Vol.2, No.6, 519-527 (2010) Health
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Hematopoietic stem cells from peripheral blood
the perspective of non-mobilized peripheral blood
Vassilios Katsares*, Zissis Paparidis, Eleni Nikolaidou, Anastasia Petsa, Iliana Karvounidou,
Karina-Alina Ardelean, Nikolaos Peroulis, Nikolaos Grigoriadis, John Grigoriadis
Biogenea-Cellgenea Ltd, Trade Centre “Plateia”, Thessaloniki, Greece; *Corresponding Author: vkatsare@gmail.com
Received 1 February 2010; revised 21 February 2010; accepted 23 February 2010.
The peripheral blood is a major source of he-
matopoietic stem cells. Almost for two decades
the peripheral blood has been mobilized, in or-
der to enhance the CD34+ concentration. The
isolated stem cells from the mobilized periph-
eral blood are used as an alternative, or in addi-
tion to bone marrow derived stem cells. In this
paper, a new perspective is being discussed;
the use of non-mobilized peripheral blood as an
alternative source for hematopoietic progenitor
cells. The number of isolated hematopoietic stem
cells is evaluated using flow cytometry. The vi-
ability can be evaluated using the trypan blue
exclusion test, the flow cytometry or automated
assays. The isolated hematopoietic stem cells
could be used for ex vivo expansion either in
static systems or in proper bioreactor systems,
prior to cryopreservation and/or transplantation.
Keywords: Non-Mobilized Peripheral Blood;
Hematopoietic Stem Cells; Ex Vivo Expansion
Since the early 1990s, peripheral blood progenitor cells
collected by apheresis have largely replaced bone mar-
row as a source of hematopoietic stem cells for autolo-
gous transplantation [1]. Peripheral blood cells produce
more rapid hematopoietic recovery, thereby leading to
reduced costs [2-5]. Furthermore, although follow-up is
more limited in the PBSC group than in the BM group,
no evidence was found that the use of PBSC was associ-
ated with an increased risk of chronic GVHD compared
to results with BM [6].
Hematopoietic stem cells are normally found in very
limited numbers in the peripheral circulation (less than
0.1% of all nucleated cells). It is logical that progenitor
cells circulate in the periphery, as this ensures an even
distribution of hematopoiesis within the BM [7]. CD34
antigen expression is used as a surrogate marker for he-
matopoietic stem cells and enumeration of CD34+ cells
has been used to quantify progenitor and stem cell con-
tent [8].
PBSCs represent a subpopulation of all CD34+ cells
(CD34+/CD38–) found in the circulation [9]. CD34+ cell
viability was measured by an established flow cytomet-
ric method [10]. The method is based on triple staining
with anti-CD34-PE, anti-CD45-FITC, and the viability
marker 7-actinomycin D, and it allows the calculation of
the absolute numbers of viable CD34+ cells. Recently, a
new rapid and accurate method has been developed for
the viability evaluation based on luminometry [11].
PBPCs are usually harvested and stored in liquid N2 un-
til reinfusion. Storage at this low temperature will block
all enzymatic pathways and metabolism in the cell [12].
Cryopreservative(s) must be added to the PBSC before
freezing in order to protect the cells. The concentration
of the cryoprotectant and the rate at which the cells are
frozen are the main factors governing the survival of the
cells. Thereafter the cells are stored in liquid N2 [13].
Due to their different cell membrane composition and
higher osmotic inactive volume, CD34+ cells are better
protected from hypertonic shock and ice crystal forma-
tion and should be more resistant to cryopreservation
damage than the remaining nucleated cell population [14].
