Enhancing in vitro specification of definitive hematopoiesis from hemogenic endothelium through genetic and epigenetic modifications

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The discovery and transplantation of hematopoietic stem cells (HSCs) is a success story of regenerative medicine. To meet the demand for HSCs, numerous attempts have been made to either expand the cells ex vivo or to generate them de novo from pluripotent stem cells. However, to date, these approaches remained largely unsuccessful.Using a genetic screening, the Daley lab has identified seven transcription factors, which in combination with growth factors are able to differentiate human pluripotent stem cells through an endothelial intermediate (hemogenic endothelium) in HSCs. The aim of this research project is to increase the efficiency of the differentiation protocol in order to generate sufficient cell numbers for subsequent down-stream applications such as drug screenings. For this, a reporter cell line will be used, which expresses a fluorescent protein under the control of the HSC-specific Runx1 promoter, thus allowing the identification of successfully specified HSCs. The goal is to identify essential transcription factors and to reduce the number of required factors for the specification into HSCs. Next, the efficiency of hematopoietic stem cell specification is to be increased by optimizing the transgene expression using a polycistronic lentiviral vector. Further, an siRNA screening should be performed to identified epigenetic modulators which inhibit the process of specification. Finally, the mechanisms by which the transcription factors and epigenetic modulators determine hematopoietic cell fate shall be deciphered by global gene expression analysis and transcription factor binding studies. In the long run, insights into the molecular mechanisms of HSC specification will allow generation of mature HSCs from pluripotent stem cells, which can serve as a potential source for clinical applications.