Vox Sanguinis (2009) 96, 256–265
ORIGINAL PAPER
© 2008 The Author(s)
Journal compilation © 2008 International Society of Blood Transfusion
DOI: 10.1111/j.1423-0410.2008.01138.x
Characterization and comparison of bone marrow and
peripheral blood mononuclear cells used for cellular therapy
in critical leg ischaemia: towards a new cellular product
Blackwell Publishing Ltd
J.-C. Capiod,1 C. Tournois,2,3 F. Vitry,4 M.-A. Sevestre,5 S. Daliphard,2 T. Reix,5 P. Nguyen,2,3 J.-J. Lefrère1 & B. Pignon3,6
1
Laboratory of Haematology, University Hospital of Amiens, Amiens, France
Laboratory of Haematology, University Hospital of Reims, Reims, France
3
EA3801, Institut Fédératif de Recherche 53, Laboratoire d’Hématologie, Centre Hospitalier Universitaire Reims, Hôpital Robert-Debré, Reims Cedex, France
4
Department of Methodology, University Hospital of Reims, Reims, France
5
Department of Vascular Surgery, University Hospital Amiens, Amiens, France
6
Department of Cell Therapy, University Hospital Reims, Reims, France
2
Background and Objectives Autologous transplantation of either bone marrow (BM)
or peripheral blood (PB) mononuclear cells (MNC) induces therapeutic angiogenesis in
patients with peripheral arterial occlusive disease. Yet, the precise nature of the cellular
product obtained from BM or PB and used in these therapeutic strategies remains unclear.
Materials and Methods We have analysed the characteristics of BM-MNC and PB-MNC
collected without mobilization and implanted in patients with critical limb ischaemia
in a clinical trial of cellular therapy including 16 individuals treated by BM-MNC and
eight by PB-MNC. These MNCs were characterized by cell counts, viability assessment
and enumeration of leucocyte subsets, CD34 stem and endothelial progenitor cells
(EPCs) (CD34+/CD133+/VEGF-R2+) by flow cytometry. Mean fluorescence intensity
ratios were determined for CD34, CD133 and VEGF-R2 markers. All analyses were
simultaneously performed in two laboratories.
Results Accuracy and reliability between both laboratories were achieved. BM-MNCs
and PB-MNCs were quantitatively and qualitatively heterogeneous and quite different
from each other. Stem cells and EPCs were significantly more present in BM- compared
to PB-cell products, but with similar mean fluorescence intensity ratios. A weakly positive
correlation was observed between CD34+ cell counts and EPCs levels, confirming the
specificity of cell identification.
Received: 11 August 2008,
revised 28 October 2008,
accepted 12 November 2008,
published online 15 December 2008
Conclusion A great variability was observed in cell product characteristics according
to their origin and also between individuals. These data stress the necessity of optimal
characterization of cell products especially in multicentric clinical trials.
Key words: cellular therapy, cellular product, critical leg ischaemia, endothelial
progenitor cells, therapeutic angiogenesis.
Correspondence: Claire Tournois, EA3801, Institut Fédératif de Recherche 53, Laboratoire d’Hématologie, Centre Hospitalier Universitaire Reims, Hôpital
Robert-Debré, 51092 Reims Cedex, France
E-mail: [email protected]
Statement of equal author contribution: Jean-Claude Capiod and Claire Tournois contributed equally to this study.
Conflict of interest: all authors disclose conflict of interest of any kind related to this manuscript. Authors had full access to the data.
Abbreviations: 7-AAD, 7-aminoactinomycin; BM, bone marrow; BMI, body mass index; CLI, critical limb ischaemia; ECD, phycoerythrin-texas red;
ECs, endothelial cells; EPCs, endothelial progenitor cells; FC, flow cytometry; GFR, glomerular filtration rate; MDRD, modification of diet in renal disease;
Med, median; MGG, May Grünwald Giemsa; PB, peripheral blood; PCy5, phycocyanin 5; PCy7, phycocyanin7; VEGF-R2, vascular endothelial growth
factor receptor 2.
256
Cell therapy in critical limb ischaemia
Introduction
Several studies have shown that autologous transplantation
of either bone marrow (BM) or peripheral blood (PB) mononuclear cells (MNC) could be effective in inducing angiogenesis
in patients with peripheral arterial occlusive disease [1–6].
Experimental studies have shown that circulating BMderived endothelial progenitor cells (EPCs) can be mobilized
and incorporated into sites of active ischaemia to increase
postnatal neovascularization [7,8], and that circulating BMderived EPCs are able to differentiate into mature endothelial
cells (ECs) [9]. The mechanisms regulating the differentiation
of BM-derived EPCs, their mobilization and their homing to
ischaemic sites remain partially unknown [10], but the
implantation of BM-MNC [11] and of PB-MNC [12] into
ischaemic tissues (through the intramuscular local transplantation of these cells) induces collateral vessel formation in
animals, suggesting that this cell therapy could constitute a
new strategy for therapeutic angiogenesis.
