Em vista das limitações éticas em torno da obtenção de células-tronco de fetos humanos, o cão é uma alternativa para estes estudos. Além disso, a terapia celular proporciona novas expectativas para o tratamento na espécie. Realizamos o estudo comparativo das células isoladas de saco vitelino, fígado e medula óssea de fetos caninos. As células foram analisadas microscopicamente e ultra estruturalmente. O imunofenótipo das células foi avaliado através de marcadores. Caracterizamos a plasticidade, o cariótipo e o potencial teratogênico destas células. Após expansão as células progenitoras formaram colônias com morfologia fibroblastóide. As células progenitoras do saco vitelino e medula óssea são compostas por: células com alta proporção núcleo-citoplasma e células com citoplasma rico em organelas, enquanto que as células progenitoras do fígado eram semelhantes à célula epitelial e células ricas em organelas. As células-progenitoras dos três tecidos fetais foram positivas para os anticorpos nestina e vimentina, mas negativas para CD45 e CD13. Células progenitoras de medula óssea foram positivas para o marcador CD44. Células progenitoras do fígado e medula óssea expressaram a proteína citoqueratina-18, enquanto as do saco vitelino expressaram ve-caderina. Células positivas para Oct3/4 foram detectadas em todas as células progenitoras. As células-progenitoras do saco vitelino e medula óssea diferenciaram-se em tecidos ósseo, cartilaginoso e muscular; já as do fígado para tecido ósseo e muscular. Nenhum tipo celular diferenciou-se em adipócitos. As células progenitoras da medula óssea diferenciaram em células semelhantes a neurônios. Sugere-se a presença de progenitores semelhantes a células mesenquimais e epiteliais. Todas as células mantiveram o cariótipo estável e não formaram tumores. Células progenitoras de medula óssea apresentaram maior capacidade de proliferação e diversidade de diferenciação. Sugere-se que estas células são possíveis candidatas para a terapia celular. / The use the human fetuses for stem cells isolation have ethical limitations. In this context the dog is an excellent candidate to fetal stem cells. Furthermore, these cells can be used in cell therapy of canine diseases We aimed at isolation and comparative characterization of progenitor cells from yolk sac, liver and bone marrow of canine fetuses. Cells were characterized using stem cells antibodies. Differentiation assays as well as karyotype analysis were performed. Teratogenic properties this cells were evaluated. After establishment of primary culture, best proliferation potential was observed in bone marrow progenitor cells. Bone marrow and liver progenitor cells were more efficient in CFU-F assay, then yolk sac progenitor cells. Evidenced by TEM cells with a high nuclear-to-cytoplasmic ratio and cells with cytoplasm rich in organelles. Cells isolated from liver showed epithelial-like morphology and cytoplasm rich in organelles. The yolk sac, liver bone marrow cells reacted positively with nestin and vimentin, being negative to CD45 and CD13 antibodies. Additionally bone marrow progenitor cells were positive to CD44. Bone marrow and liver progenitor cells reacted positively with cytokeratin 18. Yolk sac progenitor cells were positive to ve-cadherin. A few Oct3/4 positive cells were found in yolk sac, liver and bone marrow. Yolk sac and bone marrow progenitor cells showed successful osteogenic, chondrogenic, myogenic differentiation. Differentiation liver progenitor cells were able to bone and muscle cells. The bone marrow progenitor cells were able to produce neuron-like cells. None of progenitor cells showed adipogenic differentiation. The study suggests the presence of mesenchymal-like and epithelial-like progenitor cells. All the karyotype remained and failed to induce the formation of tumors. Stem cells from bone marrow showed high diversity of differentiation than other cell types. It is suggested that these cells are possible candidates for cell therapy.