A computer-controlled freezer is used for the cryo-
preservation. In order to ensure rate-controlled freezing
an optimized program is developed and adjusted ac-
In controlled rate freezing, the concentrated stem cells
are frozen down at a rate of 1-20C/min up to a tempera-
V. Katsares et al. / HEALTH 2 (2010) 519-527
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
ture point of about –40˚C. Then, the freezing process
down to a target of –120˚C is performed at a faster pace,
about 3-5˚C/min. For PBSCs the controlled rate freezing
process is considered standard [15,16], and was in dif-
ferent reports found to be superior to uncontrolled
freezing approaches. This procedure is time consuming
and requires staff with a specific expertise. Hence, the
use of uncontrolled rate freezing in which the specimen
is first cooled down to –4˚C and then directly deposited
into a freezer at –80˚C or put into liquid phase nitrogen
has been evaluated. Several reports [17-19] established
that the uncontrolled method is safe and reveals compa-
rable results to the controlled rate process for PBSCs. A
controlled study performed by Perez-Oteyza et al. [20]
showed that the controlled and uncontrolled rate freezing
approach are comparable in terms of viability testing and
that only a statistically significant decrease in the CFU-
GM clonality assay could be detected in the uncontrolled
freezing situation. Recent studies suggested that uncon-
trolled freezing is also a viable approach for UCB stem
cells [21,22].
As a cryoprotectant, a solution containing 50% DMSO
in HAES-steril® 10% is used. Prior to freezing, a part of
pre-cooled cryosolution (with 50% DMSO) is mixed
with three parts of the buffy coat (pre-cooled), to achieve
DMSO concentration of 5 or 10% in the final solution.
Cryopreservation is then carried out in aliquots in cryo-
genic vials.
Current protocols for cryopreservation of PBPCs are
usually based on the use of 10 percent DMSO in the
freezing medium [23]. HPCs can be preserved with 5%
DMSO, and such autografts can be safely used for stem
cell rescue even after long-term nitrogen storage [24].
Several techniques for the thawing procedure have
been proposed. The standard method is warming in a
water bath at 37˚C until all ice crystals disappear [19]. A
German study compared the thawing of cryopreserved
units in a warm water bath with dry heat applied by gel
pads at 37˚C. The viability and clonogenic potential
were comparable, with a trend towards less infectious
contamination in the dry method [25]. Different studies
examined the preservation of function when thawed
units were incubated at 0-37˚C [19,26].
Akkök et al. [25] suggest that even simple single-
wash DMSO depletion causes significant CD34+ cell
loss. Despite a beneficial impact on the frequency of
adverse effects during and after stem cell infusion, this
time-consuming procedure caused a delayed PLT recov-
ery and increased requirement for PLT transfusions. The
CD34+ cell loss, however, was never critically low, that
is, never lower than 2 × 106 per kg. They concluded that
single manual washing of autografts is a simple and safe
procedure that decreases the frequency of adverse events
during and after stem cell infusion. The procedure should
be recommended especially for patients with an in-
creased risk of serious toxicity, for example, patients
with cardiac amyloidosis.
Typical doses of CD34+ stem cells used for PBSCs are
2 × 106 cells/kg of recipient body weight or greater.
Doses lower than this threshold is associated with pro-
longed cytopenias and increased early mortality [27].
The use of higher doses of CD34+ cells may lead to
quicker engraftment, particularly when doses are greatly
increased [28,29]. Platelet recovery appears to be more
sensitive to CD34+ doses than neutrophil recovery [29].
Efforts to enrich PBSCT by ex-vivo CD34+ cell selection
(positive selection) have resulted in increased rates of
GVHD, possibly by altering the cytokine expression
patterns of transplanted cells or changing lymphocyte
subsets delivered with the graft [30].
Autologous stem cell grafting has been used with
varying degrees of success in chronic myelogenous leu-
kemia (CML) [31,32], acute leukemia [33], myelodys-
plasia [34], and multiple myeloma [35].
Niwa et al. [36] reported successful autologous pe-
ripheral blood stem cell transplantation with a double-
conditioning regimen for recurrent hepatoblastoma after
liver transplantation.
Nevskaya et al. [37] with preliminary results sug-
gested the feasibility of therapeutic angiogenesis by lo-
cal implantation of CD34+ and MNC from PB for Sys-
temic Sclerosis ischemic ulcers. Improved endothelial
function, stimulatory effects on circulating endothelial
precursors kinetics and augmentation of microcircula-
tory blood flow may contribute to therapeutic potential
of the implanted cells.