The questions of which cells and which optimal effective
quantity to implant are crucial as long as the precise neovascularization mechanisms are not completely elucidated.
However, in most clinical trials, the exact composition of the
cellular product used remains elusive and is obviously highly
variable. In fact, several parameters such as the origin of the
MNC (BM or PB), the health status of the donor and the
preparation procedures [13] may lead to major differences in
this cellular product, which is perhaps destined to become a
new ‘transfusion product’. For this reason, it is important to
try to characterize most precisely such a product before the
implementation of future multicentre clinical trials aiming to
fully demonstrate the efficacy and safety of this cellular
therapy in critical limb ischaemia (CLI). Such an approach
would allow choosing the ‘best’ cell product in terms of
efficacy, to standardize this product by cell characterization
methods easy to perform, and to contribute to the best knowledge
of involved mechanisms. We report here our results in
characterizing BM-MNC and PB-MNC implanted in patients
with CLI in a French clinical trial. This trial was an open
bicentric prospective study performed in two academic
centres of vascular surgery and tested the safety and efficacy
of BM-MNC or non-mobilized PB-MNC implanted in individuals
with CLI. The clinical results will be presented as soon as all
patients will have achieved sufficient follow-up. However, an
interim analysis was performed that confirmed the safety and
was in favour of similar efficacy for both cell products.
Materials and methods
A prospective clinical trial was initiated in two French academic
hospitals in order to establish whether intramuscular
implantation of BM-MNC or PB-MNC can induce clinical
improvement in patients with CLI. Enrolled patients had
257
Table 1 Patient’s characteristics
Age (years)
Gender
Male
Female
BMI (kg/m2)
Smoking
Disorders
Arterial hypertension
Hypercholesterolemia
Diabetes mellitus
GFR (ml/min/1·73 m2)a
Haemoglobin (g/l)
White blood cells (109/l)
BM cell products
group (n = 16)
PB cell products
group (n = 8)
71 [45–84]
60 [37–84]
14 (87·5%)
2 (12·5%)
24·8 [20·8–30·8]
one current, 12 past,
3 never
6 (75%)
2 (25%)
22·7 [18·5–31·0]
two current, 4 past,
2 never
11 (69%)
12 (75%)
6 (37·5%)
59 [30–86]
132 [104–154]
7·2 [5·0–10·4]
6 (75%)
3 (37·5%)
1 (12·5%)
81 [59–172]
124 [100–141]
7·4 [5·6–8·9]
Quantitative variables were expressed as median and range [min–max].
a
GFR, Glomerular filtration rate (MDRD or modification of diet in renal
disease).
unilateral CLI and were not suitable candidates for surgery,
non-surgical acts or revascularization. Patients with poorly
controlled diabetes mellitus or with a history of malignant
disorder were excluded. The ethical committee of each
participating centre approved the protocol. Written informed
consent was obtained from all patients. Twenty-four patients
were included: 16 treated with BM-MNC and eight with
PB-MNC (Table 1). Patients were consecutively included as
soon as they presented with appropriate criteria and were not
selected to receive one or another type of cells. The first 16
selected patients were treated with BM-MNC; the following
ones were treated with PB-MNC. Cells were implanted 1–3 h
after preparation by 30 intramuscular injections into the
gastrocnemius of the ischaemic leg. Total injection volume
was 30 ml. In this article, we focalized on the two types
cellular products used.
Bone marrow and peripheral blood mononuclear
cell samples
For the preparation of BM-MNC, 500 ml of bone marrow was
collected under general anaesthesia through multiple
punctures of the posterior iliac crest using a Jamshidi needle.
MNC were isolated using a blood cell separator (Cobe Spectra,
version 4, Bone Marrow Processing Program, Gambro BCT,
Lakewood, CO, USA). PB-MNC were collected by cytapheresis
of one blood mass (5·1 ± 1·1 l) during 90 min with the
same blood cell separator (Cobe Spectra, version 6, autoPBSC program, Gambro BCT). In both cases, the blood cell
separator was programmed to obtain a final volume of 40 ml
© 2008 The Author(s)
Journal compilation © 2008 International Society of Blood Transfusion, Vox Sanguinis (2009) 96, 256–265
258 J.-C. Capiod et al.
MNC concentrate (30 ml for autologous re-injection and
10 ml for controls and analyses). There was no previous
mobilization by any haematopoietic growth factor. Blood cell
separator was centralized in one centre (Reims Hospital).