L’Osteoprotégérine est une cytokine soluble qui joue un rôle clé dans le métabolisme osseux et est impliquée dans la réponse immunitaire et l’hématopoïèse. Elle est associée à la dysfonction endothéliale et semble intervenir dans l’angiogenèse. Cette cytokine constituerait en fait, un trait d’union entre le tissu osseux et vasculaire. Son rôle dans la formation de la matrice osseuse est aujourd’hui bien élucidé mais son implication dans la vascularisation reste à établir. L’OPG est rapidement libérée par l’endothélium dans des conditions inflammatoires et est donc en mesure d’intervenir dans le processus de revascularisation initié par les cellules progénitrices endothéliales (PECs). Au cours de cette étude, nous avons tenté de comprendre le rôle joué par cette cytokines dans la néovascularisation induite in vitro, par une sous population de PECs appelées ECFCs (endothelial colony-forming cells), et sur la formation des néovaisseaux in vivo.Nous avons montré qu’elle agit sur la « souchitude » des cellules CD34+, potentialise les propriétés proangiogènes des ECFCs in vitro, et participe au processus angiogénique in vivo. L’OPG agit sur les ECFCs via le syndécanne-1, inhibe leur adhésion à la matrice extracellulaire, favorise leur migration et leur tubulogenèse via la voie SDF-1/CXCR4, et potentialise leur adhésion à l'endothélium activé. Les effets observés sont corrélés à la libération du SDF-1, une activation des voies de signalisation ERK1/2, Akt et mTOR et à une activation de l’intégrine αVβ3. Par ailleurs, nous avons montré que l'OPG potentialise l’effet proangiogène du FGF-2 in vivo. Elle participe également au développement tumoral et à la dissémination des métastases, probablement via l'inhibition de l'apoptose des cellules tumorales, mais aussi par la promotion de l'angiogenèse tumorale. / Osteoprotegerin is a key regulator of bone metabolism involved in the immune response, hematopoiesis, and endothelial dysfunction. It seems to be implicated in angiogenesis and may represent a link between bone and vascular system. Although its role in bone is well recognized, its involvement in vasculature remains to be established. In inflammatory conditions, OPG is constitutively released by endothelial cells and smooth muscle cells, and therefore is able to participate in blood vessels formation induced by endothelial progenitor cells (EPCs). In this study we attempted to determine, in vitro the precise role of OPG in angiogenesis process induced by a subpopulation of EPCs called “endothelial colony-forming cells” (ECFCs), and on neovessel formation in vivo.We found that OPG causes phenotype changes of ECFCs via the activation of different molecular pathways targeting cell clonogenicity, differentiation, proliferation, migration and adhesion. Our results suggest that OPG may interact with ECFCs through its binding to syndecan-1, to induce an anti-adhesive effect and thereby promoting ECFCs migration through a SDF-1/CXCR4 dependant pathway and the ERK1/2, Akt and the mTOR pathways activation. OPG can intervene on the autocrine effect of ECFCs by inducing their adhesion to activated endothelium and their tubulogenesis, and potentiate their paracrine effects by inducing SDF-1 release. Alternatively, it can promote ECFCs survival, probably, in a αVβ3 integrin-dependent manner. In vivo, OPG potentiates FGF-2 proangiogenic effects and may participate in tumour growth, invasion and metastasis, possibly through inhibition of tumour cell apoptosis but also by promoting tumour angiogenesis.

Based on data from the World Healthcare Organisation, cardiovascular diseases are the primary cause of disease-related death globally, with myocardial infarction (MI) being the most prevalent. If not treated effectively, MI can progress to heart failure (HF). With 70 million prescriptions for HF in 2014 and 515 people in the UK being hospitalised daily with MI, the British Heart Foundation calls for novel robust treatments. Even though cardiac stem cell (CSC) therapy for MI has been under investigation for more than a decade, there still has not been a consensus over the identity of the adult endogenous CSC. Recent clinical trials, using selected Ckit+ cells or the cardiosphere-derived cells (CDCs) have shown moderate results. The aim of this thesis was to develop a digestion-based method for isolation of cardiac progenitor cells (CPCs) from the mouse atria. The resulting "CTs" were isolated by collagenase/trypsin (where their name has resulted from) digestion with a prolonged period step for cell attachment. CTs were compared to isolated CDCs for their marker expression, using RT-PCR and Immunocytochemistry, showing cells with a mesenchymal phenotype which expressed SCA1 and CKIT. The CDCs had more of a fibroblast phenotype with higher Ddr2 and Wt1 expression. Using a TGF-β1 differentiation protocol, the CTs could be differentiated more effectively to a CM lineage than could the CDCs. In addition, Oleic acid (OA) supplementation stimulated the Peroxisome proliferator-activated receptor alpha pathway and led to maturation of the CT cells, both before and after differentiation. The differentiated CTs begin to express Tnnt2, while OA led to Myh7 increase and upregulated their oxidative metabolism. Finally, the CTs were more able to survive under serum-starvation than the CDCs, and transfection with miR-210 could enhance CT survival under these conditions and increased VEGF secretion. By digestion of the whole atria and allowing a prolonged time for attachment, we have developed a novel isolation protocol which generates a cell population containing a range of progenitors. Cells within this population can survive under serum starvation and can be differentiated to a CM lineage, making them a promising therapeutic population.

