Journal of Hematology - Supplements - Haematologica

22
Table 3. Cell cycle distribution of freshly harvested CD34 +
cells derived from cord blood.
G0 G1 S G2/M
% of CD34+ cells 68.4±7 27.6±2 1.6±0.5 2.4±2
Results are expressed as a mean ± SD of 7 different experiments. G0 cells
are calculated on the basis of the percentage of CD34+ cells expressing the
nuclear protein statin.
Table 4. Cell cycle distribution of CB-derived cultured
CD34+ cells after incubation with IL-3, G-CSF and SCF.*
% G0/G1 % S % G2/M
freshly harvested 95±6 1.8±0.8 3.2±1
6 hours 77.4±8 11±3 11.6±2
12 hours 82.4±6 9.1±2 8.5±3
24 hours 81±5 8.5±3 11.5±4
*CD34+ cells were incubated ia a serum-free medium in the presence of IL-
3, G-CSF (20 ng/mL each factor) and SCF (100 ng/mL) and the DNA content
assessed by cytofluorimetric assay after 6, 12 and 24 hours of incubation.
Results are expressed as a mean ±SD of 3 experiments.
progenitor cells are in the G0/G1 phase (Table 3).
To investigate the cell cycle distribution of freshly
harvested CD34 + cells further, these cells were
stained with the PE-conjugated anti-statin monoclonal
antibody S-44 and the proportion of fluorescent
cells evaluated by means of a FACStar
flow cytometer. Expression of statin, a 57-kDa
nuclear protein, has been recognized as an
unique marker of quiescent cells: 19 the protein is
found in resting (G0) cells and is rapidly downregulated
when cells progress to the G1 phase.
Thus, expression of statin can be used to identify
cells which are in the G0 phase of the cell cycle:
this can be assessed both by immunocytochemistry
and by flow cytometry. Using the latter, we
found that 68.4±7% of freshly harvested CD34 +
cells expressed statin (n=7), indicating that
about 2/3 of CD34 + cells in the G0/G1 region of
the flow cytometric DNA histograms are in the
G0 phase and the remaining are in the G1 phase
of the cell cycle (Table 3). We also assessed the
proportion of CD34 + cells in the different phases
of the cell cycle at different time points (after
6, 12 and 24 hours) during incubation in a
serum-free medium in the presence of IL-3, SCF
and G-CSF. As shown in Table 4 a discrete proportion
of CD34 + cultured cells had rapidly progressed
into the S-phase already after 6 hours of
incubation (11%) and a similar proportion was
also detectable after 12 and 24 hours of incubation
(9.1% and 7.5% respectively). Importantly,
the proportion of CD34 + cells did not change
significantly during the 24-hour culture (97.3±2%
and 95.5±3% at the beginning and after 24 hours
of incubation with growth factors, respectively;
p>0.05) confirming that the changes observed in
the cell cycle distribution were effectively dependent
on the progression through the cell cycle of
CD34 + cells. We also observed an increase in the
absolute number of CD34 + cells after the 24-
hour incubation but it did not reach a statistical
significance. This last observation suggests that
although recruited into the S-phase within 24
hours of culture, CD34+ cells require a longer
time to complete cell division.
Discussion
The objective of this study was to evaluate the
cycling status of CB-derived progenitor cells and
their kinetics of recruitment into the S-phase of
the cell cycle in response to a combination of different
hematopoietic growth factors. We studied
both the committed progenitor cells (CFU-
GEMM, CFU-GM and BFU-E, all together
defined as CFC) and the early progenitor LTC-IC
which up to now are considered the most immature
progenitor cells which can be investigated in
vitro. 25 In fact, whereas the CFC are responsible
for the short-term engraftment of a transplant,
the LTC-IC are thought to represent a more reliable
approximation of the hematopoietic stem
cells responsible for long-term engraftment. The
proportion of cells in the S-phase of the cell cycle
was determined using the Ara-C suicide technique
which has been shown to give reliable and
reproducible results when compared to the 3 H
thymidine incorporation suicide test. 12,14 Our
results show that after 24 hours of liquid culture
in the presence of FCS there was no statistical
difference between the number of CFC and LTC-
IC grown in the presence or absence of Ara-C,
clearly indicating that very few committed and
early CB-derived progenitor cells are in the S-
phase of the cell cycle. The same results were
observed when similar experiments were performed
in serum-free conditions, ruling out the
possibility that the presence of FBS could have
affected the cycling status of the progenitor
cells. 23, 24 Our results on the proportions of CFC
and LTC-IC in the S-phase confirm those of a
recent study in which the 3 H thymidine incorporation
suicide technique was used instead of the
Ara-C suicide technique 26 demonstrating that the
Ara-C approach is comparable to thymidine suicide
for the assessment of the proportion of
hematopoietic progenitor cells in the S-phase of
the cell cycle. These data were further strengthened
by our finding that 96% of the CD34 + cells
derived from CB were found in the G0/G1 phase
of the cell cycle and only 1.6% in the S-phase,
haematologica vol. 85(supplement to n. 11):November 2000