Hypoxia depletes contaminating CD45⁺ hematopoietic cells from murine bone marrow stromal cell (BMSC) cultures: Methods for BMSC culture purification

Culture expanded bone marrow stromal cells (BMSCs) are easily isolated, can be grown rapidly en masse, and contain both skeletal stem cells (SSCs) and multipotent mesenchymal progenitors (MMPs). Despite this functional heterogeneity, BMSCs continue to be utilized for many applications due to the lack of definitive and universally accepted markers to prospectively identify and purify SSCs. Isolation is widely based on adherence to tissue culture plastic; however, high hematopoietic contamination is a significant impediment in murine models. Remarkably, when cultured at a physiological oxygen tension of 1% O₂, a 10-fold reduction in CD45⁺ hematopoietic cells associated with a concomitant increase in PDGFRα⁺ stromal cells occur. This is due, in part, to a differential response of the two populations to hypoxia. In standard tissue culture conditions of 21% O₂, CD45⁺ cells showed increased proliferation coupled with no changes in cell death compared to their counterparts grown at 1% O₂. In contrast, PDGFR α⁺ stromal cells responded to hypoxia by increasing proliferation and exhibiting a 10-fold decrease in cell death. In summary, we describe a simple and reliable method exploiting the divergent biological response of hematopoietic and stromal cells to hypoxia to significantly increase the PDGFR α⁺ stromal cell population in murine BMSC cultures.