Optimal ex vivo expansion of neutrophils from PBSC CD34+ cells by a combination of SCF, Flt3-L and G-CSF and its inhibition by further addition of TPO.

Abstract

Autologous mobilised peripheral blood stem cell (PBSC) transplantation is now a standard approach in the treatment of haematological diseases to reconstitute haematopoiesis following myeloablative chemotherapy. However, there remains a period of severe neutropenia and thrombocytopenia before haematopoietic reconstitution is achieved. Ex vivo expanded PBSC have been employed as an adjunct to unmanipulated HSC transplantation, but have tended to be produced using complex cytokine mixtures aimed at multilineage (neutrophil and megakaryocyte) progenitor expansion. These have been reported to reduce or abrogate neutropenia but have little major effect on thrombocytopenia. Selective megakaryocyte expansion has been to date ineffective in reducing thrombocytopenia. This study was implemented to evaluate neutrophil specific rather than multilineage ex vivo expansion of PBSC for specifically focusing on reduction or abrogation of neutropenia.
CD34+ cells (PBSC) were enriched from peripheral blood mononuclear cells following G-CSF-mobilisation and cultured with different permutations of cytokines to determine optimal cytokine combinations and doses for expansion and functional differentiation and maturation of neutrophils and their progenitors. Results were assessed by cell number, morphology, phenotype and function.
A simple cytokine combination, SCF + Flt3-L + G-CSF, synergised to optimally expand and mature neutrophil progenitors assessed by cell number, phenotype, morphology and function (superoxide respiratory burst measured by chemiluminescence). G-CSF appears mandatory for functional maturation. Addition of other commonly employed cytokines, IL-3 and IL-6, had no demonstrable additive effect on numbers or function compared to this optimal combination. Addition of TPO, commonly included in multilineage progenitor expansion for development of megakaryocytes, reduced the maturation of neutrophil progenitors as assessed by number, morphology and function (respiratory burst activity).
Given that platelet transfusion support is available for autologous PBSC transplantation but granulocyte transfusion is generally lacking, and that multilineage expanded PBSC do not reduce thrombocytopenia, we suggest that instead of multilineage expansion selective neutrophil expansion based on this relatively simple cytokine combination might be prioritized for development for clinical use as an adjunct to unmanipulated PBSC transplantation to reduce or abrogate post-transplant neutropenia.

Full text

Page 1

BioMed Central
Page 1 of 11
(page number not for citation purposes)
Journal of Translational Medicine
Open Access
Research
Optimal ex vivo expansion of neutrophils from PBSC CD34+ cells by
a combination of SCF, Flt3-L and G-CSF and its inhibition by further
addition of TPO
Olga Tura1, G Robin Barclay*1, Huw Roddie2, John Davies2 and
Marc L Turner1
Address: 1SNBTS Adult Cell Therapy Group, Scottish Centre for Regenerative Medicine, University of Edinburgh School of Clinical Sciences, The
Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK and 2NHS Lothian University Hospitals Division, Department of
Haematology, Western General Hospital, Edinburgh EH4 2XU, UK
Email: Olga Tura - olga.tura@ed.ac.uk; G Robin Barclay* - robin.barclay@ed.ac.uk; Huw Roddie - huw.roddie@luht.scot.nhs.uk;
John Davies - john.davies@luht.scot.nhs.uk; Marc L Turner - marc.turner@snbts.csa.scot.nhs.uk
* Corresponding author
Abstract
Background: Autologous mobilised peripheral blood stem cell (PBSC) transplantation is now a standard approach in the
treatment of haematological diseases to reconstitute haematopoiesis following myeloablative chemotherapy. However, there
remains a period of severe neutropenia and thrombocytopenia before haematopoietic reconstitution is achieved. Ex vivo
expanded PBSC have been employed as an adjunct to unmanipulated HSC transplantation, but have tended to be produced using
complex cytokine mixtures aimed at multilineage (neutrophil and megakaryocyte) progenitor expansion. These have been
reported to reduce or abrogate neutropenia but have little major effect on thrombocytopenia. Selective megakaryocyte
expansion has been to date ineffective in reducing thrombocytopenia. This study was implemented to evaluate neutrophil
specific rather than multilineage ex vivo expansion of PBSC for specifically focusing on reduction or abrogation of neutropenia.
Methods: CD34+ cells (PBSC) were enriched from peripheral blood mononuclear cells following G-CSF-mobilisation and
cultured with different permutations of cytokines to determine optimal cytokine combinations and doses for expansion and
functional differentiation and maturation of neutrophils and their progenitors. Results were assessed by cell number,
morphology, phenotype and function.
Results: A simple cytokine combination, SCF + Flt3-L + G-CSF, synergised to optimally expand and mature neutrophil
progenitors assessed by cell number, phenotype, morphology and function (superoxide respiratory burst measured by
chemiluminescence). G-CSF appears mandatory for functional maturation. Addition of other commonly employed cytokines, IL-
3 and IL-6, had no demonstrable additive effect on numbers or function compared to this optimal combination. Addition of TPO,
commonly included in multilineage progenitor expansion for development of megakaryocytes, reduced the maturation of
neutrophil progenitors as assessed by number, morphology and function (respiratory burst activity).
Conclusion: Given that platelet transfusion support is available for autologous PBSC transplantation but granulocyte
transfusion is generally lacking, and that multilineage expanded PBSC do not reduce thrombocytopenia, we suggest that instead
of multilineage expansion selective neutrophil expansion based on this relatively simple cytokine combination might be
prioritized for development for clinical use as an adjunct to unmanipulated PBSC transplantation to reduce or abrogate post-
transplant neutropenia.
Published: 30 October 2007
Journal of Translational Medicine 2007, 5:53doi:10.1186/1479-5876-5-53
Received: 11 July 2007
Accepted: 30 October 2007
This article is available from: http://www.translational-medicine.com/content/5/1/53
© 2007 Tura et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Page 2

