Year 4 (NCE)

The major shortcoming in generating functional hematopoietic stem cells (HSC) from pluripotent stem cells is the lack of adequate markers for specification and maintenance of functional human HSCs. To overcome this hurdle, we have generated fluorescent reporter hESC lines for a set of novel human HSC regulators that we have identified. As these factors are localized in the nucleus, it is not possible to observe their expression using conventional methods without killing the cell. We have now  finalized the targeting and have progressed with validating the expression of the fluorescent reporters for the HSC regulators. By comparing single cell RNA seq data from hESC derived cells to human early developmental tissues, we have shown that the expression patterns for these genes and their reporters matches with that in early human developmental hematopoietic tissues, with each HSC regulator being expressed in the most undifferentiated HSPC, and selectively in other cell types (HLF also in hepatocytes, MLLT3 and HIF3 also in erythroid cells, HOXA5, HIF3a and MYCT1 also in distinct endothelial cell, HIF3A in mesenchymal stroma). Our functional studies on these factors have revealed distinct roles in HSC biology for each one of them. Our studies also suggest that these hESC reporter lines will  become  valuable for investigators working on optimizing stem cell differentiation and culture for other tissues, as many of these reporter genes are not only restricted to HSC. Using these reporter lines to monitor the induction of HSC “stemless’ program, we are making progress in optimizing differentiation conditions toward generating functional HSCs for transplantation.