Philosophiae Doctor - PhD / Epidemiological studies have highlighted that leukaemia can be considered as the most prominent malignancy after radiation exposure during childhood. The lifetime risk on radiation-induced leukaemia for a given dose is 3 – 5 times higher for children compared to adults. The high risk at a young age is related to the elevated sensitivity of the red bone marrow where haematopoietic stem and progenitor cells (HSPCs) are located. HSPCs self-renewal capacity and long-life span increase their susceptibility to DNA damage accumulation, making them a major target of radiation-induced carcinogenesis. Proton beam therapy (PBT) is increasingly used to treat paediatric brain tumours due to its dose sparing properties compared to conventional X-ray based radiotherapy.

Haemopoietic stem and progenitor cells from bone marrow and cord blood are well characterised with respect to their phenotype, growth in clonal assays, responsiveness to cytokine stimulation, receptor profile and their ability to sustain multilineage engraftment of receptive hosts in animal models of transplantation and of course, clinically in the treatment of some haemopoietic and immunological disorders. It is generally accepted that cells bearing the CD34+ phenotype are enriched for the most primitive of haemopoietic stem cells that possess the cardinal features of self-renewal and multipotency. However, the molecular mechanisms, the spectrum of expressed genes that give rise to the physical characteristics of haemopoietic progenitor cells are not well understood. Furthermore, although CD34+ cells from different sources (bone marrow, cord blood, mobilised peripheral blood) share many common features, there are also significant differences. (For complete abstract open document)

Acute myocardial infarction (MI) is the end result of many cardiovascular diseases and is one of the leading causes of death in the western world. Cell therapy, using circulating angiogenic cells (CACs) or CD34+ cells from peripheral blood, is one approach under investigation for restoring blood flow and function to the ischemic heart. However, the numbers of CACs and CD34+ circulating cells are inversely proportional to the severity of cardiovascular disease and age; therefore, there is a need to increase their numbers and/or function for therapy. One possibility is to enhance the therapeutic potential of the cells with the use of a biomaterial. In this study, we used a collagen matrix to culture human CD34+ circulating cells, and evaluated the effect of the matrix on CD34+ cell properties and function. The matrix was able to successfully increase proliferation, migration, CD34+ phenotype and branching in an angiogenesis assay. These functional benefits may be associated with the sonic hedgehog (Shh) pathway. The collagen matrix was previously shown to enhance the function of healthy CACs, but its ability to do the same for CACs from coronary artery disease patients is unknown. In this study, the matrix was shown to enhance the viability, proliferation and angiogenic potential of patient CACs. Furthermore, gene expression for integrins and Shh pathway components in the sub-population of CD34+ cells was similar between patient and healthy donors when isolated from CACs. This work provides insight into the mechanisms for the observed matrix-enhanced function of therapeutic CACs and CD34+ cells from both healthy and CAD patient donors.

No description available.

