Disruption of the Osteoblast Niche by G-CSF Is Associated with Hematopoietic Stem Cell Quiescence and Loss of Long-Term Repopulating Activity: Role of Cdkn1a

• Published in: Blood 2008-11, Vol.112 (11), p.2447-2447
• Main Authors: Gopalan, Priya K., Christopher, Matthew J., Greenbaum, Adam M., Link, Daniel C.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V112.11.2447.2447

The bone marrow microenvironment plays a key role in regulating hematopoietic stem cell (HSC) function. In particular, bone marrow stromal signals contribute to the maintenance of HSC quiescence, a property that is thought to be associated with long-term repopulating activity. We previously reported that G-CSF treatment disrupts the osteoblast niche by inducing osteoblast apoptosis and inhibiting osteoblast differentiation. In this altered bone marrow microenvironment, we also showed that the number of HSCs in the bone marrow after G-CSF treatment (as defined by CD34− Kit+ Sca+ lineage-cells or CD150+ CD48− CD41− lineage-[SLAM] cells) was unchanged and that the HSCs were more quiescent than HSCs from untreated mice. However, despite the quiescent phenotype, there was a marked loss of HSC long-term repopulating activity. To define mechanisms for this phenotype, we first asked whether G-CSF acts directly on HSCs to inhibit their long-term repopulating activity. Bone marrow chimeras containing wild type and G-CSFR−/− cells were established and treated with G-CSF. The contribution of G-CSFR−/− cells to hematopoiesis remained stable for at least 3 months after G-CSF treatment, demonstrating that the effects of G-CSF on HSC function are not direct. We next performed RNA expression profiling on sorted SLAM cells, a cell population highly enriched for HSCs. These data showed that expression of Cdkn1a (p21cip1/waf1) was increased in HSCs harvested from G-CSF treated mice. To define the contribution of Cdkn1a to HSC quiescence and loss of repopulating activity following treatment with G-CSF, Cdkn1a−/− mice (inbred on a C57BL/6 background) were studied. Wild-type or Cdkn1a−/− mice were treated with G-CSF for 7 days and pulse labeled with bromo-deoxyuridine (BrdU), and the percentage of SLAM cells that labeled with BrdU was determined. Consistent with our previous observations, treatment of wild-type mice with G-CSF resulted in a significant decrease in the percentage of BrdU+ SLAM cells in the bone marrow. In contrast, in Cdkn1a−/− mice, no change in the percentage of BrdU+ SLAM cells after G-CSF treatment was observed [10.08 ± 2.26% (untreated); 10.96 ± 2.80% (G-CSF treated); p = NS]. To assess HSC function, competitive repopulation assays were performed using untreated or G-CSF treated bone marrow from wild type or Cdkn1a−/− mice. Surprisingly, G-CSF had a similar deleterious effect on HSC repopulating activity in both wild type and Cdkn1a−/− mice. Collectively, th