PRIMITIVE HUMAN HEMATOPOIETIC-CELLS DISPLAYING DIFFERENTIAL EFFLUX OFTHE RHODAMINE-123 DYE HAVE DISTINCT BIOLOGICAL-ACTIVITIES

Human bone marrow (BM) CD34(+) cells were stained with the vital dye, rhodamine 123 (Rh123), and analyzed for their biological properties ba sed on the level of dye retention. Heterogeneous rhodamine staining is seen within the CD34(+) population, and the staining patterns differ dramatically between fetal BM (FBM), adult BM (ABM) and mobilized peri pheral blood (MPB). Kinetic analysis of the efflux of Rh123 from ABM C D34(+) cells showed that efflux of Rh123 was most rapid from the most primitive Thy-1(+) subset. The efflux of Rh123 could be inhibited by v erapamil, suggesting that rhodamine efflux from primitive hematopoieti c cells is primarily due to the P-glycoprotein (P-gp) pump or another intracellular transport system affected by verapamil. When four CD34() subpopulations were plated onto SyS1 BM stromal cell cocultures afte r 1 to 2 weeks, only wells plated with CD34(+)Thy-1(+)Rh123(lo) (low-l evel Rh123 retention) or CD34(+)Thy-1(+)Rh123(mid) (mid-level Rh123 re tention) cells maintained greater than 50% of cells in an uncommitted CD34(+)33(-) stage. CD34(+)Lin(-) (lineage-negative) cells were fracti onated based on Rh123 dye staining into Rh123(hi) (high-level Rh123 re tention), Rh123(mid), and Rh123(lo) and deposited as single cells into long-term SyS1 BM stromal cell cultures. The Rh123(mid) fraction had immense early proliferative activity in vitro, but lost the ability to form cobblestone areas after 5 to 6 weeks in culture. In contrast, th e Rh123(lo) fraction proliferated more slowly but sustained long-term in vitro hematopoiesis as evidenced by continued cobblestone area-form ing cells (CAFC) activity for at least 6 weeks. The Rh123(hi) fraction showed a plating efficiency similar to that of the Rh123(lo) or Rh123 (mid) fractions but did not extensively proliferate in vitro and did n ot show evidence of CAFC activity, We predicted from these in vitro re sults that the Rh123(lo) subset possesses long-term engrafting potenti al. Indeed, on transplantation into the SCID-hu bone assay, all long-t erm engrafting potential and multilineage differentiation potential re sided within the Rh1231(lo-mid) but not Rh123(hi) subset. Furthermore, human marrow subpopulations derived from chimeric sheep after in uter o transplantation with CD34(+)Thy-1(+)Lin(-) cells were reisolated bas ed on Rh123 staining. Again, CD34(+)Lin(-) subsets showing Rh123(lo-mi d) had long-term growth in culture, whereas Rh123(hi)CD34(+)Lin(-) cel ls did not. These results show that, after injection of CD34(+)Thy-1()Lin(-) cells into an in utero microenvironment, primitive CD34(+) cel ls maintain a Rh123 phenotype that correlates with their in vitro CAFC activity. Thus, Rh123 staining is an effective way to define function al subsets of primitive hematopoietic cell populations. (C) 1996 by Th e American Society of Hematology.