Journal of Translational Medicine 2007, 5:53http://www.translational-medicine.com/content/5/1/53
Page 2 of 11
(page number not for citation purposes)
Background
Restoration of haematopoiesis by autologous transplanta-
tion of haematopoietic stem cells (HSC) following myelo-
ablative chemotherapy has become standard treatment
for a number of malignant disorders. Use of cytokine
mobilised peripheral blood stem cells (PBSC) has gener-
ally reduced the period of post transplant neutropenia
and thrombocytopenia compared to use of bone marrow
HSC. Identification of mobilised PBSC by CD34+ expres-
sion and collection by leukapheresis has demonstrated
that the period of neutropenia and thrombocytopenia
may be shortened by increasing the dose of CD34+ cells
transplanted. However there still remains a period of clin-
ically significant neutropenia and thrombocytopenia
which cannot be reduced by increasing CD34+ cell doses.
This is probably related to the minimum time required for
adequate post transplant expansion and maturation of
relevant HSC in vivo. Several groups have therefore inves-
tigated ex vivo expansion of PBSC prior to transplantation,
to attempt to further reduce or abrogate post transplant
neutropenia and thrombocytopenia, and which has been
the subject of a number of recent commentaries and
reviews[1-5].
CD34+ cells are heterogeneous and include primitive
multipotent stem cells and more mature lineage-commit-
ted haematopoietic progenitors. When purified they can,
by themselves, restore haematopoiesis and therefore con-
tain all necessary cell types although these cannot readily
be discriminated by phenotype[6-9]. The availability of
recombinant cytokines has allowed investigation of the
role of different cytokines in driving proliferation and
maturation of CD34+ cells with different haematopoietic
potential, and investigation of the use of different combi-
nations of cytokines for expansion of HSC for different
clinical objectives. To date, none of these ex vivo protocols
has been adopted for routine clinical use although some
have demonstrated clinical potential, especially with
regard to reduction of neutropenia[10-15]. Most do not
compare favourably on a cost-benefit basis to conven-
tional support for HSC transplantation such as transfu-
sion of blood or blood components. However, support
for neutropenia by allogeneic donor granulocyte transfu-
sion is not routinely available[16-19], unlike platelet
transfusion support for thrombocytopenia, and neutro-
penic patients remain at risk from life-threatening infec-
tion. In this context it may be informative to examine
specific expansion of neutrophil precursors from autolo-
gous PBSC, as an intended adjunct to unmanipulated
autologous PBSC transplantation, to determine whether a
simple cytokine combination might achieve this when
expansion of other HSC elements such as long term
repopulating cells or megakaryocyte precursors is not
required.
A number of studies have examined ex vivo expansion of
the more mature progenitor component of PBSC thought
to be responsible for short term reconstitution of neu-
trophils and platelets, where such manipulated cells
would be given together with standard unmanipulated
PBSC which ensure long term sustained haematopoiesis.
In most cases the ex vivo protocols have aimed to achieve
simultaneous expansion of both neutrophil and meg-
akaryocyte precursors to address the dual problems of
neutropenia and thrombocytopenia. In many such
instances the combinations of cytokines used are com-
plex, and often there is no systematic substantiation of the
requirement for each cytokine in the protocol. The sim-
plest protocols aimed at simultaneous neutrophil and
megakaryocyte precursor expansion from mobilised
autologous CD34+ cells have employed only three
cytokines, namely stem cell factor (SCF) as a proliferative
stimulus together with granulocyte colony stimulating
factor (G-CSF) to drive neutrophil maturation and throm-
bopoietin (TPO) to drive megakaryocyte maturation[10-
13,15]. These ex vivo expanded PBSC have reduced neutro-
penia when used to supplement unmanipulated autolo-
gous PBSC infusions, but with few exceptions have not
demonstrated any significant effect on thrombocytope-
nia. More complex general expansion protocols, incorpo-
rating or derived from protocols intended to expand
multipotent long-term reconstituting HSC, have not been
tested clinically. Expansion protocols aimed specifically
only at megakaryocyte generation are safe but have so far
not achieved significant reduction of thrombocytopenia
when applied clinically[20-22]. It therefore seems that
while reduction of thrombocytopenia remains elusive for
autologous PBSC engraftment, ex vivo expanded PBSC as
a supplement to unmanipulated PBSC might provide fea-
sible reduction of neutropenia if convenient and cost
effective expansion protocols can be determined.
We have investigated optimal cytokine combinations for
ex vivo expansion of neutrophil precursors by examining a
group of cytokines employed in a number of reports of ex
vivo expansion of PBSC. We initially examined cytokines
alone and in combination for maximal proliferative
expansion of PBSC CD34+ cells, then we examined the
supplementary cytokine combinations required for matu-
ration of the expanded cells by assessing expression of
aspects of neutrophil morphology, phenotype and func-
tion.
Methods
Purification of CD34+ cells
Venous blood samples (10 mL) rich in HSC were collected
in heparin from patients immediately following cell-sepa-
rator leukapheresis collection of G-CSF mobilised PBSC
for autologous transplant. Mononuclear cells were sepa-
rated by buoyant density centrifugation over Ficoll-Histo-

Page 3

Journal of Translational Medicine 2007, 5:53http://www.translational-medicine.com/content/5/1/53
Page 3 of 11
(page number not for citation purposes)
paque (1.077 g/ml; Sigma Diagnostics, UK) and washed
twice in phosphate buffered saline (PBS), and counted.
The CD34+ fraction was enriched using either negative
selection (Rosette-Sep, Stem Cell Technologies, UK) or
positive selection (MACS CD34 isolation Kit; Miltenyi
Biotec, Surrey, UK), according to the manufacturers
instructions. The efficiency of the purification of CD34+
cells was verified by flow cytometry phenotyping analysis.
Flow cytometry analysis
Whole blood, CD34+ enriched cells, or cells after culture
expansion were phenotyped by flow cytometry. Cells were
stained with PerCP-conjugated anti-human CD45 and PE-
conjugated anti-human CD34, fixed and gated for CD45+
CD34+ cells with a low side scatter, according to the
ISHAGE CD34 enumeration protocol[23]. Unmanipu-
lated and cultured cells were also analysed for expression
of surface markers specific for subpopulations of haemat-
opoietic cells. The MAbs included anti-CD34-PE and
CD41-FITC (Becton Dickinson, Oxford, UK) and anti-
CD16b-FITC (Immunotech, UK). Appropriate isotype
negative controls were compared with unstained samples,
and since these did not differ unstained samples were
used to establish positive stain boundaries. 50–100 ul of
the sample were stained with the appropriate antibodies
for 30 minutes in the dark, any erythrocytes were lysed,
and cells were washed with PBS at 200 g. The cells were
resuspended in Cell Fix (Becton Dickenson) and events
were collected by a FACS Calibur System equipped with
CellQuest software (Becton Dickenson) and analysed
offline using FCS Express software (De Novo Soft-
ware[24])
MPO intracytoplasmic stain
Anti-myeloperoxidase, MPO-7 MAb (Dako Cytomation
Ltd, Cambridgeshire, UK) strongly labels the cytoplasm of
mature and immature neutrophils. Monocytes are weakly
positive while eosinophils are unreactive. 50 ul of sample
was stained with anti-CD34-PE and anti-CD45-PerCP, as
above. The sample was fixed using 100 ul DAKO
Intrastain Reagent A (fixation), and incubated and
washed with PBS. 100 ul of the DAKO Intrastain Reagent
B (permeabilisation) and 5–10 ul of the anti-MPO-FITC
cytoplasmic antibody was added, mixed, washed and
resuspended in an appropriate fluid for flow cytometry
analysis.
Ex vivo expansion cultures
Iscove's modified Dulbecco's medium (IMDM) (Invitro-
gen, Paisley, UK) supplemented with 10% FCS (Sigma,
UK) and 1% antibiotic (Pen/Strep, Invitrogen) was used
for cultures. CD34+-enriched PBSC (2 × 105 total cells per
ml) were plated in 24-well plates in 1 ml medium with or
without various cytokines. The following recombinant
purified human cytokines were used in these studies: flt-3
ligand (Flt3-L), stem cell factor (SCF), interleukin 3 (IL-3),
interleukin 6 (IL-6) and thrombopoietin (TPO) from
PeproTech Ec Ltd, London, UK. Recombinant human
granulocyte colony-stimulating factor (G-CSF; lenogras-
tim) was a gift from Chugai Pharma, UK. Cells were cul-
tured in 100 ng/ml SCF, 10 ng/ml Flt3-L unless otherwise
stated, with or without 100 ng/ml G-CSF and with or
without 100 ng/ml TPO. Cells were cultured in a fully
humidified atmosphere of 5%CO2 in air, at 37C for 14
days. Following vigorous pipetting aliquots were removed
for cell counts, differential morphology, flow cytometry
analysis and colony assays. CD34+ numbers were calcu-
lated from total cell numbers and CD34+ proportions:
CD34+ expansion was expressed as a "fold" expansion
over starting CD34+ numbers, where fold expansion is the
final number of cells after expansion divided by the initial
CD34+ numbers before culture.
Giemsa staining and morphological analysis
The morphology of the cells was determined using a
standard Wright-Giemsa-stained cytospin preparations
(Thermo Shandon, UK). Neutrophils were scored accord-
ing to their maturity appearance on advice from experi-
enced clinical haematologists, and was principally based
on the complexity of their nuclear morphology.
Neutrophil chemiluminescence assay
Bioluminescence assays were carried out to measure the
superoxide respiratory burst activity of CD34+ derived
cells. This was measured by detecting luminol-amplified
chemiluminescence responses to phorbol myristate ace-
tate (PMA) (Sigma). In a 96-well opaque microplates, 100
ul of isolated mature peripheral blood neutrophils or
expanded cultured progenitors at 0.5 × 106 cells/ml in
IMDM without phenol red and without FCS were plated
with 100 ul of luminol (1 mM) (Sigma) and 100 ul of
PMA (1 ug/ml final concentration), and the emitted light
activity (relative light units, rlu) was measured on a micro-
plate luminometer (Labsystems Luminoskan) at 37C
every 3 minutes over 90 minutes.
Colony assays
Colony forming cells (CFC) were monitored by their
growth in methylcellulose (MC) medium. 50 ul of cells
were plated at 1.5 × 104 cells in 500 ul of MC containing
cytokines (MethoCult H4435; Stem Cell Technologies,
UK). Cells were plated in 24 well-plates. Colonies (groups
of >100 cells) formed were scored according to appear-
ance and counted after 14 days. Due to low cell numbers
of CD34+ enriched cells (from 10 mL blood samples), day
0 CFC were measured on mononuclear cell isolates before
CD34+ enrichment whereas day 14 CFC were measured
on cultured CD34+ enriched cells, which by day 14 of cul-
ture are all CD34+ in the cytokine combinations studied.
The results are expressed for each colony type as a percent

