Isolation of spleen-colony forming cells (CFU-s) using wheat germ agglutinin and rhodamine 123 labeling.

Mouse pluripotent hemopoietic stem cells could be enriched 100 to 200-fold by a procedure consisting of three steps: 1) equilibrium density centrifugation, 2) light-activated cell sorting on the basis of light scatter characteristics and fluorescence due to wheat germ agglutinin binding, 3) cell sorting after subsequent rhodamine 123 staining. The new isolation procedure does not make use of antibodies with mouse-strain restricted applicability, which were employed in earlier described methods. Therefore, it is more versatile. It is also faster due to diminished incubation time. Rhodamine 123 can also be used as a photosensitizer. The experimental conditions were, however, designed to prevent this action of the dye. Between 80% and 100% of the selected spleen-colony forming cells survived the labeling and sorting treatments. The procedure enriches for two types of stem cells. The rhodamine-dull fraction contains stem cells that form spleen colonies in lethally irradiated mice at 12-16 days and no spleen colonies at 8 days after transplantation. The rhodamine-bright fraction contains stem cells that give day-8 and day-12 spleen colonies. These latter cells, however, have a low radioprotective capacity and it can be argued that these are not self-renewing pluripotent stem cells. The heterogeneity of day-12 CFU-s (colony-forming unit spleen) that can be detected after labeling with rhodamine 123 has been observed earlier after treatment of bone marrow donor mice with 5-fluorouracil, and has led to the postulation of pre-CFU-s and a "generation-age" hypothesis for stem cells. Our presently sorted rhodamine dull cells resemble such pre-CFU-s.