Enrichment of murine hemopoietic clonogenic cells by multivariate analyses and sorting

Multivariate analyses, dual beam flow cytometry and sorting, and list mode data processing were used to distinguish and enrich committed and pluripotent stem cells in mouse bone marrow. These cells were discriminated on the basis of their forward angle and perpendicular light scatter characteristics and their Hoechst 33342 fluorescence intensity. Myeloid committed progenitors (CFU-GM) and spleen colony-forming units (CFU-S) (day 9 and day 13) were enriched 100-fold by sorting on the basis of high forward angle light scatter, intermediate perpendicular light scatter, and very low HO fluorescence intensity. Approximately 10% of the sorted cells formed colonies in the CFU-GM assay and 2% formed CFU-S colonies. Morphologic analysis of the sorted subpopulation revealed 92% blast immature cell types. The DNA distribution of the sorted subpopulation, assessed by propidium iodide staining, indicated that 98% of the progenitor-enriched subpopulations contained 2N DNA content. This separation procedure offers a simple method to obtain preparations highly enriched in clonogenic cells in one pass through the cell sorter.