Study of the requirement of the CCBE1 growth factor in the generation of cardiac myocytes from hES cells

Drago, Rita Catarina Vaz

http://hdl.handle.net/10400.1/3482

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Authors

Drago, Rita Catarina Vaz

Advisor(s)

Belo, José António

Bernardo, Andreia

Abstract(s)

In normal development, cells from the totipotent zygote give rise to the pluripotent inner cell mass and epiblast cells, which subsequently become more and more restricted, and ultimately, originate all the differentiated cells of the human body. In this Master thesis a chemically defined protocol based on signalling present in vivo was used to differentiate in vitro a human embryonic stem cell (hESCs) line, with the intent of recapitulating cardiac development. Along the differentiation protocol, the role of CCBE1 (Collagen and Calcium-Binding EGF-like domain 1) was studied. CCBE1 is a growth factor which has already been reported in mouse as being expressed and potentially required for the generation of cardiac precursor cells. As such, this study focused on three main topics: the characterization, function and regulation of CCBE1 in hES cells. Regarding characterization, CCBE1 shows an expression pattern similar to the one previously reported in mouse ES cells, i.e. it is highly expressed in pluripotency, when cells start to differentiate it is downregulated, and its mRNA and protein levels rise progressively as cells differentiate towards cardiac precursors. There is a strong association between mRNA and protein expressions, as determined by quantitative PCR analysis and immunocytochemistry assays. As for CCBE1 function, this was assessed by knocking down (KD) CCBE1 in hESCs using shRNA. CCBE1 KD cells lost their ability to differentiate properly into cardiac precursors, as evidenced by the downregulation of cardiac precursors genes. Additionally, pluripotency markers genes were also deregulated upon CCBE1 KD. The pattern of CCBE1 expression raised some questions about its regulation in pluripotency and cardiac precursor cells. To investigate this, Chromatin Immunoprecipitation (ChIP) assays were performed with an antibody against a pluripotency protein (NANOG), one against an early mesoderm protein (BRACHYURY), and one against a cardiac protein (ISL1). The results revealed binding of NANOG, BRA and ISL1 to distinct CCBE1 regulatory regions and at different differentiation timings. This work contributed to a further understanding of the complex process of human heart development by studying CCBE1, which is required for differentiation of cardiac mesoderm precursors.