No description available.

Angiogenic cell therapy is currently being developed as a treatment for coronary artery disease (CAD); however, endothelial dysfunction (ED), commonly found in patients with CAD, impairs the ability for revascularization to occur. We hypothesized that culture on a collagen matrix will improve survival and function of circulating progenitor cells (CPCs) isolated from a mouse model of ED. Overall, ED decreased the expression of endothelial markers in CPCs and impaired their function, compared to normal mice. Culture of CPCs from ED mice on collagen was able to increase cell marker expression, and improve migration and adhesion potential, compared to CPCs on fibronectin. Nitric oxide production was reduced for CPCs on collagen for the ED group; however, CPCs on collagen had better viability under conditions of serum deprivation and hypoxia, compared to fibronectin. This study suggests that a collagen matrix may improve the function of therapeutic CPCs that have been exposed to ED.

Of the thousands of dividing progenitor cells (PCs) generated daily in the adult brain only a very small proportion survive to become mature neurons through the process of neurogenesis. Identification of the mechanisms that regulate cell death associated with neurogenesis would aid in harnessing the potential therapeutic value of PCs. Apoptosis, or programmed cell death, is suggested to regulate death of PCs in the adult brain as overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, enhances the survival of new neurons. To directly assess if Bcl-2 is a regulator of apoptosis in PCs, this study examined the outcome of removal of Bcl-2 from the developing PCs in the adult mouse brain. Retroviral mediated gene transfer of Cre into adult floxed Bcl-2 mice eliminated Bcl-2 from developing PCs and resulted in the complete absence of new neurons at 30 days post viral injection. Similarly, Bcl-2 removal through the use of nestin-induced conditional knockout mice resulted in reduced number of mature neurons. The function of Bcl-2 in the PCs was also dependent on Bcl-2-associated X (BAX) protein, as demonstrated by an increase in new neurons formed following viral-mediated removal of Bcl-2 in BAX knockout mice. Together these findings demonstrate that Bcl-2 is an essential regulator of neurogenesis in the adult hippocampus.

During spinal cord injury (SCI), glutamate excitotoxicity and astrocytic scar formation can impede repair. In a preliminary study we found that ketamine, a N-methyl-D-aspartate (NMDA) receptor non-competitive antagonist, can contribute to functional recovery post SCI. Therefore, we investigated the cellular basis for this recovery with respect to neural progenitor cells using an in vitro cell culture model. We examined whether ketamine and glutamate influenced the proliferation, differentiation, and migration of differentiating endogenous neural progenitor cells (NPCs) found in the subventricular zone and spinal cord. Our study illustrates that the post functional recovery may have been due to ketamine’s influence on delaying spinal cord NPCs derived astrocyte maturation and migration while increasing radial glial cell migration. These results are promising since ketamine administration may help alleviate some of the adverse affects glutamate has on the NPCs found in the spinal cord following SCI.

Proper central nervous system development is critical for survival and depends on complex intracellular and extracellular signaling to regulate neural progenitor cell growth and differentiation; however, the mechanisms that mediate molecular crosstalk between pathways during neurogenesis are not fully understood. Here, we explored the integration of the Hedgehog (Hh) signaling pathway with the two critical developmental pathways, Receptor Tyrosine Kinase (RTK) and Notch signaling, in the growth and maintenance of neural progenitors in the developing neuroretina. We found combined and sustained RTK and Hh signaling was sufficient to establish long-term retinal progenitor cell (RPC) cultures and these cells maintained neurogenic and gliogenic, but not retinogenic, competence in vitro and in vivo. In addition, we identified crosstalk between Notch and Hh signaling, where Notch is required for Hh-mediated proliferation and Gli protein accumulation, and gain-of-function of Notch is sufficient to extend the window of Hh responsiveness in a subset of Müller glia. Both Hh-RPC monolayer establishment and Notch mediated Hh-responsiveness required Gli2. Taken together, we identified molecular cross-communication between the Hh pathway and two major pathways, Notch and RTK, during retinogenesis, advancing our understanding of mechanisms that influence Hh to control neural progenitor growth.

