%0 Thesis
%9 Doctoral
%A Abeyewickreme, A.
%B UCL Institute of Child Health
%D 2009
%F discovery:18691
%I UCL (University College London)
%P 304
%T The effect of bone morphogenetic protein 4 on haematopoietic stem cells
%U https://discovery.ucl.ac.uk/id/eprint/18691/
%X 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.