Bone Marrow Osteoblast Ablation Enhances Short-Term Hematopoietic Stem Cells Without Altering Long-Term Hematopoietic Stem Cell Populations and Accelerates Leukemia Development

Hematopoietic stem cells (HSCs) within the bone marrow (BM) microenvironment reside in close proximity to endosteal osteoblasts (OBs). Although OBs have been considered to provide a HSC niche, other studies suggest that perivascular mesenchymal cells or endothelial cells may be the primary HSC niches, and the specific role of OBs in regulation of HSCs requires further clarification. Moreover, the role of OBs in regulating leukemic stem cells (LSC) is even less well studied. To address these questions, we used a conditional OB ablation mouse model (Col2.3Δtk) in which a truncated version of the herpes simplex virus thymidine kinase (Δtk) is expressed under an OB-specific promoter. In these mice, daily intraperitoneal (IP) administration of ganciclovir (GCV) leads to production of a toxic DNA base analogue in OBs, resulting in their death. We crossed Col2.3Δtk mice with Col2.3GFP mice that specifically express GFP in OBs to facilitate assessment of OB ablation. We confirmed that 4 weeks of GCV administration resulted in ablation of endosteal OBs in this model using both immunofluorescence microscopy and flow cytometry analysis. OB ablation was associated with reduced BM cellularity (Δtk⁺ 3.7e7±3.0e6, Δtk^- 4.8e7±3.8e6 per 4 lower extremity bones, p=0.04), but did not alter spleen (SP) cellularity (Δtk⁺ 5.1e7±5.3e6, Δtk^- 6.3e7±7.4e6 cells per SP, p=0.19). OB ablation was also associated with significantly increased numbers of cells with long-term HSC (LTHSC) phenotype (Lin^-Kit⁺Sca-1⁺Flt3^-CD150⁺CD48^-) in both the BM (Δtk⁺ 6490±1315, Δtk^- 4236±922 per 4 lower extremity bones; p=0.03) and SP (Δtk⁺ 980±473, Δtk^- 96±40 per SP; p=0.04). Significant increases in common myeloid progenitor (CMP) (Δtk⁺ 145114±43608, Δtk^- 82200±26754; p=0.002) and granulocyte/monocyte progenitor (GMP) (Δtk⁺ 51411±17349, Δtk^- 20206±9279, p=0.003, p=0.02) numbers were seen in SP of OB-ablated mice, whereas significant alterations in other hematopoietic populations in BM, SP or PB were not seen. We performed limiting-dilution competitive repopulation assays to determine the functional LTHSC potential of BM cells from OB-ablated and control mice. OB-ablated mice demonstrated a higher frequency of short-term repopulating cells compared to LTHSCs from non-ablated mice (5 weeks: Δtk⁺ 1 in 4,941; Δtk^- 1 in 17,351 BM cells) but similar long-term engraftment (15 weeks: Δtk⁺ 1 in 22,853; Δtk^- 1 in 23,137 BM cells). Transplantation of BM cells from primary transplant recipients into secondary recipients demonstrated similar long-term engraftment potential after second transplant. These results suggest that despite increased numbers of phenotypic LTHSCs in OB-ablated mice, the long-term repopulating and self-renewing capacity of BM cells remains unchanged in OB-ablated mice, but on the other hand there is an increase in functional short-term repopulating capacity. Next, to examine the role of OBs in regulation of Chronic Myelogenous Leukemia (CML) stem cells, we crossed the Col2.3GFPΔtk mice with an inducible transgenic BCR-ABL mouse model of CML (ScltTA-BCR/ABL). In these mice withdrawal of tetracycline results in induction of BCR-ABL expression in HSCs and development of a CML-like myeloproliferative disorder. GCV administration to achieve OB ablation was initiated one week prior to BCR-ABL induction by tetracycline withdrawal, and was continued for the duration of the experiment. CML development was monitored by checking blood counts every 2 weeks after induction and mice were followed for survival. We observed significantly accelerated development of CML in OB-ablated versus non-ablated mice, with 50% of the OB-ablated mice dying within 47 days of CML induction, whereas >50% of the non-ablated mice survived to day 73 (p=0.017). Collectively, these studies suggest that BM OBs are not essential for maintenance of long-term repopulating and self-renewing HSCs, but regulate the expansion of short-term HSCs in the BM. Our studies also indicate an important and previously unrecognized role for OBs in regulating the leukemogenicity of CML LSCs.