Osteoblast Sorting and Intracellular Staining of CXCL12

Background

The bone marrow niche is a highly organized microenvironment with stroma cells that engage in direct cell-cell interaction with hematopoietic stem cells (HSC) that regulate HSC quiescence, differentiation, and mobilization (Anthony and Link, 2014; Mendelson and Frenette, 2014; Morrison and Scadden, 2014). Multiple cell types in HSC niche may contribute to niche functional support in distinct but maybe overlapping ways. These cells include but are not limited to osteoblasts, osteoclasts, CXCL12-abundant reticular (CAR) cells, Nestin⁺ stroma cells, leptin receptor⁺ (LepR⁺) stroma cells, endothelial cells, macrophages, megakaryocytes, neuronal, and the non-myelinating Schwann cells. Most HSCs are found in the trabecular region of bone marrow, suggesting an important HSC supporting role of the endosteum as well as factors made by osteoblasts and other cells in the endosteum (Kiel et al., 2005; Lo Celso et al., 2009). The majority of long-term HSCs are located in close vicinity of the sinusoid in close contact with LepR⁺ and CXCL12^high niche cells, indicating a perivascular niche composed by endothelial or perivascular cells (Kiel et al., 2005; Sugiyama et al., 2006; Acar et al., 2015). In addition to the perivascular niche associated with sinusoid, mesenchymal cells that surround arterioles in the bone marrow are also important for the maintenance of quiescent HSCs (Kunisaki et al., 2013). Osteoblasts are specialized endosteum-lining cells that are terminally differentiated products of mesenchymal stem cells. Characterization of murine primary osteoblasts has been achieved through culture of outgrowth of collagenase treated bone and retrospective functional analysis (Bakker and Klein-Nulend, 2011). However, culture-based analysis has been complicated by the heterogeneity of the tissue. Immunophenotyping of murine osteoblasts based on defined CD markers is a rapid and prospective approach of phenotypic analysis of osteoblasts in various disease processes. Isolated osteoblasts through flow-based sorting are especially suitable for downstream applications such as gene expression analysis.

The chemokine CXCL12 (also known as stromal-derived factor, SDF-1) together with its receptor CXCR4 are highly expressed by CAR cells but also by osteoblasts and endothelial cells. The CXCL12-CXCR4 axis is important for hematopoietic stem cell retention to their niches (Sugiyama et al., 2006). CXCL12 in the vascular niche has also been shown play a critical role in supporting leukemia-initiating cell (LIC) activity (Pitt et al., 2015). Using a mouse T-ALL model, we reported that leukemia development was accompanied by the drastic suppression of the endosteum-lining osteoblast population. We further showed that aberrant Notch activation negatively regulates the expression of CXCL12 and osteoblastic progenitor differentiation. Here we describe the procedure of sorting mouse bone marrow osteoblastic cells and the procedure of staining intracellular CXCL12 (Wang et al., 2016).