-assisted MSC delivery reduced acute apoptosis and significantly improved infarct size, left ventricular ejection fraction, LV volumes, wall motion and scar formation at 6 weeks post AMI. In addition, this thesis investigated the paracrine factors secreted by MSC, in particular focusing on the effect on cardiac repair of a novel MSC-paracrine factor SPARCL1. In summary this work provides new insight into the mechanism by which MSC may be deleterious when delivered by an IC route and a means of abrogating this effect. Moreover we present new data on the MSC secretome with elucidation of the challenges encountered using a single paracrine factor cardiac repair strategy.

Eyes open to stem cells: safety trial may pave the way for cell therapy to treat retinal disease in patients

A clinical trial using human embryonic stem cell (hESC) therapy for an inherited retinal degenerative disease is about to commence. The Advanced Cell Technology (ACT) trial will treat patients with Stargardt's macular dystrophy using transplanted retinal pigment epithelium derived from hESCs. Currently, no effective treatment is available for Stargardt's disease so a stem cell-based therapy that can slow progression of this blinding condition could represent a significant breakthrough. While there are some hurdles to clear, the ACT trial is a fine example of translational research that could eventually pave the way for a range of stem cell therapies for the retina and other tissues.

Lipidomic analysis of mesenchymal cells candidates for cell therapy

Mesenchymal stromal cells are adult stem cells found mostly in the bone marrow. They have immunosuppressive properties and they have been successfully applied as biological therapy in several clinical trials regarding autoimmune diseases. Despite the great number of clinical trials, MSCs’ action is not fully understand and there are no identified markers that correlate themselves with the immunomodulatory power. A lipidomic approach can solve some of these problems once lipids are one of the major cells’ components. Therefore, in this study cells’ lipidome was analysed and its deviations were evaluated according to the medium of culture and to the presence of pro-inflammatory stimuli, mimicking physiological conditions in which these cells are used. This was the first study ever made that aimed to analyse the differences in the phospholipid profile between mesenchymal stromal cells non-stimulated and stimulated with proinflammatory stimulus. This analysis was conducted in both cells cultured in medium supplemented with animal serum and in cells cultured in a synthetic medium. In cells cultured in the standard medium the levels of phosphatidylcholine (PC) species with shorter fatty acids (FAs) acyl chains decreased under pro-inflammatory stimuli. The level of PC(40:6) also decreased, which may be correlated with enhanced levels of lysoPC (LPC)(18:0) - an anti-inflammatory LPC - observed in cells subjected to TNF-α and IFN-γ. Simultaneously, the relative amounts of PC(36:1) and PC(38:4) increased. TNF-α and IFN- γ also enhanced the levels of phosphatidylethanolamine PE(40:6) and decreased the levels of PE(38:6). Higher expression of phosphatidylserine PS(36:1) and sphingomyelin SM(34:0) along with a decrease in PS(38:6) levels were observed. However, in cells cultured in a synthetic medium, TNF-α and IFN-γ only enhanced the levels of PS(36:1). These results indicate that lipid metabolism and signaling is modulated during mesenchymal stromal cells action.

Bone tissue engineering using foetal cell therapy.

Different cell sources for bone tissue engineering are reviewed. In particular, adult cell source strategies have been based on the implantation of unfractionated fresh bone marrow; purified, culture expanded mesenchymal stem cells, differentiated osteoblasts, or cells that have been modified genetically to express rhBMP. Several limiting factors are mentioned for these strategies such as low number of available cells or possible immunological reaction of the host. Foetal bone cells are presented as an alternative solution and review of actual treatments using these cells is presented. Finally, foetal cells used specifically for bone tissue engineering are characterised and potentially interesting therapeutic options are proposed.

Somatic Cell Therapy: A Genetic Rescue for a Tattered Immune System?

Étude de cas / Case study

Optimization Studies to Improve MSC-based Cardiac Cell Therapy : Cytokine Preconditioning and Nanoparticle Coupling