Background and objectives: In 2006 Shinya Yamanaka successfully reprogrammed mouse fibroblasts back to an embryonic stem cell-like state (called induced pluripotent cells, iPS cells) using retrovirus to introduce four genes that encode critical transcription factor proteins (Oct4, Sox2, KLF4, and c-Myc). This ability to reprogram has promising future applications in clinical and biomedical research for study of diseases, development of candidate drugs and to support therapeutic treatments in regenerative medicine. However, the clinical applications have to meet GMP requirements without the risk of insertional mutagenesis associated with retrovirus. Chromatin modifying agents are widely used in many protocols to generate iPS cells and culture of blood CD34+ cells with chromatin-modifying agents can lead to an increase in marrow repopulating cells and in the case of valproic acid increased erythroid cell colony formation. We undertook research to help understand what effects these reagents have on mobilised peripheral blood (mPB) CD34+ cells and optimised the expansion medium protocol to facilitate reprogramming work. This project aims to utilize peripheral blood mononuclear cells (MNC), one of the most easily accessible tissues to generate iPS cells using an efficient non-viral, feeder cell free methodology, with the ultimate goal of moving this methodology towards clinical use. Materials and Methods: G-CSF mobilised peripheral blood, buffy coat, cord blood and fetal liver were obtained from patients and donors under informed consent and ethics committee approval. Haematopoietic stem/progenitor cells CD34+ or CD133+) isolated by magnetic separation were flow cytometry sorted into CD34+/CD133+, CD34+/CD133-, and CD34-/CD133+ sub-populations and their lineage potential were assessed in colony forming unit assays. The effect of epigenetic modifiers valproic acid and 5-aza-2-deoxycytidine used singly or in combination with each other and with IL3 on phenotype and lineage potential of cultured CD34+ cells from mobilised peripheral blood were assessed by flow cytometry and colony-forming unit assays. Prior to reprogramming mononuclear cells from peripheral blood or CD34+ cells from blood were expanded in culture medium supplemented with stem cell factor (SCF), Fms-related tyrosine kinase 3 ligand (Flt3L) and Interleukin- 3 (IL-3) for several days. Actively proliferating cells were reprogrammed by electroporation using episomal vectors with an oriP/EBNA-1 backbone to deliver five reprogramming genes, Oct4, Sox2, Lin28, L-Myc, and Klf4. Electroporated cells were seeded onto matrigel coated plates immediately after transfection or were reseeded after three days’ culture. Subsequently, cells were cultured in specific medium on different days. When iPS colonies appeared, they were picked and cultured as for ES cells. Once established, iPS cell lines were immunophenotyped using flow cytometry and immunofluorescence and their potential to differentiate into the three germ layers was assessed in vitro. Results and Conclusion: The largest subpopulation of CD34+ cells was CD34+/CD133+ population which was essentially committed to myeloid colony production, while much smaller CD34+/CD133- subpopulation had a greater capacity to generate erythroid colonies. Optimised cytokine cocktail for expansion of CD34+ cells included IL-3, important in improving expansion and maintaining functionality of CD34+ cells. The optimised cytokine cocktail comprised 100 ng/ml SCF, 10 ng/ml Flt3L, and 20 ng/ml IL-3, which maintained CD34+ cells and MNC in an active proliferating state. In addition, valproic acid and IL3 were found to act synergistically, to increase the numbers of CD34+/CD36+ positive cells. However, we found that an apparent increase in red cell colony formation actually resulted from a decrease in white cell colonies, so no overall increase in red cell colonies was seen when equivalent numbers of CD34+ cells were plated. Proliferating MNC maintained in optimised cytokine cocktail were amenable to electroporation for the effective delivery of episomal transcription factors (Oct4, Sox2, Klf4, L-Myc, and Lin28) within a backbone of oriP/EBNA-1. We successfully developed an efficient and simple method for reprogramming MNC from fresh or frozen samples to generate induced pluripotent cells using episomal vectors in a feeder-free system without any requirement for small molecules and the highest reprogramming efficiency is 0.033% (65 colonies from 2 ◊ 105 seeding MNC). The cytokine cocktail and reprogramming methods work better in CD34+ cells from cord blood or fetal liver, and we obtained 148 iPS colonies from 105 seeding cells (0.148%) at most. In addition, fibroblasts from adult and fetal liver can be successfully reprogrammed using the same reprogramming method. The use of episomal vectors with an oriP/EBNA-1 backbone to deliver reprogramming genes, and efficient electroporation were the most important factors in efficiency of the reprogramming process. In addition, it is pivotal to initiate transfection when cells are actively proliferating. The iPS cell lines we generated maintained the successful expression of ES markers including Oct4, Nanog, SSEA3. SSEA4, TRA-1-60 and TRA-1-81, and had the capacity to successfully differentiate into cell types of ectoderm, mesoderm and endoderm layers in vitro.

