Difference in rhodamine 123 staining between human and murine hematopoietic stem and progenitor cells

Rhodaminel23 (Rhl23) has been extensively used to separate murine hematopoietic stem and progenitor cells. Long term repoptuating pluripotent stem cells in mice were found to be Rhl23 negative. Based on the expérience with mice, we attempted to use Rh 123 to separate human stem and progenitor cells from umbilical cord Mood and mobilized peripheral blood. We used MACS separation as pre-cnrichment step, and then incubated the cells with Rhl23, followed by different post-incubation periods to allow efflux of the dye out of the cell. As final step, cells were stained with antiCDM antibody and analyzed and sorted by FACS. Immediately post incubation, 1-2% of the CD34+ cells became Rhl23 dull, while after 0 min., 40-50% of them were Rh 123 dull. Incubation of the cells in the presence of verapamil resulted in blocking of the efflux, indicative of the presence and function of the P-gp pump. We did not get distinct subpopulations of Rhl23 bright, dull and negative cells in human cells in contrast to mouse cells. Prolonged post-incubation of the cells, up to 3 hours, resulted in shift of RM23 bright to dull, but without clear distinction between the populations. However, in extended long-term human cultures on stroma, sorted Rhl23 most negative cells still showed a high incidence of cobblestone area forming il (CAFQ after 13 weeks. Rhl23 bright cells only formed cobblestone areas for 4 weeks, and Rh 123 dull cells for up to 8 weeks. In the light of these results we speculate that differences in Rbl23 binding to mitochondria, and/or differences in the function of the P-gp between human and mouse, are responsible for the difference in staining patterns. In spite of these differences, the CAFC assay indicates that also in human, Rhl23 may be of use to separate primitive and more committed stem cells.