| Summary: | Culture of hESCs on murine stromal layers or in stromal free conditions as embryoid bodies results in low levels of haematopoietic cells. Here it is demonstrated that overexpression of the transcription factor HOXB4 considerably augments haematopoetic development of hESCs. Stable HOXB4 expressing hESC clones were generated by lipofection and could be maintained in the undifferentiated state for prolonged passages. Moreover, differentiation of hESCs as embryoid bodies in serum containing medium without the use of additional of cytokines led to sequential expansion of first erythroid then myeloid and monocytic progenitors. These cells retained the capacity to develop into formed blood elements during in vitro culture. Consistent with the development of committed haematopoietic cells the expression of transcription factors known to be critical for haematopoietic development was observed. The successful use of enforced gene expression to promote the differentiation of hESCs into a terminally differentiated tissue is thus demonstrated, thereby revealing an important role for HOXB4 in supporting their in vitro development along the haematopoietic pathway. Once a method for producing significant numbers of haematopoietic cells from hESCs in vitro was established, hESCs were used to study the role of stem cell leukaemia gene (SCL) in human blood and endothelial development. hESC lines were generated in which the expression of SCL, under control of an enhancer previously shown in mice to target expression to blood and endothelial progenitors, was evident through green fluorescent protein (GFP) expression. The enhancer directed GFP expression inhuman K562 cells and some differentiated progeny of HOXB4 transfected hESCs. However a more thorough assessment of GFP positive cells was hindered by problems with transgene silencing.
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