Vascular disease is the leading cause of mortality and morbidity in the western world, and account for the 1 of every 3 death’s in the US, but a cure for vascular disease is yet to be realized. Hematopoietic stem progenitor cells (HSPCs) are mobilized from bone marrow and have the innate propensity to accelerate vascular repair by reendothelialization and revascularization of ischemic areas. The vasoreparative ability of HSPCs is largely due to their capacity to home to the areas of hypoxia and their sensitivity to hypoxia plays a critical role in the vasoreparative functions of these cells. The discovery of vasoreparative potential of HSPCs resulted in a breakthrough approach of cell-based therapies for the treatment of ischemic vascular diseases. However, success of this approach is essentially dependent on the number of cells that could be collected from an individual. Therefore, novel mechanism-based strategies are needed to enhance the outcomes of autologous cell-based therapies in poor mobilizers and older adults. Recent evidence of a potential role of the vasoprotective axis of the renin angiotensin system (RAS) in HSPCs functions offers a breakthrough. Angiotensin-(1-7), the primary mediator of the protective functions which acts on Mas receptor (MasR), is generated by angiotensin converting enzyme-2 (ACE2). In this study, we tested the effects of hypoxia on stimulation of vasoreparative potential of HSPCs and in upregulation of ACE2 and MasR. Importantly, we delineated the molecular mechanism of hypoxic exposure in regulation of ACE2 and MasR in a HIF1α- dependent manner and hypoxic exposure induced shedding of the membrane bound ACE2 in HSPCs. We used luciferase, a reporter assay, cell-based assays, gene/protein expression studies and pharmacological strategies in human and mouse HSPCs to test our hypotheses. To verify the biological significance of hypoxia, we performed in vivo studies in mice and humans, which recapitulated the in vitro observations on vascular protective axis of RAS in HSPCs. Collectively, these studies provided mechanistic insights into hypoxic regulation of vascular protective axis of RAS in HSPCs and also provided compelling evidence for the clinical use of hypoxia as a promising approach for enhancing the vasoreparative outcomes of cell-based therapies. / American Heart Association grant, 13SDG16960025 / National Institutes of Health, National institute of Aging (NIA), 1R01AG056881

<p>The recent and dramatic rise in allergic disease, coupled with the manifestation of the disease within the first years of life, suggests that <em>in utero</em> events are likely critically important to the inception of allergy. Epidemiological and experimental evidence suggest that both genetic predisposition and prenatal environmental exposures (e.g., <em>in utero</em> microbial exposures) play a role in modulating neonatal immunity and subsequent development of allergy. Of relevance to the work in this thesis, reports suggest that bacterial agents can directly alter myelopoiesis and, in connection to allergy, we have previously shown that cord blood (CB) progenitors from high-atopic risk infants demonstrate altered hematopoietic responses. However, whether CB progenitor cell hematopoietic responses are directly altered by microbial stimulation, and what effect maternal atopy has on these responses are unclear. Therefore, this thesis examines the influences of bacterial lipopolysaccharide (LPS) stimulation (innate immunity), maternal atopy, and adaptive immune stimuli (representative of an atopic milieu) on CB progenitor cell eosinophilopoiesis. We show that CB progenitors from healthy, pregnant women respond to LPS through increased eosinophil-basophil (Eo/B) colony forming units (CFU) via the mitogen-activated protein kinase (MAPK) signalling pathway (Chapter 2), whereas the presence of maternal atopy (as defined by skin prick test positivity) is associated with reduced CB CD34⁺ cell LPS-induced Eo/B CFU formation (Chapter 3). To investigate the potential mechanism of reduced eosinophilopoiesis in high-atopic risk infants, CB progenitors stimulated with IL-4 (a surrogate <em>ex vivo</em> for maternal atopy), but not IL-13, demonstrate reduced LPS-induced MAPK activation and Eo/B CFU formation (Chapter 4). This novel work provides insight into mechanisms relating to the influence of maternal atopy and/or potential intrauterine exposures (e.g., prenatal cytokines) on the responsiveness of CB progenitor cells to LPS, which may be of key importance for the development of atopic illnesses. These observations may help in the generation of novel biomarkers and therapeutic targets for childhood atopy.</p> / Doctor of Philosophy (PhD)

