CD14+CD34low Cells With Stem Cell Phenotypic and Functional

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CD14+CD34low Cells With Stem Cell Phenotypic and Functional
CD14ⴙCD34low Cells With Stem Cell Phenotypic and
Functional Features Are the Major Source of Circulating
Endothelial Progenitors
Paola Romagnani,* Francesco Annunziato,* Francesco Liotta, Elena Lazzeri, Benedetta Mazzinghi,
Francesca Frosali, Lorenzo Cosmi, Laura Maggi, Laura Lasagni, Alexander Scheffold, Manuela Kruger,
Stefanie Dimmeler, Fabio Marra, Gianfranco Gensini, Enrico Maggi, Sergio Romagnani
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Abstract—Endothelial progenitor cells (EPCs) seem to be a promising tool for cell therapy of acute myocardial infarction,
but their nature is still unclear. We show here that EPCs obtainable from peripheral blood (PB) derive from the
adhesion-related selection in culture of a subset of CD14⫹ cells, which, when assessed by the highly-sensitive
antibody-conjugated magnetofluorescent liposomes (ACMFL) technique, were found to express CD34. These
CD14⫹CD34low cells represented a variable proportion at individual level of CD14⫹ cells, ranging from 0.6% to 8.5%
of all peripheral-blood leukocytes, and constituted the dominant population among circulating KDR⫹ cells. By using
the ACMFL technique, virtually all CD14⫹ cells present in the bone marrow were found to be CD14⫹CD34low
double-positive cells. EPCs, as well as purified circulating CD14⫹CD34low cells, exhibited high expression of
embryonic stem cell (SC) markers Nanog and Oct-4, which were downregulated in a STAT3-independent manner when
they differentiated into endothelial cells (ECs). Moreover, circulating CD14⫹CD34low cells, but not CD14⫹CD34⫺
cells, proliferated in response to SC growth factors, and exhibited clonogenicity and multipotency, as shown by their
ability to differentiate not only into ECs, but also into osteoblasts, adipocytes, or neural cells. The results of this study
may reconcile apparently contradictory data of the literature, showing the generation of PB-derived EPCs from either
CD34⫹ or CD14⫹ cells. We suggest that the use of this previously unrecognized population of circulating
CD14⫹CD34low cells, which exhibit both phenotypic and functional features of SCs, may be useful in improving
cell-based therapies of vascular and tissue damage. (Circ Res. 2005;97:314-322.)
Key Words: endothelial progenitor cells 䡲 monocytes 䡲 CD14⫹CD34low cells 䡲 Nanog
I
Thus, the recovery of a sufficient number of these cells for
therapeutic treatments usually requires the mobilization of
bone marrow (BM)-derived CD34⫹ or CD133⫹ populations
by administration of granulocyte colony-stimulating factor
(G-CSF);5,7 however, this treatment has been questioned,
because of the high risk of adverse events in subjects with
vascular disorders.8 On the other hand, adherence-related
selection of cultured PBMNCs allows for the recovery of a
number of EPCs sufficient for therapeutic treatments, suggesting that EPCs can also originate from circulating populations other than CD34⫹ or CD133⫹ progenitors.1–5 Accordingly, conventional cytofluorimetric techniques of
PBMNCs-derived EPCs have shown that these cells consist
of a population sharing monocytic and EC markers, but not
the classic stem cell (SC) markers CD34 and CD133.1–5,9,10
Thus, the precise nature of cells effective in clinical trials4,5
remains unclear.
nfusion of endothelial progenitor cells (EPCs) augments
neovascularization of tissue after ischemia providing a
novel therapeutic option.1–3 Indeed, these cells have been
successfully used to repair tissue damage in both murine
experimental models3–5 and in preliminary trials performed in
humans experiencing acute myocardial infarction.4,5 EPC
populations are usually derived from peripheral blood mononuclear cells (PBMNCs) cultured in presence of vascular
endothelial growth factor (VEGF), and identified as a population of adherent cells expressing both acLDL and Ulexlectin. However, controversy exists with respect to the true
nature and origin of circulating EPCs. Although it is well
known that CD34⫹, or CD133⫹ progenitors, purified by the
immunomagnetic technique and cultured in the presence of
VEGF, can generate endothelial cells (ECs) and exhibit
revascularization properties in vivo,1–7 these cells represent a
very small subset of PBMNCs, ranging from 0.02% to 0.1%.
Original received December 15, 2004; resubmission received April 28, 2005; revised resubmission received July 5, 2005; accepted July 6, 2005.
From the Center for Research (P.R., F.A., F.L., E.L., B.M., F.F., L.C., L.M., L.L., S.D., F.M., G.G., E.M., S.R.), Transfer and High Education
DENOTHE, University of Florence, Italy; the Deutches Rheuma Forschungszentrum (A.S., M.K.), Berlin, Germany; and the Department of Molecular
Cardiology (S.D.), University of Frankfurt, Germany.
*Both authors contributed equally to this work.
