The Resolution, Enrichment, and Organization of Normal Bone Marrow High Proliferative Potential Colony-forming Cell Subsets on the Basis of Rhodamine-123 Fluorescence

Cell sorting on the basis of rhodamine-123 (Rh123) fluorescence has been used in conjunction with negative immunomagnetic selection to analyze the high proliferative potential colony-forming cell (HPP-CFC) compartment of normal murine bone marrow and to resolve and enrich HPP-CFC subpopulations responsive to different combinations of the hemopoietic growth factors interleukin 1 alpha (IL-1 alpha), interleukin-3 (IL-3), and colony-stimulating factor 1 (CSF-1). HPP-CFC with a specific requirement for IL-1 alpha plus IL-3 plus CSF-1 in order to proliferate were resolved and enriched on the basis of their low Rh123 retention (Rh-dull), whereas HPP-CFC that grew in the presence of IL-3 plus CSF-1, IL-3 alone, or CSF-1 alone were Rh-bright. Further addition of IL-1 alpha to IL-3 plus CSF-1 stimulated few additional HPP-CFC in the Rh-bright fraction. Our data confirm the value of Rh123 as a probe for the dissection and analysis of the primitive hemopoietic stem cell (PHSC) compartment. These data also show that the Rh123 staining characteristics of IL-1 alpha plus IL-3 plus CSF-1-responsive HPP-CFC are consistent with the hypothesis that these HPP-CFC are closely related to PHSC with long-term reconstituting capacity in vivo and that they are among the most primitive progenitors yet detected in clonal agar culture.