Increasingly, cell-based therapy with autologous progenitor populations, such as endothelial progenitor cells (EPC), are being utilized for treatment of vascular diseases. However, both the number and functional capacity are diminished when cells are derived from patients with established risk factors for coronary artery disease (CAD). Herein, we report that inhibition of glycogen synthase kinase 3 (GSK) can improve both the number and function of endothelial progenitor cells in patients with CAD or diabetes mellitus (DM) leading to greater therapeutic benefit. Specifically, use of various small molecule inhibitors of GSK (GSKi) results in a 4-fold increased number of EPCs. Moreover, GSKi treatment improves the functional profile of EPCs through reductions in apoptosis, improvements in cell adhesion through up-regulation of very-late antigen-4 (VLA-4), and by increasing paracrine efficacy by increasing vascular endothelial growth factor (VEGF)secretion. Therapeutic improvement was confirmed in vivo by increased reendothelialization(RE) and reductions of neointima (NI) formation achieved when GSKi-treated cells were administered following vascular injury to CD-1 nude mice. Because cell-based therapy is technically challenging, we also tested a strategy of local delivery of GSKi at the site of arterial injury through GSKi-eluting stents. In vitro, GSKi elution increased EPC attachment to stent struts. In vivo, GSKi-eluting stents deployed in rabbit carotid arteries resulted in systemic mobilization of EPCs, improved local RE, and important reductions in in-stent NI formation. Finally, we tested the ability of GSKi to improve EPC-mediated arterial repair in patients with DM. As in patients with CAD, GSKi treatment improved EPC yield and diminished in vitro apoptosis. Utilizing a proteomics approach, we identified Cathepsin B (catB) as a differentially regulated protein necessary for reductions in apoptosis. Indeed, antagonism of catB prevented GSKi improvements in GSKi treated EPC mediated arterial repair in a xenotransplant wire injury model. Thus, our data demonstrates that GSKi treatment results in improvements in EPC number and function in vitro and in vivo resulting in enhanced arterial repair following mechanical injury. Accordingly, GSK antagonism is an effective cell enhancement strategy for autologous cell-based therapy with EPCs from high risk patients such as CAD or DM.

Recent advances in cellular reprogramming have enabled the generation of embryoniclike cells from virtually any cell of the body. These inducible pluripotent stem cells (iPSCs) are capable of indefinite self-renewal while maintaining the ability to differentiate into all cell types. Nowhere will this technology have a greater impact than in the ability to generate disease and patient-specific cell lines. Here we explore the capacity of human iPSCs reprogrammed from peripheral blood endothelial progenitor cells lines to differentiate into atrial myocytes for the study of patient specific atrial physiology. Methods and Results: Late outgrowth endothelial progenitor cells (EPCs) cultured from clinical blood samples provided a robust cell source for genetic reprogramming. Transcriptome analysis hinted that EPCs would be comparatively more amenable to pluripotent reprogramming than the traditional dermal fibroblast. After 6 passages, EPCs were transduced with a doxycycline inducible lentivirus system encoding human transcription factors OCT4, SOX2, KLF4 and Nanog to permit differentiation after removal of doxycycline. The high endogenous expression of key pluripotency transcripts enhanced the ease of iPSC generation as demonstrated by the rapid emergence of typical iPSC colonies. Following removal of doxycycline, genetically reprogrammed EPC-iPSC colonies displayed phenotypic characteristics identical to human embryonic stem cells and expressed high levels of the pluripotent markers SSEA-4, TRA1-60 and TRA1-81. After exposure to conditions known to favor atrial identity, EPC- iPSC differentiating into sheets of beating cardiomyocytes that expressed high levels of several atrial-specific expressed genes (CACNA1H, KCNA5, and MYL4). Conclusions: EPCs provide a stable platform for genetic reprogramming into a pluripotent state using a doxycycline conditional expression system that avoids reexpression of oncogenic/pluripotent factors. Human EPC-derived iPSC can be differentiated into functional cardiomyocytes that express characteristic markers of atrial identity.