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

造血干细胞（hsc）的发现和移植是再生医学的一个成功案例。为了满足对造血干细胞的需求，已经进行了许多尝试，要么是体外扩增细胞，要么是从多能干细胞重新生成细胞。然而，迄今为止，这些方法在很大程度上仍然不成功。通过基因筛选，Daley实验室确定了七种转录因子，它们与生长因子结合，能够通过造血干细胞中的内皮中间体（造血内皮）分化人类多能干细胞。本研究项目的目的是提高分化方案的效率，以便为后续的下游应用（如药物筛选）产生足够的细胞数量。为此，将使用一种报告细胞系，该细胞系在hsc特异性Runx1启动子的控制下表达一种荧光蛋白，从而能够鉴定成功指定的hsc。目标是确定必要的转录因子，并减少进入造血干细胞的规格所需因子的数量。下一步，利用多顺反子慢病毒载体优化转基因表达，提高造血干细胞的鉴定效率。此外，应进行siRNA筛选，以确定抑制规范过程的表观遗传调节剂。最后，转录因子和表观遗传调节剂决定造血细胞命运的机制将通过全球基因表达分析和转录因子结合研究来破解。从长远来看，深入了解造血干细胞的分子机制将使多能干细胞产生成熟的造血干细胞，这可以作为临床应用的潜在来源。

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.