Hematopoietic stem cells from peripheral blood the perspective of non-mobilized peripheral blood

doi:10.4236/health.2010.26078

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Vol.2, No.6, 519-527 (2010) Health

doi:10.4236/health.2010.26078

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.

ABSTRACT

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

1. INTRODUCTION

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].

2. CHARACTERIZATION OF

HEMATOPOIETIC STEM CELLS

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].

3. CRYOPRESERVATION AND THAWING

OF PBPCS

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-

cordingly.

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

520

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.

4. THERAPEUTIC DOSES OF PBSC

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.

5. COST ANALYSIS

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

521

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

costs.

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.

6. EX VIVO EXPANSION OF HSC

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

522

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

expansion.

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

[81].

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

523

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].

7. AUTHORS’ CONTRIBUTION

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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GLOSSARY OF ABBREVIATIONS AND

INITIALISMS

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