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Primitive human hematopoietic cells displaying differential efflux of the
rhodamine 123 dye have distinct biological activities
N Uchida, J Combs, S Chen, E Zanjani, R Hoffman and A Tsukamoto
SyStemix Inc, Palo Alto, CA 94304, USA.
Human bone marrow (BM) CD34+ cells were stained with the vital dye,
rhodamine 123 (Rh123), and analyzed for their biological properties based
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 peripheral blood
(MPB). Kinetic analysis of the efflux of Rh123 from ABM CD34+ cells showed
that efflux of Rh123 was most rapid from the most primitive Thy-1+ subset.
The efflux of Rh123 could be inhibited by verapamil, suggesting that
rhodamine efflux from primitive hematopoietic 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 after 1 to 2 weeks, only wells plated with CD34+
Thy- 1+Rh123lo (low-level Rh123 retention) or CD34+Thy-1+Rh123mid
(mid-level Rh123 retention) cells maintained greater than 50% of cells in
an uncommitted CD34+33- stage. CD34+Lin- (lineage-negative) cells were
fractionated based on Rh123 dye staining into Rh123hi (high-level Rh123
retention), Rh123mid, and Rh123lo and deposited as single cells into
long-term SyS1 BM stromal cell cultures. The Rh123mid 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, the Rh123lo
fraction proliferated more slowly but sustained long-term in vitro
hematopoiesis as evidenced by continued cobblestone area-forming cells
(CAFC) activity for at least 6 weeks. The Rh123hi fraction showed a plating
efficiency similar to that of the Rh123lo or Rh1123mid fractions but did
not extensively proliferative in vitro and did not show evidence of CAFC
activity. We predicted from these in vitro results that the Rh123lo subsets
possesses long-term engrafting potential. Indeed, on transplantation into
the SCID-hu bone assay, all long-term engrafting potential and multilineage
differentiation potential resided within the Rh123lo-mid but not Rh123hi
subset. Furthermore, human marrow subpopulations derived from chimeric
sheep after in utero transplantation with CD34+Thy-1+Lin- cells were
reisolated based on Rh123 staining. Again, CD34+Lin- subsets showing
Rh123lo-mid had long-term growth in culture, whereas Rh123hiCD34+Lin- cells
did not. These results show that, after injection of CD34+Thy- 1+Lin- cells
into an in utero microenvironment, primitive CD34+ cells maintain a Rh123
phenotype that correlates with their in vitro CAFC activity. Thus, Rh123
staining is an effective way to define functional subsets of primitive
hematopoietic cell populations.
Volume 88,
Issue 4,
pp. 1297-1305,
08/15/1996
Copyright © 1996 by The American Society of Hematology
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