Functional dissection of hematopoietic stem cell population by a stemness-monitoring system with NS-GFP transgene

Hematopoietic stem cells (HSCs) in a steady state are efficiently purified by the combination of several cell surface markers, however, such markers do not consistently reflect HSC activity. In this study, we successfully isolated functional HSCs by a unique stemness monitoring system using a transgenic mouse, in which green florescence protein (GFP) is driven by promoter/enhancer region of nucleostemin (NS) gene. We found that phenotypically defined long-term (LT)-HSC population exhibited the highest level of the NS-GFP intensity, whereas it was remarkably down-regulated during differentiation in vitro and in vivo. Importantly, among the LT-HSC population, the NS-GFPhigh cells significantly exhibited higher repopulating capacity than NS-GFPlow cells. Gene expression analysis revealed that 9 genes, including Vwf and Cdkn1c (p57), are highly expressed in NS-GFPhigh cells, which may represent signature of functional HSCs, i.e., stemness signature. When LT-HSCs suffered from remarkable stresses, such as transplantation or irradiation, NS-GFP intensity was downregulated, suggesting that NS-GFP accurately reflects HSC function. Finally, we found that functional HSCs were identified as NS-GFPhigh cells in CD34+ LSK cells, which has been recognized as progenitor cells without long-term reconstitution ability. Thus, high NS-GFP intensity represents stem cell characteristics in hematopoiesis and this system is useful for identification of uncharacterized HSCs. The LSK cells from NS-GFP transgenic mice were sorted, in which GFP is expressed under the control of particular region of NS promoter (NS-GFP).The murine LSK cells were fractionated into 4 subpopulations based on NS-GFP level , (a) NS-GFP1+, (b) -NS-GFP2+, (c) NS-GFP3+ and (d) NS-GFP4+