Samples of the end-product were collected and diluted
1 : 2 in citrate–citric acid–dextrose to avoid cellular aggregates
(ACDA, Baxter Healthcare, Deerfield, IL, USA). A final volume
of 2 ml was dispatched at stable ambient temperature in the
laboratories of the two participating centres (laboratory A for
Amiens Hospital; laboratory B for Reims Hospital). Cell
analyses were simultaneously performed in these laboratories,
within 3 h after preparation of the cell product.
Mononuclear cell characterization
Cell counts
Mononuclear cell counts were performed with an XE-2100™
Sysmex counter (Roche Diagnostics, Meylan, France) in
laboratory A, and a Gen’s® counter (Beckman Coulter,
Villepinte, France) in laboratory B. Cytospins were prepared
using Cytospin 3 Shandon® cytocentrifuge with a volume of
50 μl cells adjusted at a concentration of 1 · 109/l, dried and
stained with May Grünwald Giemsa before examination in
light microscopy. Cell count was used to calculate positive
cell quantification from the percentage of positive cells
obtained by flow cytometry (FC) analysis.
Characterization of mononuclear cell in flow cytometry
A three-colour FC analysis was performed by both laboratories
on Epics XL® analysers (Beckman Coulter) to determine the
proportion of CD2+ cells (T lymphocytes and natural killer
cells), CD19+ cells (B lymphocytes), and CD14+ (monocytes)
cells in the cell products. PE-CD2/FITC-CD45, PE-CD19/
FITC-CD45 and PE-CD14/FITC-CD45 (Beckman Coulter)
were used. Cell suspensions adjusted at 5 × 105 per tube were
incubated for 10 min, at room temperature, in the dark, with
10 μl monoclonal antibodies and 20 μl 7-AAD (7-aminoactinomycin) viability dye (Beckman Coulter). After red cell
lysis with Versalyse Lysing Solution® (Beckman Coulter), a
count was performed for each relevant marker among
20 × 103 viable (7-AAD negative), CD45+ cells.
Characterization of stem and progenitor cells in flow
cytometry
CD34+ stem cells. CD34+ stem cell analysis was performed
according to the reference method [14] using PE-CD34
(Clone 581, epitope class III, Beckman Coulter) and
FITC-CD45 anti-human monoclonal antibodies, the 7-AAD
viability dye (Beckman Coulter), PE-IgG1 and FITC-IgG1
(Beckman Coulter) as isotypic controls, and StatusFlow
Pro™ control as target values. The same protocol as for
quantifying MNC was used among 75 × 103 viable cells.
CD34+ cell enumeration was also ensured during the first
step of EPCs quantification with phycocyanin 5 (PCy5)CD34.
Endothelial progenitor cells. Endothelial progenitor cells
were defined as CD34+/CD133+/VEGF-R2+ cells [15–17].
Four-colour labelling was used to assess the co-expression of
CD34, CD133 and VEGF-R2. This allowed the concomitant
enumeration of CD34+, CD34+/CD133+ [18], CD34+/
VEGF-R2+ cells, and CD34+/CD133+/VEGF-R2+ cells. After a
10-min incubation at room temperature of 2 × 106 BM or PBMNC per tube with 5 μl of FcR Blocking Reagent (Miltenyi
Biotec, Bergisch Gladbach, Germany), the cells were processed
for four-colour FC analysis. Filters and photodetectors were
similar in both laboratories for measuring light emitted by
FITC, PE and PCy5, and different for the fourth fluorescence
that was phycoerythrin-texas red (ECD) in laboratory A and
phycocyanin7 (PCy7) in laboratory B. The following antihuman monoclonal antibodies were used: ECD-CD45 (Clone
J33, Beckman Coulter) in laboratory A and PCy7-CD45
(Clone J33, Beckman Coulter) in laboratory B, PCy5-CD34
(Clone 581, epitope class III, Beckman Coulter), and PEVEGF-R2 (R&D System, Wiesbaden, Germany). To identify
CD133+ cells, an indirect method was used with a first
incubation with biotinylated CD133 (Clone AC133, epitope 1,
Miltenyi Biotec) followed by a second with FITC-antibiotin
(Miltenyi Biotec). For each sample, seven tubes were prepared
with, respectively, four isotopic controls (one for each
fluorescence wavelength), one for autofluorescence assessment and two test tubes for positive cell analysis. Two 30-min
incubation periods were performed at 4°C in the dark: first
with CD45, CD34, CD133 and/or VEGF-R2, then with
antibiotin-FITC. After each incubation, the cells were washed
at room temperature in phosphate-buffered saline (PBS;
Dulbecco ohne Ca/Mg, Biochrom AG seromed®, Berlin,
Germany) by centrifugation for 5 min at 200 g. Cell suspensions
were then incubated with Versalyse Lysing Solution® and
IOTest 3® Fixative Solution (Beckman Coulter) following
the manufacturer’s instructions. After a final wash with 5 ml
PBS and centrifugation for 5 min at 200 g, the cells were
resuspended in 1 ml PBS with 25 μl IOTest 3® Fixative
Solution (Beckman Coulter). The gating strategy included
exclusion of platelets, debris, nude nuclei (megakaryocytic and
erythroblastic cells) and non-viable cells, and positive selection
of CD34+/CD133+, CD34+/VEGF-R2+ and CD34+/CD133+/
VEGF-R2+ cells among 400 000 relevant cells (more than
500 000 total events).
