Orientador: Márjorie de Assis Golim / Resumo: O sistema de qualidade é de suma importância em laboratórios clínicos para avaliação de processos analíticos de maneira que os resultados liberados sejam verdadeiros. Para a metodologia de imunofenotipagem celular por citometria de fluxo as amostras devem ser frescas e os exames realizados preferencialmente dentro de 48 horas. É relevante utilizar amostras de controle de qualidade internos (CQI) padronizadas, de modo que possam ser repetidas rotineiramente, como referencial de qualidade. No Brasil, poucos serviços comercializam amostras preservadas para uso como CQI. Deste modo, a padronização in house com validação de processo para obtenção de amostras que possam ser utilizadas para esta finalidade é relevante. O objetivo deste trabalho foi desenvolver controle de qualidade interno para as rotinas de quantificação de células progenitoras hematopoéticas (CPH), utilizando solução preservante e avaliar a reprodutibilidade e estabilidade ao longo do tempo. Foram preparadas soluções preservantes contendo diferentes concentrações de anticoagulantes e fixadores, e destas, foi selecionada uma composição, originalmente padronizada neste estudo. Foram utilizados 5mL de sangue periférico, sendo este acrescido da solução a ser testada. Imediatamente, realizou-se a quantificação das populações de CPH em tubo Trucount®, usando anti-CD45, anti-CD34 e 7-AAD, conforme indicado pelo fabricante. A leitura foi realizada em citômetro de fluxo modelo FACSCalibur®-BD, para obtenção dos valores abs... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The quality system is of paramount importance in clinical laboratories for evaluating analytical processes in order to consider true the released results. The samples must be performed fresh preferably within 48 hours for the cell immunophenotyping methodology by flow cytometry. It is relevant to use standardized internal quality control (IQC) samples, thus they could be repeated routinely, as a quality benchmark. In Brazil, only a few services commercialize preserved samples for use as IQC. Therefore, it is relevant to use in-house standardization with process validation to obtain samples that can be used for this purpose. The objective of this work was to develop an IQC for a daily routine quantification of hematopoietic stem cells (HSCs) by using a preservative solution and to evaluate the reproducibility and stability over time. Preservative solutions containing different concentrations of anticoagulants and fixatives were prepared, and from these, a composition was selected, which was previously originally standardized in this study. 5mL of peripheral blood were used, which was added to the solution to be tested. The HSCs populations were immediately quantified in a Trucount® tube, using anti-CD45, anti-CD34 and 7-AAD, as indicated by the manufacturer. The reading was performed in a flow cytometer model FACSCalibur®-BD in order to obtain the absolute values of HSCs on day zero, 7, 21, 35 and 49. During this period, the samples were kept refrigerated (2 to 8ºC). The value... (Complete abstract click electronic access below) / Mestre

Tissue regeneration is a complex and highly orchestrated process dependent on cells with the potential to restore structures and functions and on controlling factors from the tissue microenvironment. Hematopoietic tissue has a high ability to regenerate, which is attributed to the presence of stem cells, but the regeneration of severely damaged adult tissue is still only partially understood. Hematopoietic tissue provides a unique opportunity to study tissue regeneration due to its well-established steady- state structure and function, easy accessibility, advanced research methods, and well-defined embryonic, fetal, and adult stages of development. Embryonic/fetal liver hematopoiesis and adult hematopoiesis recovering from damage share the needto expand populations of progenitors and stem cells in parallel with increasing production of mature blood cells. We analyzed adult hematopoiesis in mice subjected to a submyeloablative dose (6 Gy) of gamma radiation, in which only a few cells with reconstituting capacity survived. We targeted the period of regeneration characterized by the renewed massive production of mature blood cells and the ongoing expansion of immature hematopoietic cells. Cells from the top of the hematopoietic hierarchy, hematopoietic stem cells, and multipotent progenitors are almost missing...