Page 4

Journal of Translational Medicine 2007, 5:53http://www.translational-medicine.com/content/5/1/53
Page 4 of 11
(page number not for citation purposes)
of total CFC and are intended for qualitative interpreta-
tion, but for approximate quantitative guidance are also
given as colony numbers per thousand cells with CD34+
cell proportions and fold increase.
Results
CD34+ cell enrichment
CD34+ cells were in general enriched to a purity of around
15–30% using the Rosette-Sep (negative-selection)
method or 60–90% using the Miltenyi (positive-selec-
tion) method. Better total yields of CD34+ cells were
obtained with the negative-selection method which was
used in most cases, but similar outcomes were obtained
with CD34+ cells enriched by the positive selection
method and these were included in the results.
PBSC expansion
We examined doses and combinations of the cytokines to
expand CD34+ PBSC in 14-day cultures. Initially SCF and
Flt3-L were examined alone and in combination (Figure
1a &1b). SCF and Flt3-L, each at 100 ng/ml, gave similar
expansion when used alone, but appeared to synergise
when used in combination in that the expansion (up to
over 20-fold in some cases) exceeded the summed expan-
sion with either alone. In general the combination of SCF
(100 ng/ml) with 10 ng/ml Flt3-L was superior to the
combination of SCF with 100 ng/ml Flt3-L (Fig 1b), but
this did not reach significance (p = 0.156, Wilcoxon
signed rank test) in the five patients studied. The combi-
nation of 100 ng/ml SCF with 10 ng/ml Flt3-L was
adopted for further studies with other cytokines.
Increase in CD34+ cells in 14-day cultures with different doses of SCF, Flt3-L and G-CSF alone and in combination
Figure 1
Increase in CD34+ cells in 14-day cultures with different doses of SCF, Flt3-L and G-CSF alone and in combina-
tion. (a) Responses of a representative patient's CD34+ cells to different combinations of SCF and Flt3-L showing the greatest
increase with the combination of 100 ng/ml SCF and 10 ng/ml Flt3-L. (b) Responses of five different patients showing that in
general 10 ng/ml Flt3-L gives a greater response than 100 ng/ml Flt3-L in combination with 100 ng/ml SCF. (c) Responses of a
representative patient's CD34+ cells to 100 ng/ml SCF + 10 ng/ml Flt3-L added at day zero, alone or with 100 ng/ml G-CSF
added at day zero or day 7. (d) Range of five patients' responses to 100 ng/ml SCF + 10 ng/ml Flt3-L + 100 ng/ml G-CSF (these
are a different five patients from those shown in b).
no SCF 10 ng/ml SCF 100 ng/ml SCF
0
5
10
15
20
25
10 ng/ml Flt3-L
100 ng/ml Flt3-L
no Flt3-L
CD34+ fold increase
0 10 100
0
5
10
15
20
25
30
35
SCF/Flt3-L (GCSF on day 0)
SCF/Flt3-L (GCSF on day 7)
no SCF/Flt3-L
ng/ml GCSF
CD34+ fold increase
100 ng/ml SCF
10 ng/ml Flt3-L
100 ng/ml GCSF (day 0)
(a)
(c)
(d)
No cytokines
100 SCF
100 SCF 10 Flt3-L
100 SCF 100 Flt3-L
(b)