The cost method involved two sets of data: a data set
including patient-related or direct costs, and a data set
including nonpatient-related or indirect costs [38]. The
patient-related costs comprise the followings: 1) hospi-
talization and basic medical service, including medical
and nursing staff; 2) pharmacy and blood products; 3)
procedures such as operating theatre, leukapheresis and
cryopreservation. On the other hand, the indirect costs
comprise the clinical service department costs, for in-
stance, radiology, clinical chemistry, pharmacy and the
nonclinical service departments such as transportation,
housekeeping and kitchen services.
A study by Mishra et al. [38] reported a cost analysis
using mobilized peripheral blood at 2001 prices and the
V. Katsares et al. / HEALTH 2 (2010) 519-527
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costs had been recalculated into US$ by using the ex-
change rates of 1st January 2001. The mean cost for the
mobilization/cryopreservation phase per patient was
US$ 6544 (range 5114-7273). The mean cost of high
dose chemotherapy followed by hospitalization was US$
25616 (13978-43277). This amounts to total running
costs of US$ 32160 (19092-50550). Taken together,
staffing, medication and blood products contributed to
74% of total costs. On average, 53% of total costs com-
prised staff costs, ranging from 39 to 76%. Personnel
resources varied from one center to another, from US$
12608 to US$ 26038 per patient. Pharmacy and blood
products contributed 16 and 5%, respectively, of the total
A study by van Agthoven [39] documented total costs
of PBSC transplantation at Euro 33742. The author ap-
plied a unit cost method where staff costs accounted for
42% of the transplant cost. This relatively large differ-
ence in staff costs between Van Agthoven and Mishra et
al. is notable, and may indicate there are cost variations
between different countries, for example, related to
wages. Van Agthoven [39] reported a remarkably low
cost per patient for the stem cell harvesting and cryopre-
servation procedures, an average of €4982, and a blood
component cost (during the induction chemotherapy
regimen, harvesting and transplantation phase), respec-
tively, of €904, €376 and €1680, a total of €2960.
In another study, Ghosh et al. [40] reported a PBSCT
cost for patients with plasma cell leukemia that ranged
from US$ 20000 to US$ 25000. The major part of the
costs related to hospitalization, growth factors, blood
products, collection and cryopreservation of PBSC.
Hopefully, the use of non mobilized peripheral blood
could eliminate the cost, since there are no mobilization
drugs and no any special equipment required.
The CD34+ surface antigen, which is a glycoprotein ex-
pressed on early progenitor cells is present on less than
0.1% of the mononuclear cells in peripheral blood [41].
Many studies have shown that the minimum acceptable
dose of HPCs for successful transplantation ranges be-
tween 2 – 5 × 106 CD34+ cells per kg of recipient weight
[42]. Furthermore, transplantation of higher doses of
CD34+ cells seems to improve haematopoietic recovery
and overall survival [43,44]. To try and overcome the
problem of low progenitor cell dose, ex vivo expansion
of CB-derived cells has been attempted. The true test of
this method is whether an expansion technology will be
able to provide a reliable, reproducible increase in the
number of progenitor cells available from a single unit
of CB, resulting in superior rates of engraftment and
overall survival in adult patients. A significant hurdle of
presently available methods for graft production is the
ability to generate an expanded population of committed
hematopoietic progenitor cells without compromising
the numbers of less differentiated progenitor cells
(CD34+ CD38 or CD34+ Lin cells), which are impor-
tant functional hematopoietic repopulating cells [45].
In order to consistently achieve an adequate cell dose,
the processing methods must minimize cell losses. Each
additional manipulation of a cellular product potentially
leads to further loss of cells. In most studies, CD34+ cell
selection is done before initiating cell culture [46], but
the CD34+ cell selection itself is associated with a sub-
stantial loss of progenitor cells. This cell loss, which
may not be significant for smaller children, may become
critical in reaching a suitable dose for transplant in older
children and adults [47].
To achieve adequate cell doses, many researcher used
different ex vivo expansion protocols, either the tradi-
tional way, or using a bioreactor system. Beshlawy et al.
[48] used three cytokine combinations, i.e. cell factor
alone, IL-3 alone, and both stem cell factor and IL-3.
Interleukin-3 enhances the amplification of early and
committed progenitor cells without impairing the
long-term engraftment of stem cells [49].