South Africa is in dire need of a public umbilical cord blood stem cell bank (UCB SCB). A severe shortage of genetically compatible samples for BM transplantation precludes the majority of South Africans from receiving the relevant medical care. UCB is a viable alternative to BM but is currently disposed of post-delivery. UCB could furthermore serve as a resource of genetically compatible haematopoietic progenitor cells (HPCs) that could be used in gene therapy approaches directed towards a cure for HIV-1. Knowing whether HIV-1 affects or infects primitive HPCs is vital to determine the course of action for transplantation of UCB-derived genetically resistant HPCs. Collecting and storing UCB in a public UCB bank could thus serve as a vital resource of genetically compatible samples for BM transplantation. It was thought that the high incidence of HIV-1 in South African patients and the persistent stigma surrounding HIV-1 would be problematic for collecting sustainable numbers of UCB units and subjecting units to compulsory screening for infectious diseases. This was however, not the case. In the South African context, we are faced with unique and rich challenges relating to cultural and religious differences that are further augmented by linguistic constraints and educational insufficiencies. Nevertheless, the majority of patients within the interviewed patient cohort were supportive of the idea of establishing a public UCB SCB in SA and were willing to undergo additional HIV-1 screening. The Ultrio-Plus® assay was verified in this study for screening UCB units for HIV-1 and could be used in routine analyses of UCB units prior to banking. Conflicting results in the literature exist with regard to HIV-1’s ability to infect or affect haematopoietic progenitor cells. Results from this study revealed that HIV-1 was not only able to affect HPCs’ ability to form colonies in vitro, but was also capable of infecting CD34+ HPCs in some individuals. These results substantiate the theory that some CD34+ HPCs serve as viral reservoirs which could account for residual viraemia in patients on antiretroviral therapy. Results suggest that allogeneic transplantation of HIV-1 infected individuals with UCB-derived, genetically modified HPCs, should be pursued. / Thesis (PhD)--University of Pretoria, 2012. / Immunology / unrestricted

Haematopoietic stem and progenitor cells (HSPCs) are crucial for lifelong blood cell production. We analysed the cell cycle and cell production rate in HSPCs in murine haematopoiesis. The labelling of DNA-synthesizing cells by two thymidine analogues, optimized for in-vivo use, enabled the determination of the cell cycle flow rate into the G2-phase, the duration of the S-phase and the average cell cycle time in Sca-1+ and Sca-1- HSPCs. The determination of cells with 2n DNA content and labelled during the preceding S-phase was used to establish the cell flow rates in the G1-phase. Our measurements revealed a significant difference in how Sca-1+ and Sca-1- HSPCs self-renew and differentiate. The division of Sca-1+ progenitors led to the loss of the Sca-1 marker in about half of newly produced cells, corresponding to asymmetric cell division. In contrast both Sca-1- progenitors, arising from mitotic cell division, entered a new round of the cell cycle. This corresponds to symmetric self-renewing cell division. The novel data also enabled us to estimate the cell production rates in the Sca-1+ and in three subtypes of Sca-1- HSPCs. We focused on adult murine erythroid differentiation in the next part of our study. We introduced an original flow cytometry approach for identifying and studying erythroid...

Der Cortex von Säugetieren ist der Hirnbereich, der fundamental für höhere kognitive Funktionen wie Lernen, Gedächtnis, Aufmerksamkeit und komplexes Denken ist. Die Entwicklung des Cortex wird von neuralen Vorläuferzellen gesteuert, die schnell proliferieren, um ihren Pool zu expandieren, bevor sie zu differenzierenden Zellteilungen wechseln, um alle Neuronen zu generieren, aus denen der reife sechs schichtige Neokortex besteht. Der schrittweise Wechsel von Selbsterneuerung zu Neurogenese ist ein zeitlich regulierter Prozess, dessen Fehler schwere lebenslange kognitive Erkrankungen verursachen können. Aus diesem Grund ist es enorm wichtig zu verstehen, welche Faktoren die Schicksalsentscheidung der neuralen Vorläuferzellen regulieren. In den letzten zwei Jahrzehnten haben mehrere Studien die Wichtigkeit von nicht-kodierenden RNAs, wie lange nicht-kodierende und micro RNAs, für diese zeitliche Regulierung hervorgehoben. Mithilfe der Generierung einer kombinatorischen RFP/GFP Reporter Mauslinie, die die Isolierung von proliferierenden und differenzierenden Vorläuferzellen und neugeborenen Neuronen erlaubt, wurde berichtet, dass die lange nicht-kodierende RNA Miat als ein Regulator des neuralen Vorläuferzellen-Schicksals mittels Spleißen fungiert. Die Arbeit dieser Thesis zeigt, dass die Überexpression von Miat den Wechsel der neuralen Vorläuferzellen von proliferierenden zu neurogenen Zellteilungen verzögert und etabliert eine Strategie, um Miat-gespleißte Ziele auf Einzelpopulationslevel während der Corticogenese zu entdecken. Außerdem wurde die doppelte Reporter Mauslinie genutzt, um einen umfassenden und kompletten Katalog von micro RNAs, die in neuralen Vorläuferzellen und Neuronen exprimiert sind, zu erstellen. Dies führte zur Identifizierung von miR-486-5p als ein neuer Regulator der neuralen Vorläuferzellen-Schicksalsentscheidung.