Page 5

Journal of Translational Medicine 2007, 5:53http://www.translational-medicine.com/content/5/1/53
Page 5 of 11
(page number not for citation purposes)
In a series of tests, 100 ng/ml G-CSF was superior to 10
ng/ml G-CSF (not shown). Addition of 100 ng/ml of G-
CSF to the SCF/Flt3-L combination gave a further syner-
gistic expansion of the enriched CD34+ cells (to up to over
30-fold in some cases) (Figure 1c and 1d), whereas G-CSF
alone showed low expansion potential. The addition of G-
CSF to the SCF/Flt3-L combination at the start of cultures
(day 0) was in general superior to the addition of G-CSF
midway (day 7) through cultures, which implies that G-
CSF synergises with SCF/Flt3-L during early proliferative
expansion rather than acting only to differentiate and
mature cells expanded by SCF/Flt3-L alone. In 9 patients
where SCF/Flt3-L alone or in combination with G-CSF at
day 0 has been tested there is a wide range of expansion
responses (3 to 34 fold for the latter combination), but in
all cases there was superior expansion with SCF/Flt3-L/G-
CSF than with SCF/Flt3-L (p = 0.0019, Wilcoxon signed
rank test). The comparison of expansion by addition of G-
CSF at either day 0 or day 7 of the 14 day cultures did not
reach significance (p = 0.062, Wilcoxon signed rank test).
Other cytokines which were tested in combination with
optimal SCF/Flt3-L/G-CSF were IL-3, IL-6 and TPO. Either
IL-3 or IL-6 alone slightly lowered the optimal SCF/Flt3-L/
G-CSF expansion, but not significantly, whereas together
they slightly enhanced the optimal SCF/Flt3-L/G-CSF
expansion, but not significantly (not shown). Since these
effects were not overt, and for the sake of simplicity for
any implementation of clinical protocols for cell expan-
sion, we decided that inclusion of IL-3 and IL-6 gave no
worthwhile advantage over SCF/Flt3-L/G-CSF alone for
generation of neutrophil progenitors. TPO in combina-
tion with SCF/Flt3-L/G-CSF slightly reduced the expan-
sion seen with SCF/Flt3-L/G-CSF alone, presented below.
Phenotypes of expanded cells
After 14 days stimulation with SCF/Flt3-L, all cells stained
with anti-CD34 (Figure 2a). The majority of cells stained
to medium fluorescence intensity, with a small popula-
tion of CD34-bright cells. When G-CSF was added to SCF/
Flt3-L at day 0 there was a dimming of the major popula-
tion medium-fluorescence-intensity CD34-staining peak,
and disappearance of the bright CD34+ population (solid
line, b). When G-CSF was added to SCF/Flt3-L at day 7
there was a similar dimming of the major population
medium-fluorescence-intensity
which appeared less dimmed, and retention of the bright
CD34+ population (dotted line, b), which implied the
effect is less than addition of G-CSF at day 0. In no case
were there any obvious surviving CD34-negative cells at
14 days, except when G-CSF was used alone without SCF/
Flt3-L (not shown because of very low cell numbers, not
suitable for histogram representation).
CD34-staining peak
We used expression of CD16b as a non-labile neutrophil-
specific marker. Cells cultured with SCF/Flt3-L/G-CSF
showed a small shift up in CD16b staining compared to
unstained and isotype controls or compared to cells cul-
tured with SCF/Flt3-L alone, but all were dim compared to
mature peripheral blood neutrophils which in our hands
stain brightly with CD16b (not shown). There was a
major shift up in intracytoplasmic myeloperoxidase
expression in cells cultured with SCF/Flt3-L/G-CSF com-
pared to cells cultured with SCF/Flt3-L alone (not shown).
Morphology
CD34+ cells expanded with SCF/Flt3-L alone and stained
in Wright-Giemsa remained morphologically undifferen-
tiated, with no clear indication of development of granu-
locytic morphology. When G-CSF was added there was an
extensive development of neutrophil morphology. Seg-
mented cells in different stages of neutrophil maturation
were found and around 80% of the cells were in a quite
mature stage with apparent nuclei segmentation, lobula-
tion, and granularity in the cytoplasm (see below under
Effects of TPO where these studies were repeated with/
without addition of TPO).
Colony assays
Methocult colony assays were carried out for some PBSC
blood samples on cells before culture (day 0) and at day
14 following cytokine expansion culture (Table 1).
Because of limited cell numbers following CD34+ cell
Change in CD34 expression of cultured cells
Figure 2
Change in CD34 expression of cultured cells. Anti-
CD34-PE staining of 14-day cultured cells stimulated with
100 ng/ml SCF + 10 ng/ml Flt3-L with or without 100 ng/ml
G-CSF. (a) SCF + Flt3-L alone; (b) SCF + Flt3-L with G-CSF
(solid line = day 0; dotted line = day 7); (c) no anti-CD34,
isotype control (for SCF + Flt3-L alone, but controls for the
other cultures show overlapping peaks). Gated on intact cells
(forward/side scatter) and CD45 staining.

Page 6

Journal of Translational Medicine 2007, 5:53http://www.translational-medicine.com/content/5/1/53
Page 6 of 11
(page number not for citation purposes)
enrichment, phenotype, but not CFC assays, were carried
out on CD34+ enriched cells before culture. Instead pre-
expansion CFC assays were performed on the mononu-
clear cells before CD34+ enrichment. We therefore can
only directly judge the qualitative results (CFC frequen-
cies), but have included available data as some indirect
quantitative guidance. Before expansion, the predomi-
nant colonies generated were erythroid BFU-E, but after
culture with SCF/Flt3-L or SCF/Flt3-L + G-CSF the cells
showed a pronounced shift to myeloid commitment.
There was a marked shift towards CFU-GM and away from
BFU-E in all expanded cultures. CFU-GM were consist-
ently higher and BFU-E lower in cultures with G-CSF com-
pared to SCF/Flt3-L alone, but the switch to CFU-GM with
SCF/Flt3-L alone compared to unexpanded cells is almost
as large as that with SCF/Flt3-L + G-CSF, and may indicate
that most expanded cells are myeloid committed, even
without addition of G-CSF.
Neutrophil superoxide activity
An important index of neutrophil function is the produc-
tion of superoxide radicals upon activation, which we
measured here by detecting luminol-amplified chemilu-
minescence responses to phorbol myristate acetate
(PMA). We compared the respiratory burst activity of
peripheral blood neutrophils from healthy adult donors
with the respiratory burst activity of CD34+ enriched
mobilised PBSC expanded in SCF/Flt3-L alone or
expanded in SCF/Flt3-L with G-CSF. CD34+ PBSC cul-
tured with SCF/Flt3-L alone show no bioluminescence
response to PMA, whereas those cultured with SCF/Flt3-L
and G-CSF respond like mature healthy neutrophils (see
below under Effects of TPO where these studies were
repeated with/without addition of TPO).
Effects of TPO on optimal expansion and maturation of
CD34+ enriched mobilised PBSC
The addition of TPO to the above SCF/Flt3-L/G-CSF neu-
trophil expansion/differentiation cocktail was tested by
examining cell expansion, morphology and function. The
addition of TPO to SCF/Flt3-L showed no significant
change in expansion compared to SCF/Flt3-L alone. The
addition of TPO to SCF/Flt3-L/G-CSF slightly reduced or
had no effect on the expansion with SCF/Flt3-L/G-CSF
alone (Figure 3). This reduction did not reach significance
(p = 0.0625, Wilcoxon signed rank test, n = 5) in the small
number of paired samples tested, but may indicate a trend
which would become significant in a larger sample. Cer-
tainly TPO offered no advantage over SCF/Flt3-L/G-CSF
alone in terms of cell expansion, and could be omitted for
the sake of culture simplicity on these grounds alone.
Cells expressing CD41 were found in small numbers in
cultures to which TPO was added, but were not found in
any cultures when TPO was omitted (not shown). When
cells were counted and scored according to their degree of
neutrophil differentiation, cells stimulated with SCF/Flt3-
L/G-CSF + TPO were less differentiated with less mature
neutrophil morphology than cells cultured with SCF/Flt3-
L/G-CSF alone (Figure 4). Myeloperoxidase (MPO) activ-
ity is found in mature neutrophils and myelocyte progen-
itors. The CD34+ cells cultured with SCF/Flt3-L/G-CSF
with TPO show a marginal decline in intensity of expres-
sion of MPO compared with the cells cultured without
Table 1: Colony formation by day 0 mononuclear cells and by day 14 expanded CD34+ cells.
% BFU-E % CFU-GM + % CFU-GBFU-E per thousand cells CFU-GM per
thousand cells
% CD34 in CFC assay CD34 fold
expansion in culture
Day 0
exp 28
exp 29
exp 32
74
74
72
26
26
28
35.9
8.6
5.0
12.4
3.0
1.9
2.6
1.2
0.4
Day 14
exp 28 SF
exp 28 SFG
10
5
90
95
3.0
0.8
28.5
16.1
100
100
17.5
33.4
exp 29 SF
exp 29 SFG
18
9
82
91
1.1
0.4
4.9
4.5
100
100
1.8
2.8
exp 32 SF
exp 32 SFG
13
0
87
100
0.67
0
4.7
4.7
100
100
10.8
32.4
CFC comparisons in 3 sets of mobilised PBSC before culture (day 0: mononuclear cell isolates, not CD34+ enriched) and after culture (day 14:
expanded CD34+ enriched cells) with SCF/Flt3-L (SF) and with SCF/Flt3-L + G-CSF (SFG). The proportional (percent) results were used for
qualitative comparisons. Some indication of quantitative responses can be obtained from CFC numbers, which may relate to CD34+ numbers
allowing for CD34+ expansion.

