Bmi-1-Green Fluorescent Protein (GFP)-Knock-In Mice Reveal the Dynamic Regulation of Bmi-1 Expression in Normal and Leukemic Hematopoietic Cells.

The ability to self-renew is essential for all kinds of stem cells regardless of tissue types. One of the best candidate genes involved in conferring self-renewal capacity is Bmi-1, which has been proven to be essential for the maintenance of both normal adult hematopoietic and leukemia stem cells as well as adult neural stem cells. Furthermore, it has been reported that Bmi-1 is expressed at high level in immature hematopoietic cells and its expression is down-regulated along with cell differentiation. In order to investigate the possible role of Bmi-1 expression as a marker of stem or progenitor cells in several types of normal or malignant tissues, we generated two types of Bmi-1-green fluorescent protein (GFP)-knock-in mice, in which GFP was expressed under the endogenous transcriptional regulatory elements of the Bmi-1 gene. In one strain, GFP was inserted into the transcription start site, resulting in the heterozygous allele for Bmi-1. In another strain, GFP was expressed as a fusion protein with Bmi-1 from the targeted allele. Using these two types of targeted reporter mice, we demonstrate that Bmi-1 expression levels are well-correlated with cell differentiation in normal hematopoietic cells. Bmi-1 is expressed in CD150+(or Flk2/Flt3−) CD34− c-kit+ lin- Sca-1+ (KLS) cells, which is highly enriched with long-term hematopoietic stem cells (LT-HSCs), at its highest levels and down-regulated upon commitment to differentiation. The Bmi-1high/lineage-marker- cells contained 33.8% of KLS cells, indicating that HSCs can be highly enriched according to the Bmi-1 expression levels combined with lineage-markers. Then, we analyzed the Bmi-1 expression levels in acute myeloiud leukemia (AML) models induced by the combination of TEL/PDGFβR and AML1/ETO, or chronic myelogenous leukemia (CML) models induced by P210 BCR/ABL. Bmi-1 is also expressed at its highest levels in undifferentiated KLS leukemia cells. In addition, the Bmi-1 expression levels in leukemic KLS cells are similar to normal KLS cells, suggesting that the Bmi-1 over-expression in leukemia samples might simply reflect the accumulation of immature cells. Furthermore, in several other non-hematopoietic tissues, cells could be separated into distinct subpopulations with differential Bmi-1 expression. Thus, these mice allow for the isolation of viable Bmi-1-expressing cells and have the potential to become a useful tool for understanding the role of Bmi-1 in normal and cancer stem cells in multiple tissue types.