The regenerative potential of adult stem cell populations within the human body bears great promises for their use in regenerative medicine. The bone marrow (BM) harbours two different types of adult stem cells, haematopoietic stem and progneitor cells (HSPCs) and multipotent mesenchymal stromal cells (MSCs), which are tightly regulated in their distinct anatomically defined niches by multiple cues such as cytokines, cell-cell contacts, the extracellular matrix (ECM) and the physical microenvironment. The ex vivo expansion of these cells for applications in regenerative therapies is of great interest and several biomaterial approaches attempt to mimic the natural BM niche and its components to control stem cell maintenance and differentiation. However, as of now the complexity of such stem cell niches is hard to recapitulate. Towards this goal, this work was focussing on the ECM environment of BM stem cells and was set out to engineer improved in vitro culture systems. MSC themselves are one of the most important cell types within the BM that secrete and construct ECM-networks and thereby shape the microenvironment of the residing cells. The potential of primary human BM-MSC to secrete ECM in vitro has been exploited to generate niche-like ECM surrogates in a robust and versatile format. Application of decellularisation regimes allowed the fabrication of complex matrices which demonstrated suprastructural, compositional and physicochemical properties compareable to those of the native BM-ECM environment. Reliable stability and reproduciblity was achieved by a dedicated procedure of maleic anhydride co-polymer-mediated covalent binding of fibronectin and subsequent anchorage of cell-secreted ECM molecules. As a result of the high reproducibility, a complete proteomic register of ECM molecules was obtained in combination with determining the complex fibrillar and soft gel-like characteristics of MSC-derived matrices. Based on the established BM niche-like substrate, the impact of extracellular matrices on MSC and HSPC ex vivo behavior has been explored. Both cell types demonstrated strong adhesion to ECM substrates and depicted a changed cellular morphology upon contact with native ECM structures compared to standard culture substrates or simple ECM protein coatings, indicating an intense interplay between the cell and the microenvironment. MSC that re-grew into their own matrices have shown advantageous proliferation and cytokine secretion levels as well as enhanced differentiation intensity (upon differentiation induction) compared to MSC that were cultured on less complex substrates. Similarly, HSPC were also instructed for enhanced expansion on MSC-derived matrices without exhaustion of stem cell-marker expressing progenitor cells. The efficiency of these matrices was related to their ability to mimic the native composite suprastructure, ligand nano-topography, molecular composition and physical properties of natural BM ECM environments. The data obtained within this thesis set the ground for a more rational design of artificial stem cell niches with defined and distinct properties, offering exciting options for the in-depth analysis and understanding of stem cell regulation by exogenous cues.

No description available.

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.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13464号 / 論医博第2251号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 金子 新, 教授 江藤 浩之, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Stem cell therapy may present an effective treatment for metastatic brain cancer and glioblastoma. Here we posit the critical role of a leaky blood-brain barrier (BBB) as a key element for the development of brain metastases, specifically melanoma. By reviewing the immunological and inflammatory responses associated with BBB damage secondary to tumoral activity, we identify the involvement of this pathological process in the growth and formation of metastatic brain cancers. Likewise, we evaluate the hypothesis of regenerating impaired endothelial cells of the BBB and alleviating the damaged neurovascular unit to attenuate brain metastasis, using the endothelial progenitor cell (EPC) phenotype of bone marrow-derived mesenchymal stem cells. Specifically, there is a need to evaluate the efficacy for stem cell therapy to repair disruptions in the BBB and reduce inflammation in the brain, thereby causing attenuation of metastatic brain cancers. To establish the viability of stem cell therapy for the prevention and treatment of metastatic brain tumors, it is crucial to demonstrate BBB repair through augmentation of vasculogenesis and angiogenesis. BBB disruption is strongly linked to metastatic melanoma, worsens neuroinflammation during metastasis, and negatively influences the prognosis of metastatic brain cancer. Using stem cell therapy to interrupt inflammation secondary to this leaky BBB represents a paradigm-shifting approach for brain cancer treatment. In this review article, we critically assess the advantages and disadvantages of using stem cell therapy for brain metastases and glioblastoma. / National Institutes of Health / Revisión por pares