Page 7

Journal of Translational Medicine 2007, 5:53http://www.translational-medicine.com/content/5/1/53
Page 7 of 11
(page number not for citation purposes)
TPO (not shown). However, since MPO stains most of the
stages of the committed neutrophil differentiation path-
way then an effect by TPO on the stage of neutrophil mat-
uration achieved might not be clearly detected with this
method. Neutrophil superoxide activity is a good indica-
tor of neutrophil functional maturity. CD34+ cells cul-
tured with SCF/Flt3-L/G-CSF show a maximal superoxide
response to PMA, similar to fresh mature peripheral blood
neutrophils. Cells cultured with SCF/Flt3-L without G-
CSF show little superoxide response whether or not they
are cultured with TPO. When TPO is added to CD34+ cells
cultured with SCF/Flt3-L/G-CSF, the superoxide response
is reduced, but not abolished (Figure 5). In this respect,
TPO appears antagonistic to the effect of G-CSF on pro-
moting maturation of neutrophil superoxide activity in
the expanded CD34+ cells.
Discussion
These studies indicate that SCF and Flt3-L synergise to
provide a considerable proliferative stimulus to expand
PBSC CD34+ cells in culture, which is not significantly
increased by supplementary addition of IL-3, IL-6 or TPO
alone or in combination. Addition of G-CSF to SCF and
Flt3-L further expands total PBSC CD34+ cells and drives
neutrophil differentiation and maturation. The resultant
cells have reduced expression of CD34, express neutrophil
morphology, and acquire neutrophil function as assessed
by myeloperoxidase activity and superoxide generation in
response to PMA. Supplementary addition of TPO to SCF/
Flt3-L/G-CSF reduces maturation of neutrophil morphol-
ogy and function, and has a slight inhibitory effect on
Morphological changes in 14-day cultured cells from four patients stimulated with SCF/Flt3-L/G-CSF alone or SCF/Flt3-L/G-CSF + TPO
Figure 4
Morphological changes in 14-day cultured cells from
four patients stimulated with SCF/Flt3-L/G-CSF
alone or SCF/Flt3-L/G-CSF + TPO. Cells (200 per slide)
were scored as 0 (no morphological change, resembling lym-
phocytes) and from 1 to 3 on degree of developing neu-
trophil morphology. Any cells with non-neutrophil
morphological changes were classed as others. Bars repre-
sent mean and standard error; lines connect the scores for
each cytokine cocktail so that curve shifts can be seen.
3210 others
0
10
20
30
40
50
SCF / Flt3-L / G-CSF
SCF / Flt3-L / G-CSF / TPO
neutrophil morphology score
percent of total cells
Effect of TPO on expansion of CD34+ cells from five patients
Figure 3
Effect of TPO on expansion of CD34+ cells from five patients. (a) Effect of TPO on expansion in SCF + Flt3-L alone and
in SCF + Flt3-L + G-CSF. Bars represent mean and standard error. (b) Individual patient's responses in SCF + Flt3-L + G-CSF
with or without TPO. These are different patients from those shown in figure 1.
SCF/Flt3-LSCF/Flt3-L + G-CSF
0
5
10
15
20
25
no TPO
+ TPO
fold increase from CD34+
SCF/Flt3-L + G-CSF
SCF/Flt3-L + G-CSF + TPO
(a)(b)

Page 8

Journal of Translational Medicine 2007, 5:53 http://www.translational-medicine.com/content/5/1/53
Page 8 of 11
(page number not for citation purposes)
total CD34+ cell expansion which does not reach signifi-
cance. This indicates that for selective ex vivo expansion of
neutrophil precursors from PBSC, it is valuable to include
Flt3-L with SCF and G-CSF for maximal expansion, and
that supplementary inclusion of IL-3 and/or IL-6 is super-
fluous and increases the complexity and cost of protocols.
Supplementary inclusion of TPO, favoured in protocols
aimed to produce combined expansion of both neu-
trophil and megakaryocyte precursors, shows no benefit
in terms of expansion and retards the maturation of neu-
trophil precursors.
A variety of different studies have examined HSC expan-
sion ex vivo and have used a variety of parameters to meas-
ure outcomes. Because of limited sample volumes we
preferred to measure quantitative outcomes by cell count-
ing and morphological and phenotype analysis, and clo-
nogenic colony assays were only carried out on a small
number of samples, comparing day 0 colony assays on
PBSC mononuclear cells before enrichment of CD34+
cells with day 14 assays on cells cultured after CD34+
enrichment. While the results expressed in Table 1 are
indicative of CFU expansion, they are indeterminate
quantitatively and were used only for qualitative compar-
ison. Our main quantitative method is based on cell
counts and phenotype and morphology measurements as
used in some reported ex vivo PBSC expansion stud-
ies[25,26] and also as used in a study of neutrophil expan-
sion from cord blood[27]. Expression of neutrophil
enzyme activities and functional activity is also used in
two of these studies[27,25]. In our study colony assays
showed slight differences between cells expanded with
SCF/Flt3-L and cells expanded with SCF/Flt3-L/G-CSF in
that more BFU-E colonies were retained in the absence of
G-CSF, but myeloid colonies predominated in both
expansion cultures whereas erythroid colonies predomi-
nate before expansion, as has been reported by Reichle et
al[14]. Thus colony assays could not be used to sensitively
assess the effects of other supplementary cytokines on
SCF/Flt3-L/G-CSF. We did not use CD16 in this study
since it is not neutrophil specific and our observations
indicate that it is rapidly lost from mature neutrophils.
Expression of CD16b in expanded cells was dim com-
pared to mature neutrophils and differences in expression
between SCF/Flt3-L and SCF/Flt3-L/G-CSF expanded cells
were marginal and were not used to evaluate effects of
supplementary cytokines. For expression of superoxide
production addition of G-CSF to SCF/Flt3-L is necessary.
This showed a marked reduction when TPO was added to
the SCF/Flt3-L/G-CSF combination. Addition of TPO also
shifted the morphology to a less differentiated profile.
From the outset of ex vivo expansion Haylock et al[28]
promoted autologous PBSC CD34+ cell expansion for
reduction of neutropenia as an adjunct to unmanipulated
PBSC engraftment. Many subsequent studies have tar-
geted expansion of all classes of HSC, including primitive
long-term reconstituting cells and mature lineage-com-
mitted progenitors, to provide sufficient reconstitutive
graft materiel from smaller starting amounts of PBSC,
without support from unmanipulated PBSC. However,
Holyoake et al[29] demonstrated clinically that transplan-
tation solely with ex vivo expanded CD34+ cells does not
confer durable haematopoietic reconstitution, and that
unmanipulated PBSC are required for durable reconstitu-
tion. Evidently it is difficult to achieve simultaneous
expansion of committed progenitors and preservation or
expansion of more primitive multipotent HSC in the
same system. Of necessity this remains the target of advo-
cates of the use of umbilical cord blood HSC for alloge-
neic transplantation where
transplantation is not an option, since although cord
blood is rich in CD34+ cells the volumes are limited and
absolute CD34+ cell amounts are low compared to that
available from PBSC by apheresis collection[30]. As has
recently been reviewed[31] the goal of many cord blood
expansion protocols remains a single complex expansion
system delivering mature trilineage erythroid, myeloid
and megakaryocyte precursors for rapid reconstitution
with retention of multipotent stem cells for durable
reconstitution. Some cord blood studies promote the use
of multiple cords, some of which may be manipulated ex
vivo for selective lineage expansion[32-34]. The lack of
durable reconstitution by expanded cord blood cells
alone has been confirmed in animal studies[35], and indi-
cates the necessity of ensuring retention of multipotent
long-term reconstituting HSC, for which single system
protocols are still being sought[36].
autologous PBSC
PML-stimulated chemiluminescence responses of cells expanded with different cytokine combinations
Figure 5
PML-stimulated chemiluminescence responses of
cells expanded with different cytokine combinations.
PML-stimulated chemiluminescence responses of an individ-
ual patient's 14-day cultured cells stimulated with combina-
tions of SCF + Flt3-L with and without G-CSF and the effect
of TPO on these combinations. The baseline (no PMA) is for
the SCF/Flt3-L/G-CSF combination: the baselines for the
other cytokine combinations overlaid this baseline and are
not shown.
0 1020 3040
-0.1
0.1
0.3
0.5
0.7
0.9
1.1
SCF/Flt-3L+G-CSF
SCF/Flt-3L+G-CSF+TPO
SCF/Flt-3L
SCF/Flt-3L+TPO
cells alone (no PMA)
minutes
chemiluminescence (rlu)

