Journal of Hematology - Supplements - Haematologica

23
according to the flow cytometric assessment of
the DNA content. By means of this technique,
however, it is not possible to distinguish, among
the cell fraction with a DNA content typical of
the G0/G1 phase, cells which are really quiescent
(G0) from cells which are cycling (G1). Thus, we
took advantage of an anti-statin monoclonal
antibody in order to discriminate between resting
(statin positive) and cycling (statin negative)
cells. We found, in fact, that about 60% of freshly
harvested CB-derived CD34 + cells were resting,
being out of the cell cycle, whereas most of
the remaining progenitor cells was actually
cycling, being in the G1 phase of the cell cycle.
The fact that incubation of progenitor cells in
the presence of FCS for 36 hours was associated
with the progression into the S-phase of a significant
number of both CFC and LTC-IC can be
interpreted considering that these cells entering
the S-phase derive from those in the G1 phase of
the cell cycle. Our data are substantially in agreement
with previous reports which showed, by
means of the evaluation of DNA content by flow
cytometry, that more than 90% of CD34 + cells
derived from CB are in the G0/G1 phase of the cell
cycle. 27-29 However, due to the technical
approach which was used in these studies, the
whole CD34 + cell population rather than the single
types of progenitor cells were evaluated. Most
importantly, we have been able, for the first time,
to discriminate between the proportion of these
cells in the G0 and G1 phase before treatment
with cytokines.
A new observation of our work emerges from
the evaluation of the kinetics of recruitment of
CB-derived progenitor cells into the S-phase of
the cell cycle. To study the kinetics of recruitment
into the S-phase, we incubated the mononuclear
cell fraction derived from CB with a combination
of cytokines and then assessed the proportion
of CFC and LTC-IC entering the S-phase of
the cell cycle after 12, 24 and 36 hours exposure
to these cytokines. In these experimental conditions
we observed that 70% and 81% of CFC and
LTC-IC respectively entered the S-phase of the
cell cycle after 24 hours. Furthermore, more than
90% of both progenitor cells were killed by Ara-
C upon continuous exposure to G-CSF, IL-3 and
SCF (up to 36 hours) suggesting that a proportion
of both CFC and LTC-IC can be rapidly
recruited into the S-phase of the cell cycle in vitro
in the presence of these cytokines. Cytofluorimetric
assessment of the DNA content confirmed
that CB-derived CD34 + cells in the presence
of this combination of cytokines rapidly
start to progress into the S-phase. Interestingly,
our data showing that incubation of CB progenitor
cells in serum-free culture and hematopoietic
growth factors is associated with a more
rapid induction of the cell cycle in comparison to
incubation in the presence of FBS is in keeping
with results reported by Jordan and coworkers 30
who found that serum-free conditions are preferable
for activating quiescent cells.
Taken together our results are in apparent contrast
with parallel experiments performed with
bone marrow (BM)- and peripheral blood (PB)-
derived progenitor cells which showed that a
longer exposure to these cytokines (up to 48-72
hours) is needed to trigger such cells into the
cell cycle. 8 The difference between the kinetics of
recruitment into the cell cycle of CB and adult
progenitor cells can be explained assuming that
the rate of exit from the G0/G1 phase in response
to cytokines is faster for at least a proportion of
CB-derived progenitor cells than for PB- or BMderived
progenitor cells. 27 We do not have a
definitive explanation for this different behavior
but we can formulate two not mutually exclusive
interpretations: i) CB-derived progenitor cells
may respond differently to hematopoietic
growth factors: in fact, it has been shown that
the proliferative response of CB-derived CFC
and LTC-IC to SCF is higher than that of their
PB- or BM- derived counterpart; 29,31,32 ii) at least
a proportion of the progenitor cells rapidly
recruited into the S-phase is derived from those
in the G1 phase of the cell cycle, which represent
a substantial number of CB-derived CD34 + cells,
as shown by our experiments performed with
statin.
We also found that the absolute number of
CFC and LTC-IC after 24 and 36 hours of exposure
to IL-3, SCF and G-CSF (in the absence of
Ara-C) was not statistically different from their
baseline (at the beginning of incubation) number.
Moreover, we found that differentiation of
cultured CD34 + cells, if any, is minimal. In fact,
neither their proportion nor absolute number
changed significantly after 24 hours of incubation
in the presence of growth factors. This suggests
that the combination of cytokines used in
our experiments is not only able to trigger the
cell cycle of CB-derived progenitor cells but also
does not induce a decline in their number (possibly
due to their differentiation or death), at
least for 24 hours. This latter observation could
have a practical implication for example when
CB-derived hematopoietic progenitor cells
should be used as a target for retroviral mediated
gene transfer protocols.
Finally, our results can also be interpreted considering
what has been previously reported by
Gothot et al. 33 who found that in steady-state
bone marrow the turnover of primitive
hematopoietic cells is lower than that of their
committed counterpart and that a direct relationship
exists between the rate of cycling and
haematologica vol. 85(supplement to n. 11):November 2000