Functional dissection of hematopoietic stem cell populations with a stemness-monitoring system based on NS-GFP transgene expression

• Published in: Scientific reports 2017-09, Vol.7 (1), p.11442-12, Article 11442
• Main Authors: Ali, Mohamed A. E., Fuse, Kyoko, Tadokoro, Yuko, Hoshii, Takayuki, Ueno, Masaya, Kobayashi, Masahiko, Nomura, Naho, Vu, Ha Thi, Peng, Hui, Hegazy, Ahmed M., Masuko, Masayoshi, Sone, Hirohito, Arai, Fumio, Tajima, Atsushi, Hirao, Atsushi
• Format: Article
• Language: English
• Subjects: 13/100; 13/31; 38/39; 38/61; 631/1647/48; 631/532/1542; Animals; Biomarkers; CD34 antigen; Cell Differentiation - genetics; Cell Lineage - genetics; Cell Self Renewal; Cell Separation - methods; Cell surface; Colony-Forming Units Assay; Computational Biology - methods; Gene Expression; Gene Expression Profiling; Genes, Reporter; Hematopoiesis; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - metabolism; Humanities and Social Sciences; Immunophenotyping; Irradiation; Mice; Mice, Transgenic; Monitoring systems; multidisciplinary; Nucleostemin; Radiation; Recombinant Fusion Proteins; Rodents; Science; Science (multidisciplinary); Single-Cell Analysis; Stem cell transplantation; Stem cells; Surface markers; Transgenes; Transgenic mice; Transplantation
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-11909-3

Hematopoietic stem cells (HSCs) in a steady state can be efficiently purified by selecting for a combination of several cell surface markers; however, such markers do not consistently reflect HSC activity. In this study, we successfully enriched HSCs with a unique stemness-monitoring system using a transgenic mouse in which green florescence protein (GFP) is driven by the promoter/enhancer region of the nucleostemin (NS) gene. We found that the phenotypically defined long-term (LT)-HSC population exhibited the highest level of NS-GFP intensity, whereas NS-GFP intensity was strongly downregulated during differentiation in vitro and in vivo . Within the LT-HSC population, NS-GFP high cells exhibited significantly higher repopulating capacity than NS-GFP low cells. Gene expression analysis revealed that nine genes, including Vwf and Cdkn1c (p57), are highly expressed in NS-GFP high cells and may represent a signature of HSCs, i.e., a stemness signature. When LT-HSCs suffered from remarkable stress, such as transplantation or irradiation, NS-GFP intensity was downregulated. Finally, we found that high levels of NS-GFP identified HSC-like cells even among CD34 + cells, which have been considered progenitor cells without long-term reconstitution ability. Thus, high NS-GFP expression represents stem cell characteristics in hematopoietic cells, making this system useful for identifying previously uncharacterized HSCs.