Page 9

Journal of Translational Medicine 2007, 5:53http://www.translational-medicine.com/content/5/1/53
Page 9 of 11
(page number not for citation purposes)
Published PBSC expansion protocols all employ SCF as
the major direct proliferative stimulus for primitive and
committed HSC. Other cytokines are used with SCF to
provide additional direct stimuli, or synergistic stimuli,
for proliferative expansion. Early preclinical studies of ex
vivo expansion of PBSC CD34+ cells employed highly
complex cytokine mixtures[28,37]. However, Flt3-L was
not available to these investigators, and it appears that it
may substitute most other cytokine mixtures in synergis-
ing with SCF for HSC expansion. Further cytokines are
employed to stimulate mature and immature HSC to dif-
ferentiate and mature along specific lineage-committed
progenitor pathways, and may also provide proliferative
and survival signals for these committed cells. It has been
shown that certain combinations of cytokines can pro-
duce expansion of both primitive and committed haemat-
opoietic progenitors from PBSC in the same system[38]. It
may be naïve to assume that all of these processes can pro-
ceed simultaneously in the same system without competi-
tion or inhibition between different HSC expansion and
differentiation pathways. High level of complexity of
cytokine cocktails may be required to achieve multiline-
age expansion yet retain primitive multipotent HSC for
long term reconstitution, which is the objective for cord
blood. This is probably not required for selective neu-
trophil expansion only, as an adjunctive support for
unmanipulated autologous PBSC transplantation.
For PBSC the focus has largely turned to expansion of lin-
eage-committed progenitors for more rapid reconstitu-
tion, to be given as an adjunct to unmanipulated PBSC
which supply durable reconstitution. Most of the pub-
lished protocols have targeted simultaneous expansion of
both neutrophil and megakaryocyte precursors, and many
have simplified the expansion protocols for clinical appli-
cation to include only SCF, G-CSF and TPO. Most of these
have reported significant clinical reduction of neutrope-
nia when these expanded cells are given with unmanipu-
lated PBSC compared with unmanipulated PBSC
alone[15,11,10,12,13], and in some cases neutropenia
was virtually abolished[15,11]. Recent studies of PBSC
expansion by SCF, G-CSF and TPO in new medium for-
mulations have shown retention of long-term reconstitut-
ing HSC activity in vitro[39], and as well as abrogating
neutropenia when used as an adjunct to unmanipulated
PBSC these have demonstrated durable clinical haemat-
opoietic reconstitution by expanded cells alone, not as an
adjunct to unmanipulated PBSC[11].
The outcome of using adjunctive PBSC expanded with
multilineage protocols on clinical reduction of thrombo-
cytopenia ranged from no effect[10,15] to a reduction in
post-transplant platelet transfusion requirement[13,11]
or a mean reduction in thrombocytopenia duration by 1
day[12]. More complex multilineage expansion protocols
such as those used for cord blood do not appear to have
been evaluated clinically. Selective ex vivo expansion of
megakaryocyte precursors by relatively simple protocols
including SCF, TPO and IL-3[22], or SCF and TPO
only[21], showed no clinical benefit on thrombocytope-
nia reduction when compared to unmanipulated PBSC
alone when administered as an adjunct to unmanipulated
autologous PBSC. It therefore appears that thus far, for
autologous PBSC transplantation, clinical neutropenia
but not thrombocytopenia is amenable to reduction by
simple cytokine ex vivo expansion protocols.
A few studies have investigated selective ex vivo expansion
exclusively of neutrophil progenitors from PBSC[26,40,
25,14] and in two such studies the expanded cells have
been used clinically to supplement autologous PBSC.
Results were inconclusive in one clinical study[40] but
reduced neutropenia in the other[14]. These studies
employed a variety of expansion protocols, culture medi-
ums and in vitro outcome assessments, and it is difficult to
directly compare results quantitatively or qualitatively
when described by clonogenic colony numbers or by dif-
ferent total or phenotypically or morphologically distinct
cells numbers. However assessed, all agree that it is possi-
ble to expand neutrophil precursors ex vivo from PBSC.
Our results are consistent with the findings of others that
TPO is not necessary for selective neutrophil expansion
from PBSC[25,14,26,40] or cord blood[27] CD34+ cells.
Its inhibitory effect on neutrophil maturation is probably
marginal, but it is certainly superfluous for specific neu-
trophil precursor expansion. Consistent with our in vitro
observations, it has been reported that in vivo administra-
tion of TPO delays myeloid recovery following chemo-
therapy[41]. Others have suggested that megakaryocyte
expansion may be best achieved with quite different
cytokine combinations from those required for neu-
trophil precursor expansion[20,42,43]. It may be best to
focus on selective neutrophil precursor expansion as a fea-
sible and needed goal for reduction of neutropenia, and if
megakaryocyte generation is to be considered then sepa-
rate protocols should be devised to optimise this in dis-
tinct cultures rather than attempting simultaneous
neutrophil and megakaryocyte generation in the same sys-
tem. These results in preliminary small-scale laboratory
culture suggest that the combination of SCF, Flt3-L and G-
CSF should be investigated for development of protocols
suitable for clinical scale-up and GMP compliance, as a
possible cost-effective means of reducing neutropenia fol-
lowing autologous PBSC transplant which is not
addressed by availability of donor granulocyte transfu-
sion.

