Isolation of a mouse bone marrow population enriched in stem and progenitor cells by centrifugation on a Percoll...

Given the complex composition of bone marrow, a cell separation technique that results in populations enriched in progenitor cells is required for cellular differentiation and transplantation studies. In the present study, we designed a method that allows for the isolation of a progenitor-enriched population of bone marrow by exploiting the physical properties of these cells. Bone marrow aspirate was separated on a discontinuous Percoll gradient (ranging from 1.050 to 1.083 g/cm3) that resulted in the recovery of six cell fractions. The fractions were characterized by FACS and RT-PCR (reverse transcription-PCR) analyses and evaluated for their capacity to differentiate into haematopoietic and mesenchymal cells. Fraction IV, including cells with a density of 1.070-1.076 g/ml, contained 11.68% of total bone marrow cells and was enriched in c-kit+ and Sca-1+ (stem cell antigen-1) progenitor cells as compared with total bone marrow. This fraction demonstrated an increase in clonogenic capacity under specific conditions as well as a potential to generate a mesenchymal stem cell culture in a shorter period than that using bone marrow aspirate. Furthermore, this fraction lacked differentiated cell types and contained cells positive for endothelial markers, which further increases its value in cellular transplant. In conclusion, a bone marrow subpopulation that is enriched in progenitor cells and may be valuable in cellular transplant therapy can be isolated by exploiting the physical properties of these cells.