Positive cells quantification was calculated from cell
counts and percentages of cells identified by FC.
Mean fluorescence intensity (MFI) ratios were established
using the MFI of each labelled peak vs. that of the peak of
non-specific labelling obtained with the relevant isotypic
control [19]. Fluorospheres (Flowset®, Beckman Coulter)
© 2008 The Author(s)
Journal compilation © 2008 International Society of Blood Transfusion, Vox Sanguinis (2009) 96, 256–265
Cell therapy in critical limb ischaemia
were also used in both laboratories to check for the absence
of significant differences between the two instruments in the
comparable channels. The software for list mode analysis
was XL System 2® (Beckman Coulter) and CXP™ (Beckman
Coulter).
Cell controls. Two types of cell controls were tested with the
same protocol. First, PB-MNC were purified from three
samples obtained from healthy patients by density gradient
centrifugation (d = 1·077, Lymphocyte Separation Medium,
Eurobio, Les Ulis, France) and provided a control for
non-specific labelling. Second, the HEL7 erythroblast cells
(ATCC® number: TIB-180TM) [20] were used as positive
control for VEGF-R2 and CD34 labelling, respectively, and
the HT29 cell line (ATCC® number: HTB-38) for CD133
labelling [21].
Intra- and interlaboratory variation. Intralaboratory evaluation
of assay reproducibility was realized independently in the
two centres. Each laboratory tested 20 samples (11 BM-cell
products and nine PB-cell products) from healthy or CLI
patients for CD34+, CD34+/CD133+, CD34+/VEGF-R2+ and
CD34+/VEGF-R2+ cell counts. A coefficient of variation was
calculated from n repetitive measurements for each assay
(n = 4 or 5 for PCy5-CD34+ cell counts; n = 3 for CD34+/
CD133+ cell counts; and n = 2 for CD34+/VEGF-R2+ and
CD34+/VEGF-R2+ cell counts).
Laboratory reliability was determined using the Bland and
Altman representation [22]. This method led to the determination of a laboratory A and laboratory B mean difference.
This difference is considered as the systematic bias a laboratory
has compared to the other laboratory. In a second step, the
Bland and Altman representation allowed us to determine the
limit of agreement (95% confidence interval of mean difference)
observed between laboratory A and laboratory B.
259
Table 2 Laboratory reliability study (laboratory A–laboratory B) of flow
cytometry (FC) quantification of stem and progenitor cells using the Bland
and Altman summarized representation
BM and PB cell products
(laboratory A–laboratory B)
PE-CD34+ (109/l)
PCy5-CD34+ (109/l)
CD34+/CD133+(109/l)
CD34+/VEGF-R2+ (109/l)
CD34+/CD133+/VEGF-R2+ (109/l)
Mean
difference biasa
Limits of
agreement CI 95%b
–0·037
–0·030
+0·022
–0·00024
–0·000149
[–0·154; +0·079]
[–0·095; +0·036]
[–0·080; +0·124]
[–0·0031; +0·0024]
[–0·0034; +0·0031]
a
Bias correspond to the mean difference between laboratory A and
laboratory B. This represents the systematic bias observed between
laboratory A and laboratory B for a measure.
b
Limit of agreement correspond to the confidence interval (CI) 95% of the
mean difference between laboratory A and laboratory B. This represents the
range of value obtained, with 95% CI, by laboratory B for a given value
measured by laboratory A.
Reliability
PCy5-CD34 labelling being the first step in the characterization
of CD34+/CD133+/VEGF-R2+ EPCs, we compared PCy5CD34+ cell enumeration to PE-CD34+ cell enumeration: the
mean difference and 95% confidence interval between these
enumerations were +0·013% [–0·53; +0·56] and +0·006%
[–0·25; +0·26] in laboratory A and laboratory B, respectively.
Enumeration of stem and endothelial progenitor cells by FC
in both laboratories appeared highly reproducible (Table 2).
A slightly larger difference was noted for immature EPCs in
PB-MNC products, which was found to have a frequency of
about 1 × 10–5.