Page 10

Journal of Translational Medicine 2007, 5:53http://www.translational-medicine.com/content/5/1/53
Page 10 of 11
(page number not for citation purposes)
Conclusion
We have systematically investigated a number of combi-
nations of cytokines for in vitro culture expansion of
CD34+ cells from G-CSF mobilised PBSC, and have found
that a combination of SCF, Flt3-L and G-CSF synergise to
give maximal expansion and neutrophil maturation.
Addition of other cytokines commonly employed for ex
vivo PBSC expansion did not increase cell numbers or
improve neutrophil maturation, and appear superfluous.
Addition of TPO, commonly employed in multilineage
PBSC expansion to enhance megakaryocyte production,
appeared to have a moderate retarding effect on both
expansion and maturation of neutrophils from CD34+
cells, so that TPO appears at best superfluous and at worst
detrimental to ex vivo neutrophil expansion by SCF, Flt3-
L and G-CSF. While platelet transfusion is standard but
few transfusion centres offer granulocyte transfusion, and
while multilineage expansion does not reduce thrombo-
cytopenia, we recommend that ex vivo neutrophil expan-
sion from PBSC by a neutrophil specific expansion
protocol such as that above should be investigated as an
adjunct to autologous PBSC transplantation for reduction
or abrogation of post-transplant neutropenia.
Competing interests
The author(s) declare that they have no competing inter-
ests.
Authors' contributions
JD and MLT conceived of the study and attracted funding.
GRB and OT designed, coordinated and analysed the
study, and drafted the manuscript.
HR, JD and MLT authorized access to patient and blood
donor samples for the study and contributed clinical
expertise and oversight to the study design.
OT carried out cell isolation and culture, FACS and func-
tional analysis as a component of her PhD thesis studies
under the supervision of GRB.
All authors read and approved the final manuscript.
Acknowledgements
This study was supported in part by an educational grant from Chugai
Pharma UK and we wish to thank David Eves of Chugai Pharma UK for his
encouragement.
References
1.Shpall EJ, Jones RB, Bearman SI, Cagnoni PJ, Nieto Y, McNiece IK:
Peripheral blood stem cell transplantation in breast cancer.
Baillieres Best Pract Res Clin Haematol 1999, 12:219-232.
2. McNiece I, Briddell R: Ex vivo expansion of hematopoietic pro-
genitor cells and mature cells. Exp Hematol 2001, 29:3-11.
3.Paquette R, Dorshkind K: Optimizing hematopoietic recovery
following bone marrow transplantation. J Clin Invest 2002,
109:1527-1528.
4.Heike T, Nakahata T: Ex vivo expansion of hematopoietic stem
cells by cytokines. Biochimica et Biophysica Acta (BBA) - Molecular Cell
Research 2002, 1592:313-321.
McNiece I: Ex vivo expansion of hematopoietic cells. Exp
Hematol 2004, 32:409-410.
Despres D, Flohr T, Uppenkamp M, Baldus M, Hoffmann M, Huber C,
Derigs HG: CD34+ cell enrichment for autologous peripheral
blood stem cell transplantation by use of the CliniMACs
device. J Hematother Stem Cell Res 2000, 9:557-564.
Ogura M, Kagami Y, Suzuki R, Miura K, Uehira K, Kurokawa T, Ito T,
Kinoshita T, Emi N, Morishima Y, Kodera Y, Ueda R: Phase I/II trial
of cure-oriented high-dose chemoradiotherapy with trans-
plantation of CD34+ peripheral blood stem cells purified by
the immunomagnetic bead method for refractory hemato-
logical malignancies. Nagoya CD34+ PBSCT Study Group.
Cancer Chemother Pharmacol 1997, 40 Suppl:S51-S57.
Shpall EJ, Jones RB, Bearman SI, Franklin WA, Archer PG, Curiel T,
Bitter M, Claman HN, Stemmer SM, Purdy M, .: Transplantation of
enriched CD34-positive autologous marrow into breast can-
cer patients following high-dose chemotherapy: influence of
CD34-positive peripheral-blood progenitors and growth fac-
tors on engraftment. J Clin Oncol 1994, 12:28-36.
Urbano-Ispizua A, Solano C, Brunet S, de la RJ, Odriozola J, Zuazu J,
Figuera A, Caballero D, Martinez C, Garcia J, Sanz G, Torrabadella M,
Alegre A, Perez-Oteiza J, Jurado M, Oyonarte S, Sierra J, Garcia-
Conde J, Rozman C: Allogeneic transplantation of purified
CD34+ cells from peripheral blood: Spanish experience of 62
cases. Spanish Group of allo-PBT. Bone Marrow Transplant 1998,
21 Suppl 3:S71-S74.
McNiece I, Jones R, Bearman SI, Cagnoni P, Nieto Y, Franklin W,
Ryder J, Steele A, Stoltz J, Russell P, McDermitt J, Hogan C, Murphy
J, Shpall EJ: Ex vivo expanded peripheral blood progenitor cells
provide rapid neutrophil recovery after high-dose chemo-
therapy in patients with breast cancer. Blood 2000,
96:3001-3007.
Boiron JM, Dazey B, Cailliot C, Launay B, Attal M, Mazurier F,
McNiece IK, Ivanovic Z, Caraux J, Marit G, Reiffers J: Large-scale
expansion and transplantation of CD34(+) hematopoietic
cells: in vitro and in vivo confirmation of neutropenia abro-
gation related to the expansion process without impairment
of the long-term engraftment capacity. Transfusion 2006,
46:1934-1942.
Paquette RL, Dergham ST, Karpf E, Wang HJ, Slamon DJ, Souza L,
Glaspy JA: Ex vivo expanded unselected peripheral blood: pro-
genitor cells reduce posttransplantation neutropenia,
thrombocytopenia, and anemia in patients with breast can-
cer. Blood 2000, 96:2385-2390.
Prince HM, Simmons PJ, Whitty G, Wall DP, Barber L, Toner GC,
Seymour JF, Richardson G, Mrongovius R, Haylock DN: Improved
haematopoietic recovery following transplantation with ex
vivo-expanded mobilized blood cells. Br J Haematol 2004,
126:536-545.
Reichle A, Zaiss M, Rothe G, Schmitz G, Andreesen R: Autologous
tandem transplantation: almost complete reduction of neu-
tropenic fever following the second transplantation by ex
vivo expanded autologous myeloid postprogenitor cells.
Bone Marrow Transplant 2003, 32:299-305.
Reiffers J, Cailliot C, Dazey B, Attal M, Caraux J, Boiron JM: Abroga-
tion of post-myeloablative chemotherapy neutropenia by
ex-vivo expanded autologous CD34-positive cells. Lancet
1999, 354:1092-1093.
Briones MA, Josephson CD, Hillyer CD: Granulocyte transfusion:
revisited. Curr Hematol Rep 2003, 2:522-527.
Price TH: Granulocyte colony-stimulating factor-mobilized
granulocyte concentrate transfusions. Curr Opin Hematol 1998,
5:391-395.
Strauss RG: Rebirth of granulocyte transfusions: should it
involve pediatric oncology and transplant patients? J Pediatr
Hematol Oncol 1999, 21:475-478.
Yeghen T, Devereux S: Granulocyte transfusion: a review. Vox
Sang 2001, 81:87-92.
Bertolini F, Battaglia M, Pedrazzoli P, Da Prada GA, Lanza A, Soligo D,
Caneva L, Sarina B, Murphy S, Thomas T, della Cuna GR: Megakary-
ocytic progenitors can be generated ex vivo and safely
administered to autologous peripheral blood progenitor cell
transplant recipients. Blood 1997, 89:2679-2688.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.

