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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4098-4107
Influence of Cytokines on the Growth Kinetics and Immunophenotype
of Daughter Cells Resulting From the First Division of Single
CD34+Thy-1+lin Cells
Julie P. Goff,
Donna S. Shields, and
Joel S. Greenberger
From the Department of Radiation Oncology, University of Pittsburgh
and University of Pittsburgh Cancer Institute, Pittsburgh, PA.
There is a need to determine whether culture conditions may exist
for ex vivo expansion of hematopoeitic stem cells (HSC), which favor
solely proliferative self-renewal of HSC as opposed to proliferation
with differentiation. Using single cells, we studied the effects of
individual and combinations of cytokines in serum-free medium on the
kinetics of the first cell doubling and the resulting phenotype of each
of individual daughter cell. CD34+Thy-1+lin
cells were plated 1 cell per well in Terasaki plates in
serum-free medium containing cytokines. Each well containing a single
cell was monitored daily over 7 days for maintenance, division, or death. When division occurred in an individual well, the phenotype of
the daughter cells was determined by staining with anti-CD34 fluorescein isothiocyanate (FITC)- and phycoerythrin (PE)-conjugated lineage specific antibodies. The cumulative percent of wells with an
undivided single cell, wells in which the cell had divided, and wells
in which the cell had died were scored. The number of doublets with
conserved phenotype (CD34+lin) was
compared to those wells with one or more differentiated daughter cells
(CD34+lin+). Over 7 days, cells cultured in
single factors showed that between 13% (interleukin-6 [IL-6]) and
29% (thrombopoietin [TPO]) of the cells were undivided, between 13%
(IL-1) and 35% (TPO) of the cells doubled, and between 35% (TPO) and
greater than 60% (IL-11, IL-1, or hepatocyte growth factor
[HGF]) died. When combinations of cytokines were used
over 7 days, between 5% (FLT-3 ligand [FLT-3L], stem
cell factor [SCF], IL-3, IL-6, granulocyte
colony-stimulating factor [G-CSF],  nerve growth factor
[NGF]) and 22% (FLT-3L + HGF) of the cells
remained undivided, between 15% (HGF, IL-1, IL-11, G-CSF) and 68%
(SCF + TPO) of the cells had doubled and between 27% (FLT-3L + TPO) and 70% (HGF, IL-1, IL-11, G-CSF) died. The combination of FLT-3L + TPO induced the highest total percent (64.6%) of cells with
conserved phenotype (percent conserved doublets + percent with 1 cell
conserved), followed by SCF + TPO, (50%) and the combination of
FLT-3L, SCF, IL-3, IL-6, G-CSF, NGF (53%). These combinations also
produced the highest yield of cells with conserved phenotype after one
division (FLT-3L + TPO  81 cells/100 initial cells, SCF + TPO 68 cells/100 initial cells) (P = .01). Observation of the
time of the initial cell division and phenotype of the daughter cells
allowed us to identify candidate combinations of cytokines that promote
maintenance of lin cells (TPO), or recruit the primitive
cells to divide and undergo phenotypic self-renewal (FLT-3L + TPO,
SCF + TPO).
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