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Master's Dissertation
DOI
10.11606/D.17.2012.tde-14062012-131047
Document
Author
Full name
Everton de Brito Oliveira Costa
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2012
Supervisor
Committee
Fontes, Aparecida Maria (President)
Carramaschi, Lygia da Veiga Pereira
Covas, Dimas Tadeu
Title in Portuguese
Caracterização das Células-Tronco/Progenitoras Hematopoéticas obtidas de Células-Tronco Embrionárias Humanas In Vitro em Sistema de Co-Cultivo com Fibroblastos de Embriões Murinos.
Keywords in Portuguese
Célula progenitora hematopoética
Célula-tronco embrionária humana
Endotélio hemogênico
Hemangioblasto
Abstract in Portuguese
A hematopoese tem sido bem descrita em modelos murinos nas últimas décadas, contudo, trabalhos demonstrando os mecanismos da hematopoese em humanos ainda são escassos. A derivação da primeira linhagem de células-tronco embrionárias humanas (CTEhs) em 1998, gerou novas perspectivas tanto para o estudo da hematopoese na tentativa de mimetizar o que ocorre naturalmente durante o desenvolvimento embrionário, quanto para a aplicação clínica das células hematopoéticas obtidas a partir da diferenciação dessas células. Contudo, apesar de inúmeros trabalhos terem demonstradoa obtenção de células hematopoéticas a partir de CTEhs, os protocolos têm gerado quantidades variáveis de células, com baixa eficiência e com propriedades funcionais de células primitivas. Desse modo, este trabalho procurou estabelecer um modelo próprio de diferenciação de CTEhs-H1 em células progenitoras hematopoéticas para que estas pudessem ser melhor caracterizadas e obtidas de forma mais eficiente. Para isto, foi desenvolvido um sistema de diferenciação baseado no co-cultivo da linhagem de CTEh-H1 com fibroblastos de embrião de camundongo (MEFs), em meio de diferenciação suplementado soro fetal bovino (SFB) e citocinas e fatores de crescimento hematopoéticos em baixas concentrações. Como resultado, o desenvolvimento do presente trabalho permitiu o estabelecimento de um método para geração de populações mistas de células enriquecidas em CPHs positivas para o marcador CD45, o qual mostrou ser coexpresso com outros marcadores hematopoéticos (CD31, CD43, CD71 e CD38), e células hematopoéticas maduras positivas para marcadores mielóide-específicos (235a, CD14, CD15, CD16) e com características morfológicas típicas. Foi demonstrado que as células obtidas expressavam genes relativos ao sistema hematopoético (CD45, CD31, runx1, tal1, lmo2, prom1, CD34 e notch1), e possuíam potencial clonogênico in vitro da ordem de 1/574 células plaqueadas. Em adição, corroboramos os achados de que as células hematopoéticas apresentam duas origens distintas: a partir do endotelio hemogênico e a partir de células com propriedades hemangioblásticas independentes do endotélio hemogênico.
Title in English
Characterization of Hematopoietic Stem/Progenitor Cells Obtained In Vitro from Human Embryonic Stem Cells in Co-Culture System with Mouse Embryonic Fibroblasts.
Keywords in English
Hemangioblast
Hematopoietic progenitor cells
Hemogenic endothelium
Human embryonic stem cell
Abstract in English
Hematopoiesis has been well described in murine models in recent decades, however, studies demonstrating the mechanisms of hematopoiesis in humans are still scarce. The first human embryonic stem cells line (hESCs) derived in 1998, has generated new perspectives about the study of hematopoiesis as in attempting to mimic what naturally occurs during embryonic development, as for clinical application of hematopoietic cells obtained from the differentiation of these cells. However, although numerous studies have shown the production of hematopoietic cells derived from hESCs, the protocols have generated varying quantities of cells with low efficiency and functional properties of primitive stem cells. Thus, this study sought to establish our own model for hESC-H1 differentiation in hematopoietic progenitor cells so that they could be better characterized and obtained more efficiently. For this way, we developed a differentiation system based on co-culture of hESC-H1 line with inactivated mouse embryonic fibroblasts (MEFs) in differentiation medium supplemented with fetal calf serum (FCS) and cytokines and hematopoietic growth factors in low concentrations. As a result, the development of this study allowed the establishment of a method for generation of mixed population of cells enriched in hematopoietic progenitor cells positive for the marker CD45, which proved to be co-expressed with other hematopoietic markers (CD31, CD43, CD71 and CD38), and mature hematopoietic cells positive for myeloid-specific markers (235a, CD14, CD15, CD16) and morphological characteristics typical. It was shown that these cells expressed genes related to the hematopoietic system (CD45, CD31, runx1, TAL1, LMO2, prom1, CD34 and NOTCH1), and had clonogenic potential in vitro of 1/574 plated cells. In addition, we corroborate the findings that hematopoietic cells have two distinct origins: they can arise as from an hemogenic endothelium as from cells with hemangioblastic properties by an hemogenic endothelium-independent way.
 
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Publishing Date
2012-08-31
 
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