Statistical analysis
Quantitative variables were expressed as median (Med) and
range [min–max]. Comparisons between cell products were
performed using the Mann–Whitney test. Paired comparisons
used the non-parametric Wilcoxon test. Significance was set
as P < 0·05. The correlation between the different subtypes
of stem and progenitor cells used the non-parametric Spearman
test. Statistical analyses were performed using SAS software
v 8·0 (SAS Institute, Cary, NC, USA).
Results
Owing to the fact that we were dealing with very rare events,
the reliability in both laboratories was assessed. Then, using
this validated strategy of analysis, we characterized the cell
therapy products.
Viability and cell counts in bone marrow and
peripheral blood cell products
The median viability of BM cell products (n = 16) and PB
cell products (n = 8) was 95% [91–99] and 98% [94–99],
respectively, without any difference between both laboratories.
Cell counts of BM and PB cell products are presented in
Table 3. Red blood cells, platelets, total nucleated cells,
lymphocytes and monocytes were in significantly higher
concentration in PB than in BM cell products. The proportion
of remaining mature granulocytes was low, with median
values below 12·7% [2·0–47·5] and without any difference
between BM and PB cell products. As shown in Table 4, FC
analysis confirmed that CD2+ cells (T lymphocytes and
natural killer cells), CD19+ B lymphocytes and CD14+ monocytes were significantly more frequent in PB cell products.
© 2008 The Author(s)
Journal compilation © 2008 International Society of Blood Transfusion, Vox Sanguinis (2009) 96, 256–265
260 J.-C. Capiod et al.
Table 3 Cell counts of BM- and PB-cell productsa
Haematocrit (%)
Platelets (109/l)
Total nucleated cells (109/l)
Total MNCs (109/l)
Lymphocytes (109/l)
Monocytes (109/l)
Erythroblasts (109/l)
Other cellsc (109/l)
Mature granulocytes (109/l)
BM cell products PB cell products
Med [min–max] Med [min–max]
Pb
4·4 [2·4–12·6]
729 [461–1214]
37·8 [18·1–71·5]
32·4 [10·9–52·2]
18·0 [6·2–36·5]
5·7 [2·1–12·4]
2·9 [0·4–13·6]
2·2 [0–4·1]
6·2 [1·9–33·9]
0·0012
0·0002
0·0001
0·0001
0·0002
0·0001
–
–
0·32
10·0 [6·4–21·0]
1491 [916–1756]
122·9 [67·7–165·9]
108·6 [63·1–162·6]
57·9 [23·1–125·8]
42·0 [21·2–61·7]
–
–
8·8 [3·3–31·6]
a
For each sample, the value of each parameter was calculated as the mean of
results from laboratory A and laboratory B; then median, min and max were
calculated from the 16 BM- and the eight PB-cell products. Data were
obtained by combining total cell counts and differentials performed after
counting of 400 cells on May Grünwald Giemsa-stained cytospins.
b
Non-parametric Mann–Whitney test.
c
Other cells: blasts, immature granulocytes and plasma cells.
Quantification of stem and progenitor endothelial
cells in bone marrow and peripheral blood cell
products
Stem and progenitor endothelial cells being very rare events
(CD34+ cells coexpressing CD133 and VEGF-R2 was only
0·002% of total peripheral blood mononuclear cells [15]), we
used two methods for FC characterization: (i) determination of
the percentage of nucleated cells and quantification of the
absolute number of each category of cells in the cell products;
(ii) raw MFI values to quantify the cells. As the cell products
were concentrated after collection on a cell separator,
platelets, debris, nude nuclei (megakaryocytic and erythroblastic
cells) and non-viable cells were numerous and excluded by
gating (Fig. 1a, histogram 1). These events represented a
median of 25·0% (range: 6·9–49·6) of total events, with no
difference between BM and PB cell products.
