Abstract
A labeling system for tissue stem cells is a powerful tool for stem cell research and for development of stem cell-based regenerative medicine to human disease. Here we generated transgenic mice expressing green fluorescent protein (GFP) under control of enhancer/promoter activity to Nucleostemin (NS) gene. NS, a nucleolar GTP-binding protein, expresses at high levels in embryonic stem cells and neural stem cells, suggesting that NS expresses in various tissue stem cells. In fact, we found that NS highly expressed in mouse immature hematopoietic cells containing hematopoietic stem cells (HSCs) and progenitor cells compared with differentiated hematopoietic cells. To examine whether HSCs could be labeled by using enhancer/promoter activity to NS gene, we generated transgenic mice harboring putative genomic enhancer/promoter region of NS gene followed by GFP cDNA-polyA in the 3′ end (NS-GFP tg mice). Fluorescence activated cell sorting (FACS) analysis indicated that most bone marrow (BM) mononuclear cells (MNCs) showed green fluorescence in NS-GFP tg mice. To validate the relationship between fluorescent intensity of GFP and expression levels of endogenous NS mRNA, BM MNCs from NS-GFP tg mice were separated into four fractions depending on their fluorescent intensity (i.e., GFP−, GFP+, GFP++, and GFP+++) by FACS, and these cells were subjected to quantitative real-time PCR analysis. The fluorescent intensity of GFP certainly reflected the amount of endogenous NS mRNA, indicating that the enhancer/promoter region used in this study is sufficient for monitoring the expression levels of endogenous NS mRNA in vivo. We next characterized expression levels of cell-surface markers on the four fractions of BM MNCs. FACS analysis showed that the GFP+++ cells, but not the other cell populations, lost the expression of mature hematopoietic cell markers (myeloid, B cells, T cells and erythroid cells). The fluorescent intensity of GFP in c-Kit+ Sca-1+ Lineage− (KSL) cells, which represent a primitive hematopoietic cell fraction containing HSCs and progenitor cells, was much higher than that in the other cell populations, indicating that the immature hematopoietic cells highly express GFP. To investigate the biological properties of the GFP-high expressing cells, we further performed colony-forming assay in vitro and BM transplantation assay in vivo. When the sorted BM MNCs were cultured in methylcellulose medium supplemented with cytokines, GFP+++ cells showed the highest number of colony formation. To assess the repopulating capacity as stem cell function, the sorted BM MNCs were transplanted into irradiated recipient mice. Notably, only GFP+++ cells had long-term reconstitution capacity of hematopoiesis in the recipient mice on the competitive reconstitution assay. In addition, the GFP+++ cells that were sorted from the GFP+++ cells-transplanted mouse retained the reconstitution capacity after secondary BM transplantation. The reconstitutive capacity of the GFP+++ cells into multi-lineages was confirmed by detection of donor-derived B, T, and myeloid cells in the recipient mice. These results demonstrate that HSCs with self-renewal capacity and differentiation potential for multi-lineages are enriched in GFP+++ cell population. The NS-GFP tg mouse system may provide a useful tool for effective enrichment of HSCs in combination with other cell-surface markers for HSCs.
A protocol for generation of transgenic mice by manipulating spermatogonial stem cells in vivo.