Several investigators reported significantly decreased
cell viability after cryopreservation [50-51] and attrib-
uted this to the effect of thawing and washing to remove
the cryoprotectant. Laroche et al. [47] stated that thaw-
ing and washing result in loss of cells approaching 20%
when compared with pre-freeze counts, with the wash
step responsible for nearly half of this cell loss. However,
Beshlawy et al. [48], using umbilical cord blood derived
hematopoietic stem cells, detected mean fold expansion
of 6.64 ± 3.34 with stem cell factor alone, 7.38 ± 2.86
with both stem cell factor and IL-3, and 8.11 ± 4.49 with
IL-3 alone after 2 days culture of the samples frozen for
2 weeks. There were no statistically significant differ-
ences in fold expansion between the 3 cytokine combi-
nations before freezing and after 1 week and 2 weeks of
freezing. They concluded that although preservation
procedures could decrease the count and viability of cord
blood HSCs, freezing does not impair their ex vivo ex-
pansion potential; however, it results in a significant loss
of cell viability.
In a similar study, Moezzi et al. [51] used stem cell
factor, IL-3, and thrombopoietin and reported levels of
expansion of (4.2-4.7 fold) after 7 days of culture of
samples cryopreserved for 1 month.
It was shown that a combination of early- and late-
acting cytokines, including SCF, thrombopoietin (TPO),
G-CSF and IL-3, resulted in only a marginal-fold expan-
sion of late (CD34+) and early (CD34+CD38 ) progenitor
V. Katsares et al. / HEALTH 2 (2010) 519-527
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
cells, probably due the fact that the late-acting cytokines
drive the cultures mainly toward accelerated differentia-
tion [52,53].
On the other hand, cultures with only early-acting cy-
tokines (SCF, TPO, IL-6 and FLT-3 ligand) resulted in
better and prolonged expansion of both late and early
progenitors [54], which are important for short-term
early trilineage engraftment [55-57].
Peled et al. [58] suggested that TEPA supports the self
renewal division cycle without compromising differen-
tiation capacity of hematopoietic stem cells.
A number of serum-free media have been used over
the last few years with different results [59-62]. For ob-
taining sufficient numbers of progenitor cells for trans-
plant, FCS [63] and autologous plasma [60,61] have
been used in clinical expansion protocols. However,
Lam et al. [61] suggested that, with the appropriate se-
rum-free media and cytokines, FCS may be excluded in
clinical expansions. On the other hand, human plasma,
which may contain factors that promote cell maturation
[64,65] is thus unlikely to add significant value to the
It has been reported that MSC constitutively secrete
various hematopoietic cytokines, among them stem cell
factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO),
leukemia-inhibiting factor (LIF), interleukin (IL)-6, IL-7,
IL-8, IL-11, IL-12, IL-14, and IL-15 [66-68]. Addition of
MSC as a feeder layer has been shown to improve ex-
pansion of cord blood HSC, inhibit their differentiation,
and decrease their rate of apoptosis [66,69-71]. Li et al.
[66] demonstrated that bone marrow MSC can increase
human adult PBSC expansion as compared with culture
in the presence of cytokine alone.
Conventional culture systems such as T-flasks and gas
permeable blood bags are the most widely used devices
for expanding hematopoietic cells. However, such static
culture systems have several inherent limitations. Firstly,
lack of mixing results in concentration gradients for dis-
solved oxygen (DO), pH, cytokines and metabolites.
Secondly, the environmental conditions in well-plate and
T-flask are not readily monitored or controlled online.
Thirdly, static systems require repeated changes of cul-
ture medium, which significantly increases the risk of
contamination. Hence there is an urgent need for devel-
oping bioreactors for HSCs expansion, which overcomes
the limitation of mass transport, keeps culture parame-
ters constant and controls differentiation [72].
Several kinds of bioreactors have been applied in the
field of HSCs expansion, including stirred tank bioreac-
tor, fixed bed bioreactor and perfusion chamber [73-75].
