The CD45^lowCD271^high Cell Prevalence in Bone Marrow Samples May Provide a Useful Measurement of the Bone Marrow Quality for Cartilage and Bone Regenerative Therapy

Background: Bone marrow aspirates and concentrates are increasingly being used for musculoskeletal regenerative therapies, providing bone and cartilage progenitors. However, the quality of these bone marrow samples remains imprecise within clinical settings. As there is a need for the development of these therapies, a method of counting CD45lowCD271high cells was optimized and tested as an indicator of bone marrow sample quality. Methods: Bone marrow aspirates were collected from 54 donors (28 male and 26 female; median age of 48 years). The reagent concentrations were optimized for fast staining, and an acoustic-focusing flow cytometer (Attune) was used to enable automated CD45lowCD271high cell counting in bone marrow aspirates, bone marrow concentrates, and samples loaded onto a collagen scaffold. The CD45lowCD271high cell counts were compared with those obtained using another flow-cytometry-based method (LSR II) and with connective tissue progenitor (CTP) counts quantified using a colony forming unit-fibroblast (CFU-F) assay. Results: The optimized method enabled the counting of CD45lowCD271high cells within only 15 minutes. The quantified cell counts (median, 1,520; range, 96 to 20,992 cells/mL of bone marrow) were positively correlated with the CTP counts (p < 0.0001; r = 0.7237). In agreement with CFU-F and LSR II-based assays, the CD45lowCD271high cell counts quantified using the Attune-based method decreased with age in the samples from female but not male donors (p = 0.0015 and p = 0.3877, respectively). A significant increase in CD45lowCD271high cell counts was detected following bone marrow concentration (mean, 5-fold; 95% confidence interval [CI], 3.6 to 7.2-fold). Additionally, the number of CD45lowCD271high cells attached to the collagen scaffold was positively correlated with the number of progenitor cells that survived on the scaffold after 2-week culture (p = 0.0348). Conclusions: An assay for counting CD45lowCD271high cells may provide a useful measurement of bone marrow quality. While the specificity of this measurement of CD45lowCD271high cells remained low in our experimental conditions, CD45lowCD271high cell counts were positively and modestly correlated with the prevalence of CTPs.