Bone morphogenetic protein-4 (BMP4) is highly expressed at sites of haematopoietic stem cell (HSC) formation. During HSC formation in humans, BMP4 is strongly expressed by cells underlying the ventral floor of the dorsal aorta. BMP4 in combination with other factors has been shown to play a role in haematopoietic differentiation. The genes Runx1, Scl, Gata2 and Lmo2 are vital to the development of the haematopoietic system, and deletion of these genes produces an embryonic lethal phenotype due to the absence of blood formation. This study investigated whether BMP4 alone upregulates the expression of these genes. The role of BMP4 was explored during HSC development in an embryonic stem (ES) cell differentiation model and at later developmental stages using ex vivo foetal liver and bone marrow serum free cultures. Differentiating ES cells cultured in serum-free medium were found to express BMP4 and the BMP receptor endogenously. To establish a model for exogenous BMP4 addition in isolation, lentiviral vectors were used to deliver short hairpin RNA (shRNA) for sustained RNAi knockdown of endogenous Bmp4 expression during ES cell differentiation. Differentiating shRNA treated ES cells were cultured and the expression of Runx1, Scl, Gata2 and Lmo2 was measured over time by real time reverse transcription PCR. With the addition of exogenous BMP4 alone, expression of Runx1, Scl, Gata2 and Lmo2 was unchanged at days 2 and 4 but increased at day 6 of differentiation. This demonstrates that BMP4 up regulates the expression of these genes which are critical to the development of the haematopoietic system. The use of lentiviral shRNA knockdown provides a model for the control of endogenous growth factors in future investigations of growth factors in ES cell differentiation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:564697 |
| Date | January 2009 |
| Creators | Abeyewickreme, A. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/18691/ |
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