Contexte: La cardiopathie ischémique (IHD) reste une cause majeure de mortalité en Amérique du Nord. La thérapie cellulaire cardiaque (CCT) a émergé comme une thérapie prometteuse pour aider à guérir certaines malades cardiaques. Parmi les cellulaires avec propriétés pluripotentes, les cellules stromales mésenchymateuses (MSC) sont prometteuses. Cependant, plusieurs questions demeurent non résolues et certaines défis empêchent l'application clinique de la CCT se dans l'IHD, tels que le faible taux de rétention cellulaire in situ, le suivi des cellules in vivo post-implantation et post-acheminements et l`apoptose. Ici, le traitement préliminaire des MSC avec des facteurs de croissance et leur couplage avec des nanoparticules (NP) seront étudiés comme des méthodes pour optimiser MSC. Méthodes: Des MSCs provenant du rat (rMSC) et du cochon (pMSC) ont été isolés à partir de moelle osseuse. Les rMSC ont été préconditionnées avec SDF-1a, TSG-6 et PDGF-BB, et ensuite soumises à une hypoxie, une privation de sérum et a un stress oxydatif. Des études de cicatrisation ont également été effectués avec rMSCs préconditionnées. En parallèle, de nouvelles NP ferromagnétiques liées aux silicones ont été synthétisées. Les NPs ont été couplées aux pMSCs suivant leur fonctionnalisation avec l`anticorps, CD44, un antigène de surface du MSC bien connu. Par la suite, les études de biocompatibilité ont été réalisées sur pMSC-NP et en incluant des tests des processus cellulaires tels que la migration, l'adhésion, la prolifération et les propriétés de la différenciation. Résultats: Parmi toutes les cytokines testées, PDGF-BB a démontré la plus grande capacité à améliorer la survie de MSC dans des conditions d'hypoxie, de privation de sérum et en reponse au stress oxydatif. La conjugaison de NP a atténué la migration et la prolifération des pMSCs, mais n`a pas changé leur capacité de différenciation. Enfin, la complexe du MSC-NP est détectable par IRM. Conclusion: Nos données suggèrent que de nouvelles stratégies, telles que traitement préliminaire de PDGF-BB et le couplage des nanoparticules ferromagnétiques, peuvent être considérés comme des avenues prometteuse pour optimiser les MSCs pour la CCT.

A novel alternative to cryopreservation for the short-term storage of stem cells for use in cell therapy using alginate encapsulation

Efficient transport of stem/progenitor cells without affecting their survival and function is a key factor in any practical cell-based therapy. However, the current approach using liquid nitrogen for the transfer of stem cells requires a short delivery time window is technically challenging and financially expensive. The present study aims to use semipermeable alginate hydrogels (crosslinked by strontium) to encapsulate, store, and release stem cells, to replace the conventional cryopreservation method for the transport of therapeutic cells within world-wide distribution time frame. Human mesenchymal stem cell (hMSC) and mouse embryonic stem cells (mESCs) were successfully stored inside alginate hydrogels for 5 days under ambient conditions in an air-tight environment (sealed cryovial). Cell viability, of the cells extracted from alginate gel, gave 74% (mESC) and 80% (hMSC) survival rates, which compared favorably to cryopreservation. More importantly, the subsequent proliferation rate and detection of common stem cell markers (both in mRNA and protein level) from hMSCs and mESCs retrieved from alginate hydrogels were also comparable to (if not better than) results gained following cryopreservation. In conclusion, this new and simple application of alginate hydrogel encapsulation may offer a cheap and robust alternative to cryopreservation for the transport and storage of stem cells for both clinical and research purposes.

Establishment of a Brazilian Line of Human Embryonic Stem Cells in Defined Medium: Implications for Cell Therapy in an Ethnically Diverse Population

Pluripotent human embryonic stem (hES) cells are an important experimental tool for basic and applied research, and a potential source of different tissues for transplantation. However, one important challenge for the clinical use of these cells is the issue of immunocompatibility, which may be dealt with by the establishment of hES cell banks to attend different populations. Here we describe the derivation and characterization of a line of hES cells from the Brazilian population, named BR-I, in commercial defined medium. In contrast to the other hES cell lines established in defined medium, BR-I maintained a stable normal karyotype as determined by genomic array analysis after 6 months in continuous culture (passage 29). To our knowledge, this is the first reported line of hES cells derived in South America. We have determined its genomic ancestry and compared the HLA-profile of BR-I and another 22 hES cell lines established elsewhere with those of the Brazilian population, finding they would match only 0.011% of those individuals. Our results highlight the challenges involved in hES cell banking for populations with a high degree of ethnic admixture.

Graphene oxide nanoparticles as a nonbleaching optical probe for two-photon luminescence imaging and cell therapy

Lasting glow: Under femtosecond laser irradiation, graphene oxide nanoparticles (GONs) give strong two-photon luminescence (TPL; see picture). The presence of GONs also induces microbubbling, which causes cell death at an order of magnitude lower laser power than when cells are not labeled. The results show that GONs can be used for TPL-based imaging and photothermal cancer therapy.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alveolar osseous defect in rat for cell therapy. Preliminary report

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Experimental Basis and New Insights for Cell Therapy in Chronic Obstructive Pulmonary Disease

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)