Cell differential and count in bone marrow- and
peripheral blood-derived products
The population of EPCs (CD34+/CD133+/VEGF-R2+) was a
highly discrete subset with small size and homogenous
structure [forward scatter and side scatter low in FC]: median
values observed for 4 × 105 analysed cells were 56 [6–295]
and 4 [1–10] in BM and PB cell products, respectively (gate
D in histogram 5, Fig. 1a). These subsets were identified as
small clusters, clearly separable from the background and
absent in the relevant isotypic control. Results of stem and
progenitor endothelial cell quantification are presented in
Table 4. EPCs as well as CD34+, CD34+/CD133+ and CD34+/
VEGF-R2+ cells were significantly higher in BM cell products
than in PB cell products. Among CD34+ cells, 55% [27–68]
and 49% [21–82], co-expressed CD133+ in BM and PB cell
products, respectively, without any difference between both
products (P = 0·39). EPCs represented 1·2% [0·2–8·5] and
1·4% [0·3–8·0] of CD34+/CD133+ cells in BM MNC and PB
MNC products, respectively (P = 0·50). The frequency of
CD34+ cells expressing CD133+ and VEGF-R2+ was only
0·7% [0·1–3·6] and 0·8% [0·1–3·9] of the total CD34+ in BM
and PB cell products, respectively (P = 0·52). Among CD34+
cells, 0·6% [0·1–3·8] and 1·9% [0·2–3·9] co-expressed
VEGF-R2+ in BM and PB cell products, respectively
Table 4 Flow cytometry characterization of BM- and PB-cell productsa
CD2+
CD19+
CD14+
PCy5-CD34+
CD34+/CD133+
CD34+/VEGF-R2+
CD34+/CD133+/VEGF-R2+
(109/l)
(109/l)
(109/l)
%c
(109/l)
%c
(109/l)
%c
(109/l)
%c
(109/l)
BM cell products
Med [min–max]
PB cell products
Med [min–max]
Pb
14·8 [4·6–29·2]
2·8 [1·1–8·0]
4·6 [2·0–10·5]
2·27 [0·86–4·53]
0·78 [0·20–3·04]
1·21 [0·23–2·11]
0·47 [0·05–1·48]
0·017 [0·002–0·077]
0·0057 [0·0014–0·0335]
0·014 [0·002–0·074]
0·0047 [0·0009–0·0276]
54·2 [21·7–111·8]
7·0 [0·9–20·2]
37·4 [12·1–54·0]
0·09 [0·06–0·15]
0·10 [0·04–0·24]
0·04 [0·02–0·11]
0·05 [0·02–0·16]
0·002 [0·0003–0·004]
0·0023 [0·0004–0·0046]
0·001 [0·0003–0·002]
0·0011 [0·0002–0·0034]
0·0001
0·032
0·0001
–
0·0001
–
0·0002
–
0·0234
–
0·0022
a
For each sample, the value of each parameter was calculated as the mean of results from laboratory A and laboratory B; then median, min and max were
calculated for the 16 BM-MNC and the eight PB-MNC products.
b
Non-parametric Mann–Whitney test.
c
Percentage of nucleated cells.
© 2008 The Author(s)
Journal compilation © 2008 International Society of Blood Transfusion, Vox Sanguinis (2009) 96, 256–265
Cell therapy in critical limb ischaemia
261
Fig. 1 (a) Quantification of endothelial progenitor cells (EPCs) by a four-colour flow cytometry (FC) analysis in mononuclear cells (MNC). (1) Morphometry
analysis with side scatter (SS) vs. forward (FS); platelets and broken cells are excluded and relevant MNC were selected in gate K. (2) SS vs. ECD- or PCy7-CD45;
relevant MNC were selected by counting 400 000 CD45+/– cells in gate A. (3) Among relevant MNC (AK), total CD34+ cells were selected in gate B. (4) CD34+/
CD133+ cells were selected in gate C. (5) CD34+/CD133+/VEGF-R2+ cells were selected in gate D. (6) and (7) To complete this analysing strategy, two histograms
in fluorescence bi-parametric mode were used to show CD34+/VEGF-R2+ cells (histogram 6, gate H2) and CD34+/CD133+/–/VEGF-R2+/– cells (histogram 7).
(b) Mean fluorescence intensity (MFI) expression. The mean fluorescence intensity and the MFI ratio (test/isotypic control) were calculated from 100% positive
and negative cells [19].
(P = 0·086). In BM cell products, 87% [67–96] of CD34+/
VEGF-R2+ cells co-expressed CD133+ as compared to 50%
[21–86] in PB cell products (P = 0·0009).
The individual distribution of stem and progenitor cells in
the 24 cell products is presented on Fig. 2. A large dispersion
of values was observed in both BM and PB cell products.
Consequently, in some BM cell products, stem and progenitor
cell concentrations were in the range of PB cell products (see,
for example, the cell product from patient no. 8 for stem cells
and from patient no. 24 for EPCs concentrations) (Fig. 2a,b).
Whatever the origin of cell products (BM or PB), a strong
positive correlation was observed between CD34+ and
CD34+/CD133+ cell quantifications (r = 0·98, P < 0·0001).
The correlation between CD34+/VEGF-R2+ and CD34+/
CD133+/VEGF-R2+ cell concentrations was also high
(r = 0·94, P < 0·0001). In contrast, the correlation between
CD34+ and CD34+/CD133+/VEGF-R2+ cell concentrations
was only weakly positive (r = 0·52, P < 0·01) (Fig. 2a,b).