The transplantation of hematopoietic peripheral blood stem cells is one of the therapeutic choices for the treatment of various hematological and oncological diseases, extending disease-free survival and, in some cases, providing the cure of the patient. This is a retrospective study in which the medical records of patients who underwent mobilization, collection and infusion of peripheral hematopoietic stem cells for autologous transplantation were analyzed with the objective of evaluating these procedures, identifying the characteristics of the population assisted and verifying the transplant-related mortality and overall survival of transplanted patients. The study analyzes 176 patients (78F/98M) of the Hematology-Oncology Service of the University Hospital of Santa Maria (HUSM) in the period between December 1996 and December 2011. The diagnoses included: Multiple Myeloma (54), Hodgkin Lymphoma (47), Non Hodgkin Lymphoma (35), Acute Myeloid Leukemia (16) and other neoplasias (24) such as Breast Cancer, Wilms Tumor, Ewing s Sarcoma, Neuroblastoma, Amyloidosis, Testicular Tumor, Medulloblastoma, Macroglobulinemia, CNS Tumor. The median age of the patients was 42 years (1 to 69 years). Before the stem cells mobilization, all the patients had already been submitted to some kind of treatment related to the disease and 31% had received previous radiotherapy. The mobilization schema included hematopoietic growth factor (G-CSF) associated our not whit different chemotherapies. The number of mobilization and leukapheresis carried out were 203 and 474 respectively. The desired number of CD34+ cells in the collection was 2,0x106/kg, considering that the majority of patients achieved the quantity expected. The median of total nucleated cells collected was 7,01x108/kg and of CD34+ cells it was of 2,63x106/kg, with a median of two leukapheresis per patient (1 to 6). Of the 176 patients in this study 120 patients were submitted to autologous transplantation, three received two infusions each. The median of total nucleated cells infused was 6,46x108/kg and of CD34+ cells was 3,17x106/kg. After the infusion of hematopoietic stem cells, the recovery of neutrophils occurred between 7 and 28 days (median 10 days), while the platelets between 8 and 79 (median 12 days), considering that three patients did not have platelet recovery. The correlation between the quantity of CD34+ cells infused and the recovery of neutrophils and platelets was verified. Transplantrelated mortality was 5%. The probability of overall survival of the group in a year was of 79,8% and 55,1% in five years. In conclusion, this study demonstrated that the majority of the patients achieved the desired number of CD34+ cells through mobilization and collection and the results obtained with autologous transplantation were similar to those described in the literature. / O transplante de células-tronco hematopoiéticas periféricas constitui-se uma das opções terapêuticas para o tratamento de várias doenças hematológicas-oncológicas, podendo prolongar a sobrevida livre da doença e, às vezes, pode representar a cura para o paciente. Este é um estudo retrospectivo através da revisão de prontuários dos pacientes submetidos à mobilização, coleta e infusão de células-tronco hematopoiéticas periféricas para transplante autogênico, com o objetivo de avaliar estes procedimentos, identificar as características da população atendida e verificar a mortalidade relacionada ao transplante e sobrevida global dos pacientes transplantados. Foram incluídos 176 pacientes (78F/98M) do Serviço de Hematologia-Oncologia do Hospital Universitário de Santa Maria, no período de dezembro de 1996 a dezembro de 2011. Os diagnósticos incluíram: Mieloma Múltiplo (54), Linfoma de Hodgkin (47), Linfoma não Hodgkin (35), Leucemia Mielóide Aguda (16) e outras neoplasias (24), como Câncer de Mama, Tumor de Wilms, Sarcoma de Ewing, Neuroblastoma, Amiloidose, Tumor de Testículo, Meduloblastoma, Macroglobulinemia, Tumor de SNC. A mediana de idade dos pacientes foi de 42 anos (1 a 69 anos). Antes da mobilização de célulastronco, todos os pacientes já haviam sido submetidos a algum tratamento relacionado a doença de base e 31% haviam recebido radioterapia prévia. Os esquemas de mobilização incluíram fator de crescimento hematopoiético (G-CSF) associado ou não a diferentes quimioterapias. Foram realizadas 203 mobilizações e 474 leucoaféreses. O número desejado de células CD34+ na coleta foi de 2,0x106/kg, sendo que a maioria dos pacientes (89,8%) alcançou a quantidade esperada. A mediana de células nucleadas totais coletadas foi de 7,01x108/kg e de células CD34+ foi de 2,63x106/kg, com mediana de duas leucoaféreses por paciente (1 a 6). Dos 176 pacientes do estudo, 120 foram submetidos a transplante autogênico de células-tronco hematopoiéticas do sangue periférico, sendo que três pacientes receberam duas infusões cada. A mediana de células nucleadas totais infundidas foi de 6,46x108/kg e de células CD34+ foi de 3,17x106/kg. Após a infusão das células-tronco hematopoiéticas, a recuperação dos neutrófilos ocorreu entre 7 e 28 dias (mediana 10 dias), enquanto as plaquetas entre os dias 8 e 79 (mediana 12 dias), sendo que em três pacientes não houve recuperação plaquetária. Foi encontrada correlação entre a quantidade de células CD34+ infundidas e recuperação de neutrófilos e plaquetas. A taxa de mortalidade relacionada ao transplante foi de 5%. A probabilidade de sobrevida global do grupo em um ano foi de 79,8% e em cinco anos de 55,1%. Em conclusão, este estudo demonstrou que a maioria dos pacientes atingiu a quantidade desejada de células CD34+ através das mobilizações e coletas e os resultados obtidos com os transplantes autogênicos foram semelhantes aos descritos na literatura.

