Journal of Hematology - Supplements - Haematologica

haematologica 2000; 85(supplement to n. 11):18-25
original paper
Cord blood-derived hematopoietic progenitor cells: in vitro response
to hematopoietic growth factors and their recruitment into the S-
phase of the cell cycle
VITTORIO ROSTI,* LUCIA MALABARBA,° ISABELLA RAMAJOLI,° SABINA CASULA,° GAETANO BERGAMASCHI,° MARCO DANOVA,°
ROSANGELA INVERNIZZI,° ALESSANDRO PECCI,° LAURA SALVANESCHI, # MARIO CAZZOLA @
*Research Laboratory of Organ Transplantation, Clinical Immunology Unit; °Department of Internal Medicine and Medical
Therapy, Section of Internal Medicine and Medical Oncology; # Department of Immunohaematology and Blood
Transfusion; @ Institute of Hematology; IRCCS Policlinico San Matteo and University of Pavia School of Medicine, Pavia,
Italy
ABSTRACT
Background and Objectives. In the recent years
many studies on the expansion of cord blood (CB)-
derived progenitor cells have been performed,
whereas less information is available on their cycling
status. The objective of this study was to evaluate the
cycling status of CB-derived colony-forming cells
(CFC) and long-term culture-initiating cells (LTC-IC),
and their recruitment into the S-phase of the cell
cycle in response to a combination of cytokines.
Design and Methods. CB-derived CFC and LTC-IC
were first quantified by standard clonogenic assay
and long-term culture, respectively. In a second set
of experiments, CB-derived progenitor cells were
incubated with interleukin(IL)-3, stem cell factor
(SCF) and granulocyte colony-stimulating factor (G-
CSF) and their cell cycle status assessed both by the
cytosine arabinoside (Ara-C) suicide approach and
by flow cytometric DNA analysis.
Results. We found that only small proportions of both
CFC and LTC-IC were in the S-phase of the cell cycle.
These estimates were confirmed by flow cytometric
DNA analysis, which showed that 96%±2% of CBderived
CD34+ cells were in G0/G1 and only
1.6%±0.4% in the S-phase. Staining of CD34+ cells
with an anti-statin monoclonal antibody, a marker of
the G0 phase, indicated that among CD34+ cells with
a flow cytometric DNA content typical of the G0/G1
phase, 68%±7% of cells were in the G0 phase of the
cell cycle. Twenty-four hour incubation with IL-3, SCF
and G-CSF significantly increased the proportion of
cells in the S-phase for both CFC and LTC-IC without
inducing any loss in their number. Flow cytometric
DNA analysis also showed an increase of CD34+
cells in the S-phase upon continuous exposure to
these cytokines.
Interpretations and Conclusions. Our findings indicate
that: i) a small number of CB-derived CFC and
LTC-IC are in the S-phase of the cell cycle; ii) a substantial
number of CD34+ cells with a flow cytometric
DNA content typical of the G0/G1 fraction are
Correspondence: Vittorio Rosti, M.D., Medicina Interna ed Oncologia,
IRCCS Policlinico San Matteo, 2, p.le Golgi; 27100 Pavia, Italy. Phone:
+39.0382.52626 – Fax +39.0382.525222 – E-mail: virosti@tin.it
cycling, as they are found in the G1 phase of the cell
cycle; iii) 24-hour incubation with IL-3, SCF and G-
CSF can drive a proportion of progenitor cells into
the S-phase without reducing their number.
©2000, Ferrata Storti Foundation
Key words: CD34 + , cell cycle, CFC/LTC-IC, statin
In the recent years umbilical cord blood (CB)
has been regarded as an attractive source of
hematopoietic progenitor cells for bone marrow
transplantation. 1 However, its use for this
purpose has been limited mainly to pediatric
patients 2, 3 because of the low number of progenitor
cells which can be detected in each single
unit of CB. 4 As a consequence, over the last few
years, many studies have been carried out with
the aim of establishing a combination of
cytokines which would allow ex vivo expansion of
the more primitive hematopoietic progenitor
cells 5,6 in order to make this source of hematopoietic
progenitor cells available for adult
patients as well. However, no definitive agreement
on a common expansion protocol has so far been
reached. In contrast to the large body of studies
on the expansion of CB progenitor cells, less
information is currently available on the cycling
status of CB-derived hematopoietic progenitor
cells, both committed (colony-forming cells,
CFC) and early (long-term culture-initiating cells,
LTC-IC). The definition of the cycling status of
these progenitor cells can be of interest for different
reasons. For instance, retroviral gene transfer
requires the target cells to be cycling: in this
regard, CB-derived progenitor cells could be an
appealing source of cells for gene transfer protocols,
7 provided they are in the S-phase of the cell
cycle. Should these cells be in a quiescent status,
they would need to be driven into the cell cycle in
order to achieve good efficiency in gene transfer.
Moreover, in recent years the cycling status of
both CFC and LTC-IC derived from different
sources (bone marrow, peripheral blood and
haematologica vol. 85(supplement to n. 11):November 2000