Cell quantification using mean fluorescence intensity
values
When the quantification of the different subsets was
expressed as MFI ratios (Fig. 1b), the results were as indicated
in Table 5. No significant differences were observed in MFI
© 2008 The Author(s)
Journal compilation © 2008 International Society of Blood Transfusion, Vox Sanguinis (2009) 96, 256–265
262 J.-C. Capiod et al.
Table 5 Mean fluorescence intensity (MFI) expression of BM and PB cell
productsa
BM-cell products
Med [min–max]
PCy5-CD34+
CD34+/CD133+
CD34+/CD133+/
VEGF-R2+
MFI ratio 24·8 [6·8–64·9]
MFI ratio 3·5 [1·7–16·1]
MFI ratio 15·7 [5·7–39·5]
PB-cell products
Med [min–max]
Pb
31·3 [27·0–41·5]
3·9 [2·1–9·4]
18·1 [13·0–51·1]
0·46
0·80
0·35
a
For each sample, the value of each parameter was calculated as the mean of
results from laboratory A and laboratory B; then median, min and max were
calculated for the 16 BM-MNC and the eight PB-MNC products.
b
Non-parametric Mann–Whitney test.
of the three markers (observed in 14 BM MNC samples and in
three PB MNC samples), cell products could be classified in
three clusters: low, medium or bright (Fig. 2c).
Using MFI expression, the correlation between CD34+ and
CD34+/CD133+ was weakly positive according to Spearman’s
test (r = 0·46, P < 0·03). However, the correlation between
CD34+ vs. CD34+/CD133+/VEGF-R2+ MFI expression was
moderately positive (r = 0·61, P < 0·01) (Fig. 2c).
Discussion
Fig. 2 Progenitor cells among bone marrow (BM)- and peripheral blood
(PB) mononuclear cells (MNC). (a) Individual stem cell concentrations
(PCy5-CD34+ and CD34+/CD133+), (b) endothelial progenitor cell
concentrations [CD34+/VEGF-R2+ cells and immature endothelial
progenitor cells (EPCs)] and (c) their mean fluorescence intensity (MFI)
expression. For each sample, the values displayed are the mean of results
obtained in laboratory A and laboratory B. BM cell products no. 6 and no. 8
came from the same patient. BM cell product no. 16 and PB cell product
no. 19 came from the same patient. Numbers matched with the clinical trial
patient’s inclusion numbers.
expressions between BM and PB cell products; and that for
either CD34+, CD34+/CD133+ or CD34+/CD133+/VEGF-R2+
cells. However, MFI ratio comparison showed the presence of
two types of cell products: without and with a homogeneous
MFI ratio, that is, varying in the same way for CD34, CD133
and VEGF-R2 markers. Among the homogeneous MFI ratios
Precise characterization of cell products used is essential in
clinical trials of cell therapy. We report here data on BM and
PB cell products implanted in patients with CLI included in
such a trial. Owing to the potential importance of EPCs, we
focused on enumeration of these cells.
Endothelial progenitor cells are rare events [23] and their
quantification by FC is challenging. Using strict technical
conditions and following published recommendations [24],
we showed that accuracy and reliability between laboratories
can be achieved. The results presented here are considerably
strengthened by the fact that they were obtained concomitantly
in two different laboratories, demonstrating that sticking to
rigorously established standard operating procedure [25] can
provide reliable results in multicentre studies. Furthermore,
the standard deviation for relatively rare populations is
simply n–1/2 where ‘n’ does the number of events comprise
the subset. Indeed, when one collects a million events,
finding a single event in a gate is not meaningful [26]. Such
standard operating procedures and new generation instruments
could further improve EPCs quantification, mainly by
shortening both preparation and analysis time by allowing
the use of a wider range of conjugates and a faster acquisition
of the large numbers of events necessary for reliable enumeration
of such rare events as EPCs [27].
Contrary to previously reported studies, PB MNC were
collected in our trial without any previous mobilization (in
order to avoid possible side-effects reported with the use of
© 2008 The Author(s)
Journal compilation © 2008 International Society of Blood Transfusion, Vox Sanguinis (2009) 96, 256–265
Cell therapy in critical limb ischaemia
haematopoietic growth factors in patients with arterial
diseases) [28,29]. In these circumstances, significant differences
were observed according to the origin of the MNC. As
expected, the proportions of both mature and immature cell
subsets were different: mature cells (including lymphocytes,
monocytes and platelets) were in higher concentration in PB
cell products, while EPCs as well as CD34+, CD34+/CD133+,
CD34+/VEGF-R2+ were significantly higher in BM cell
products. Nevertheless, a great interindividual heterogeneity
was observed: in some BM cell products, progenitor cell
concentration was as low as in PB cell products. This variability
may be related to the patient’s cardiovascular status or the
ongoing therapy [30,31]. All these data are to be interpreted
taking into consideration that the mechanisms by which
MNCs can induce angiogenesis are not yet known. Experimental
data suggest that implanted cells, especially EPCs, are
incorporated into vascular structures [32]. If this is true, the
use of BM MNC, which are rich in stem/progenitor cells,
would be preferable. Other studies are in favour of an indirect
effect of implanted MNC that may secrete cytokines and/or
growth factors [33]. PB MNC, even collected without any
previous mobilization, would be as effective as BM MNC. The
paracrine effect of monocytes has been reported [34].