Les cellules progénitrices endothéliales formant des colonies (EFCFs) sont issues decellules CD34+ de la moelle osseuse humaine. Peu de données concernent l’expression dufacteur tissulaire (FT) lors de cette différenciation endothéliale. Outre son rôle dansl’initiation de la génération de thrombine, le FT est impliqué dans l’angiogenèse.Nous montrons que les cellules CD34+ expriment le FT mais non ses isoformes. LesECFCs expriment peu de FT à l’état basal. En revanche, leur stimulation par le TNF-α induitune augmentation de l’expression de FT, et la génération de microparticules pro-coagulantes.Nous avons analysé les modifications fonctionnelles induites par cette stimulation. Nosrésultats montrent que l’expression de FT par les ECFCs est responsable d’une activité procoagulante majeure, alors que les propriétés angiogéniques ne semblent pas affectées.L’expression du tissue factor pathway inhibitor (TFPI) a été évaluée, ainsi que la capacité desmicroparticules issues de ECFCs à générer des métalloprotéinases (MMP2-, MMP-9).Une évaluation de la stabilité chromosomique des cb-ECFCs durant leur expansion aété réalisée, mettant en évidence des anomalies de nombre, mais pas d’anomalies destructures. Les conséquences de ces résultats en termes de thérapie cellulaire appliquées auxpathologies cardio-vasculaires sont discutées. Enfin, nous évoquons la possibilité deconsidérer l’expression de FT comme un marqueur de différenciation cellulaire. / Endothelial colony-forming cells (ECFCs) can be obtained from human bone marrowCD34+ cells. In spite of the essential role of the tissue factor (TF) in coagulation triggeringand angiogenesis, its expression during endothelial differentiation is not established. We showthat CD34+ cells express TF, but not TF splicing forms. ECFCs express a small amount of TFat baseline level. In contrast, ECFCs express TF high levels of TF on response to TNF-α andcan generated highly pro-coagulant microparticles. We have examined the functionalproperties induced by TNF-α stimulation. TF expression confers to ECFCs a strong thrombingeneration capacity without influencing their non-coagulant properties. We have examinedthe co-expression of the tissue factor pathway inhibitor (TFPI) and the ability of ECFCs togenerate microparticles producing metalloproteins (MMP-2, MMP-9).We have performed an evaluation of cb-ECFCs chromosomal stability during theirexpansion. We found quantitative but no structural chromosomal abnormalities. Theconsequences of our observations in the use of cell therapy in cardiovascular diseases arediscussed. We conclude that TF expression may be considered as cell differentiation marker

Osteoblasts (OST) provide strong intrinsic growth modulatory activities on hematopoietic stem and progenitor cells via different mechanisms that include secretion of growth factors, and cellular interaction. Previously we showed that medium conditioned by mesenchymal stromal cell (MSC)-derived osteoblasts (M-OST) improve the expansion of cord blood (CB) CD34+ cells. I hypothesize that the hematopoietic supporting activity of M-OST would vary as a function of their maturation. This was tested by producing osteoblast conditioned media (OCM) from M-OST at distinct stages of maturation, and testing their growth regulatory activities in CB CD34+ cell cultures. My results showed that some of the growth promoting activity of OCM on CB cells are not dependent on the maturation status, while others are and those are largely independent of Notch signalling. In conclusion, these results provide further evidence that osteoblasts release factors that can promote the growth of immature CB progenitors in a Notch-independent way.