Page 11

Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral
Journal of Translational Medicine 2007, 5:53http://www.translational-medicine.com/content/5/1/53
Page 11 of 11
(page number not for citation purposes)
21. Decaudin D, Vantelon JM, Bourhis JH, Farace F, Bonnet ML, Guillier
M, Greissenger N, Marracho MC, Assari S, Bennaceur AL, Nemati F,
Michon J, Turhan AG, Boccaccio C: Ex vivo expansion of meg-
akaryocyte precursor cells in autologous stem cell transplan-
tation for relapsed malignant lymphoma. Bone Marrow
Transplant 2004, 34:1089-1093.
Scheding S, Bergmannn M, Rathke G, Vogel W, Brugger W, Kanz L:
Additional transplantation of ex vivo generated megakaryo-
cytic cells after high-dose chemotherapy. Haematologica 2004,
89:630-631.
Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I: The
ISHAGE guidelines for CD34+ cell determination by flow
cytometry. International Society of Hematotherapy and
Graft Engineering. J Hematother 1996, 5:213-226.
De Novo Software http://www denovosoftware com 2007 [http://
www.denovosoftware.com].
Hino M, Suzuki K, Yamane T, Sakai N, Kubota H, Koh KR, Ohta K,
Hato F, Kitagawa S, Tatsumi N: Ex vivo expansion of mature
human neutrophils with normal functions from purified
peripheral blood CD34+ haematopoietic progenitor cells. Br
J Haematol 2000, 109:314-321.
Scheding S, Meister B, Buhring HJ, Baum CM, Mc Kearn JP, Bock T,
Kanz L, Brugger W: Effective ex vivo generation of granulopoi-
etic postprogenitor cells from mobilized peripheral blood
CD34(+) cells. Exp Hematol 2000, 28:460-470.
De Bruyn C, Delforge A, Bernier M, Bron D: Ex vivo expansion of
neutrophil precursor cells from fresh and cryopreserved
cord blood cells. Cytotherapy 2003, 5:87-98.
Haylock DN, To LB, Dowse TL, Juttner CA, Simmons PJ: Ex vivo
expansion and maturation of peripheral blood CD34+ cells
into the myeloid lineage. Blood 1992, 80:1405-1412.
Holyoake TL, Alcorn MJ, Richmond L, Farrell E, Pearson C, Green R,
Dunlop DJ, Fitzsimons E, Pragnell IB, Franklin IM: CD34 positive
PBPC expanded ex vivo may not provide durable engraft-
ment following myeloablative chemoradiotherapy regi-
mens. Bone Marrow Transplant 1997, 19:1095-1101.
Piacibello W, Sanavio F, Garetto L, Severino A, Dane A, Gammaitoni
L, Aglietta M: Differential growth factor requirement of prim-
itive cord blood hematopoietic stem cell for self-renewal and
amplification vs proliferation and differentiation. Leukemia
1998, 12:718-727.
Robinson S, Niu T, de Lima M, Ng J, Yang H, McMannis J, Karandish
S, Sadeghi T, Fu P, del Angel M, O'Connor S, Champlin R, Shpall E: Ex
vivo expansion of umbilical cord blood. Cytotherapy 2005,
7:243-250.
Pecora AL, Stiff P, Jennis A, Goldberg S, Rosenbluth R, Price P, Goltry
KL, Douville J, Armstrong RD, Smith AK, Preti RA: Prompt and
durable engraftment in two older adult patients with high
risk chronic myelogenous leukemia (CML) using ex vivo
expanded and unmanipulated unrelated umbilical cord
blood. Bone Marrow Transplant 2000, 25:797-799.
Shpall EJ, Quinones R, Giller R, Zeng C, Baron AE, Jones RB, Bearman
SI, Nieto Y, Freed B, Madinger N, Hogan CJ, Slat-Vasquez V, Russell
P, Blunk B, Schissel D, Hild E, Malcolm J, Ward W, McNiece IK:
Transplantation of ex vivo expanded cord blood. Biol Blood
Marrow Transplant 2002, 8:368-376.
Jaroscak J, Goltry K, Smith A, Waters-Pick B, Martin PL, Driscoll TA,
Howrey R, Chao N, Douville J, Burhop S, Fu P, Kurtzberg J: Aug-
mentation of umbilical cord blood (UCB) transplantation
with ex vivo-expanded UCB cells: results of a phase 1 trial
using the AastromReplicell
101:5061-5067.
McNiece I, Almeida-Porada G, Shpall EJ, Zanjani E: Ex vivo
expanded cord blood cells provide rapid engraftment in fetal
sheep but lack long-term engrafting potential. Exp Hematol
2002, 30:612-616.
Peled T, Mandel J, Goudsmid RN, Landor C, Hasson N, Harati D, Aus-
tin M, Hasson A, Fibach E, Shpall EJ, Nagler A: Pre-clinical develop-
ment of cord blood-derived progenitor cell graft expanded
ex vivo with cytokines and the polyamine copper chelator
tetraethylenepentamine. Cytotherapy 2004, 6:344-355.
Brugger W, Mocklin W, Heimfeld S, Berenson RJ, Mertelsmann R,
Kanz L: Ex vivo expansion of enriched peripheral blood
CD34+ progenitor cells by stem cell factor, interleukin-1
beta (IL-1 beta), IL-6, IL-3, interferon-gamma, and erythro-
poietin. Blood 1993, 81:2579-2584.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
System.
Blood 2003,
35.
36.
37.
38.Gammaitoni L, Bruno S, Sanavio F, Gunetti M, Kollet O, Cavalloni G,
Falda M, Fagioli F, Lapidot T, Aglietta M, Piacibello W: Ex vivo
expansion of human adult stem cells capable of primary and
secondary hemopoietic reconstitution. Exp Hematol 2003,
31:261-270.
Ivanovic Z, Duchez P, Dazey B, Hermitte F, Lamrissi-Garcia I, Mazu-
rier F, Praloran V, Reiffers J, Vezon G, Boiron JM: A clinical-scale
expansion of mobilized CD 34+ hematopoietic stem and
progenitor cells by use of a new serum-free medium. Trans-
fusion 2006, 46:126-131.
Zimmerman TM, Lee WJ, Bender JG, Schilling M, Smith SL, Van Epps
DE, Williams SF: Clinical impact of ex vivo differentiated mye-
loid precursors after high-dose chemotherapy and periph-
eral blood progenitor cell rescue. Bone Marrow Transplant 2000,
26:505-510.
Geissler K, Yin JA, Ganser A, Sanz MA, Szer J, Raghavachar A, Hoelzer
D, Martinez C, Taylor K, Kanz L, To LB, Archimbaud E: Prior and
concurrent administration of recombinant human meg-
akaryocyte growth and development factor in patients
receiving consolidation chemotherapy for de novo acute
myeloid leukemia--a randomized, placebo-controlled, dou-
ble-blind safety and efficacy study. Ann Hematol 2003,
82:677-683.
van den OS, de HM, Calafat J, van der Schoot CE, von dem Borne AE:
A combination of megakaryocyte growth and development
factor and interleukin-1 is sufficient to culture large numbers
of megakaryocytic progenitors and megakaryocytes for
transfusion purposes. Br J Haematol 1999, 106:553-563.
van den OS, von dem Borne AE, de HM: Influence of medium
components on ex vivo megakaryocyte expansion. J Hema-
tother Stem Cell Res 2001, 10:193-200.
39.
40.
41.
42.
43.