Platelets are a source of growth factors [35], and products
obtained by apheresis are currently rich in platelets.
Lymphocytes could also play a role as they release numerous
factors potentially involved in angiogenesis [36]. Finally, the
quantity of MNC that should be implanted for optimal
efficacy is unknown.
There is no consensus regarding the immunophenotype of
EPCs. As CD45 expression on EPCs is controversial [37,38],
we chose to analyse CD45– and CD45+ cells. For this,
aggregated platelets and nude nuclei were excluded. In
accordance to most previous reports, we defined EPCs by the
co-expression of CD34+/CD133+/VEGFR-2+. However, it
has recently been reported that these cells, when isolated, are
unable to generate endothelial cells in culture [23]. Subsequently,
the co-expression of CD34+/VEGF-R2+ appears as the best
combination to define EPCs [39].
In our study, the cellular therapy product obtained from
BM presents analogies with cellular therapy products used in
CLI in other studies, in terms of total amount of implanted
BM MNC, of CD34+ cell amount, and of percentage of CD34+
cells among the BM-MNC [1,3,5]. The cellular therapy
products obtained from PB in our study (without previous
mobilization) was less concentrated, when compared to
cellular therapy products used (after previous mobilization
by granulocyte–colony-stimulating factor) in CLI in other
studies, in terms of total amount of implanted PB MNC, of
CD34+ cell amount, and of percentage of CD34+ cells among
the PB MNC [4,6]. In these studies, the characterization of
cellular therapy products was limited to the quantification of
total MNC and of CD34+ cells. In our study, the MFI ratio and
263
the quantification of EPCs showed a large diversity of cellular
subpopulations, but their characterization needs further studies.
We did not observe any significant difference in the
percentage of CD34+ cells co-expressing CD133 or VEGF-R2
and of CD34+/CD133+ cells co-expressing VEGF-R2 between BM and PB cell products. Conversely, the percentage
of CD34+/VEGF-R2+ cells co-expressing the CD133 marker
was significantly higher in BM than in PB cell products. This
can be explained by the fact that EPCs loose CD133 when
leaving BM and maturing [15]. Circulating CD34+/
VEGF-R2+ cells could also include mature ECs after their
detachment from the vessel wall [40]. However, mature ECs
cells have been characterized by their morphometry analysis
in FC (high size and heterogeneous structure) [41]. We thus
decided to exclude these cells in our gating strategy.
With either cell product (BM or PB), the correlation
between CD34+ cells and EPCs concentrations was weakly
positive. This has to be kept in mind when choosing the best
parameter to characterize a cell product aimed at inducing
angiogenesis. Quantification of CD34+ cells is easy and
well-standardized. However, the CD34 marker is not specific
to angiogenic stem cells.
In order to assess whether or not the epitope density on
different cell types could influence the results when
expressed as a percentage of positive cells, we analysed raw
MFI. Using the ratio defined in the material and method
section, similar MFI ratios for CD34, CD133 and VEGF-R2
were observed when comparing BM and PB cell products.
However, the MFI ratios were very different between cell
products, whether the origin was BM or blood. This variability
may indicate differences between donors. Of interest, we
noted that MFI ratios in two BM cell products that were
harvested in the same patient were similar. Besides, MFI
should allow determining the level of expression of CD34,
CD133, VEGF-R2 and possible other markers. The next step
would be to correlate the epitope density of such markers
with the functional capacity of cells [42,43]. This approach
should be challenged and validated by clinical trials.
In large multicentre trials that will be necessary to prove
the efficacy of cell therapy in CLI, it will be crucial to take
into account the characterization of the cell products used.
Owing to the fact that the involved mechanisms are not yet
known, mature as well as stem/progenitor cells have to be
considered. If EPCs turned out to be the active cells, then, a
consensual definition of these cells should be proposed. Other
stem/progenitor cells, such as mesenchymal stem cells [44]
or multipotent adult progenitor cells [45], possibly present in
cell products, may also be worthwhile to consider.
Acknowledgements
The study is registered on clinicaltrials.gov under the number:
NCT00533104. Furthermore, the authors appreciated the
© 2008 The Author(s)
Journal compilation © 2008 International Society of Blood Transfusion, Vox Sanguinis (2009) 96, 256–265
264 J.-C. Capiod et al.
contribution of Sylvie Remy, Valérie Creuza, Catherine Massé,
and Jacques Vigne. This study was funded by a clinical
research hospital programme grant (French ministry of
health, PHRC 2003).
12
13
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