It is known that hematopoietic cells are extremely sensi-
tive to shear force, hence cells may suffer some physical
damage under shear environment like in a stirred tank
bioreactor and perfusion chambers [73]. In stirred tank
bioreactor, agitation may affect cell surface marker ex-
pression, including cytokine receptors, which can have a
profound effect on which cells expand and to what ex-
tent expansion occurs [73]. A condition with low shear
but low concentration gradients is highly desirable for
hematopoietic cell expansion [72].
Rotating wall vessel (RWV) bioreactor may provide a
technical solution. The RWV bioreactor has several key
characteristic features as follows [76]: firstly, fluid flow
is near solid body and is laminar at most operating con-
ditions, which avoids the large shear stresses associated
with turbulent flow and allows introduction of controlled
and nearly homogenous shear fields; secondly, the cul-
ture medium is gently mixed by rotation, avoiding the
necessity for stirring vanes, which may damage cells by
both local turbulence at their surface and the high flow
rates created between the vessel walls and the vanes;
thirdly, there is no headspace in the RWV bioreactor
while in roller bottles, due to incomplete filling of the
vessel, the air in the headspace creates turbulence and
secondary bubble formation in the culture medium,
which are both potent sources of extra shear and turbu-
lence; finally, the RWV bioreactor supports coculture
efficiently by bringing different cell types of different
size and density together simply and efficiently. So by
optimizing the geometry of the bioreactor and opera-
tional condition, it is possible to provide a uniform and
low shear condition within the bioreactor. At the same
time concentration gradient can be minimized.
RWV bioreactors have been used to simulate micro-
gravity in space flight to study how microgravity affects
the hematopoiesis of astronauts [77,78], to proliferate
BM cells [79]. The RWV bioreactor can provide a 3D
suspension culture environment and all hematopoietic
cells are suspended in the culture medium effectively,
which overcomes the concentration gradients in T-flasks
and makes the utilization of cytokines more effective.
The National Aeronautics and Space Administration
(NASA) developed two RWV bioreactors for tissue
mass culture [80]. The slow turn lateral vessel (STLV)
bioreactor has been used to culture several kinds of cells
both on Earth and in space. The STLV was operated at
15-30 rpm on Earth and slower in space allowing a
free-fall state, reducing the shear stress. The high aspect
ratio vessel (HARV) bioreactor has a similar design, but
the rotating speed can be slower than STLV. The NASA
RWV systems have been used to study the effects of
microgravity on murine HSC and evaluating the hema-
topoietic homeostasis during long space expeditions
The elucidation of mechanisms governing self-renewal
and differentiation of HSC is needed to control the in
V. Katsares et al. / HEALTH 2 (2010) 519-527
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
vitro expansion. Results from pilot clinical trials of
transplants using expanded UCB-HSC have shown no
adverse effects in the patients. However, more clinical
trials must be conducted using expanded HSC for guar-
antying the safety [82]. Very recently, Delaney et al.
(2010) claimed that when cord blood progenitors ex-
panded ex vivo in the presence of Notch ligand we in-
fused in a clinical setting after a myeloablative prepara-
tive regimen for stem cell transplantation, the time to
neutrophil recovery was substantially shortened. This is
the first instance of rapid engraftment derived form ex
vivo expanded stem/progenitor cells in humans [83].
All authors contributed substantially to this research.
V.K., J.G., and N.G. designed research and collected the
data; V.K., Z.P., A.P., E.N., I.K., and K.A.A. performed
literature revision; V.K. analysed and interpreted data,
and wrote the manuscript. All authors drafted the manu-
script, revised it critically and approved it.
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Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
BM: Bone Marrow
DMSO: Dimethyl sulfoxide
GVHD: Graft versus Host Disease
HARV: High Aspect Ratio Vessel
hESC: human Ebryonic Stem Cell
HLA: Human Leukocyte Antigen
HPC: Hematopoietic Progenitor Cell
HSC: Hematopoietic Stem Cell
LTC-IC: Long-term culture initiating colony
NASA: National Aeronautics and Space Administration
PBSC: Peripheral Blood Stem Cell
PBSCT: Peripheral Blood Stem Cell Transplantation
PPC: Primitive progenitor cell
RWV: Rotating Wall Vessel
STLV: Slow Turn Lateral Vessel