Correspondence to Sergio Romagnani, Dipartimento di Medicina Interna, Università di Firenze, Viale Morgagni 85 Firenze 50134-Italy. E-mail
[email protected]
© 2005 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000177670.72216.9b
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EPCs Derive From Nanogⴙ CD14ⴙCD34low Cells
Romagnani et al
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In this study, we demonstrate that PBMNC-derived EPCs
appear to be CD14⫹ by using the conventional cytofluorimetric technique, but virtually all of them were also found to
express low levels of surface CD34, when assessed by
highly-sensitive flow cytometric techniques, such as the
antibody-conjugated magnetofluorescent liposomes (ACMFL)11 and the fluorescence amplification by sequential
employment of reagents (FASER). PBMNC-derived EPCs
resulted from the adherence-related selection in culture of a
subset of double-positive CD14⫹CD34low cells preexisting,
even if highly diluted, in the circulation. CD14⫹CD34low
cells constituted the dominant population among circulating
KDR⫹ cells and exhibited high expression of mRNA for
embryonic SC (ESC) markers, such as Nanog12,13 and Oct4,14 as well as for a marker of adult SCs, Bmi-1.15 The
expression of stemness markers was strongly downregulated
in a STAT-3–independent manner after in vitro differentiation of these cells into mature ECs. Finally, circulating
double-positive CD14⫹CD34low cells exhibited clonogenicity
in response to SC growth factors and gave rise not only to
ECs, but also to osteoblasts, adipocytes, or neural cells.
These data indicate that the major source of EPCs obtainable from peripheral blood (PB) is a subset of double-positive
CD14⫹CD34low cells, showing phenotypic and functional
features of multipotent SCs. These properties distinguish
them from truly differentiated monocytes, allowing their
recovery from PB for cell-based therapies of vascular damages, as well as other tissue damage.
Materials and Methods
Reagents
See online data supplement available at http://circres.ahajournals.org.
Tissues and Cells
Primary cell cultures were obtained as previously described.16,17 See
online data supplement.
Immunomagnetic Isolation of Circulating Cells
Isolation of CD14⫹ monocytes, CD34⫹ HSCs, or KDR⫹ cells was
performed by using the MACS system, as described elsewhere.18
Cell Cultures
PBMNCs (8⫻106 cells per well), CD14⫹, CD14⫹CD34⫺, and
CD14⫹CD34low cells were cultured as previously described.1–2 See
online data supplement.
Flow Cytometry Cell Analysis and Sorting
Flow cytometry analysis of cell surface molecules was performed as
detailed elsewhere.18 CD14⫹CD34low and CD14⫹CD34⫺ as well as
CD133⫹CD34⫹ or CD133⫺CD34⫹ cells were sorted by using a
FACS ARIA with the Diva.
ACMFL and FASER Techniques
The ACMFL technique was performed as detailed elsewhere.
online data supplement.
11
See
Western Blotting
STAT3 phosphorylation was detected in CD14⫹CD34low cells by
Western blotting (see online data supplement), as detailed
elsewhere.19
315
Real-Time Quantitative RT-PCR (TaqManTM)
Taq-Man RT-PCR was performed as described elsewhere.17 See
online data supplement.
Confocal Microscopy
Confocal microscopy was performed as previously described.20 See
online data supplement.
Labeling With CFDA-SE
Labeling of circulating CD14⫹CD34low or CD14⫹CD34⫺ sorted
cells with CFDA-SE was performed as described previously.18
Colony Formation and In
Vitro Multidifferentiation
Generation of clonal cell lines and in vitro differentiation into
osteoblasts, adipocytes or neural cells from sorted circulating
CD14⫹CD34low cells was preformed as described elsewhere.21,22 See
online data supplement.
Statistical Analysis
Statistical analysis were performed using SPSS software (SPSS, Inc).
See online data supplement.
Results
EPCs Derived From In Vitro Cultured Human
Adult PBMNCs Are Double-Positive
CD14ⴙCD34low Cells
Culturing human adult PBMNCs for 5 days in EGM-MV
supplemented with VEGF resulted in an adherent population
consisting mainly of acLDL⫹ Ulex-lectin⫹ cells (⬇90%), as
assessed by confocal microscopy (Figure 1A), thus matching
the previously described EPC phenotype.1–5 In agreement
with the described features of PBMNC-derived EPCs,1,2
virtually all adherent cells examined on day 5 of culture
expressed CD14 together with other markers (CD11c, CD16,
CD31, CD86, CD105, HLA-DR), in the apparent absence of
SC (CD34, CD133) markers (online Table I).
These findings were confirmed at RNA level by using Real
time RT-PCR (Figure 1B and data not shown), the only
exception being the high expression in PBMNC-derived
EPCs of CD34 mRNA (Figure 1B), which was in contrast
with the lack of surface CD34 protein (online Table I). The
marked dissociation in CD14⫹ EPCs between mRNA and
protein CD34 expression allowed the hypothesis that these
cells could display CD34 on their surface at levels below
detection sensitivity of the classic flow cytometry. To verify
this possibility, we assessed the presence of CD34 on the
same cells by a highly sensitive technique, such as ACMFL,
which can increase fluorescence signal intensity 100- to
1000-fold compared with conventional methods.11 By using
this technique, virtually all EPCs were found to express on
their surface not only CD14, but also CD34 (Figure 1C).
EPCs Derived From In Vitro Cultured PBMNCs
Result From the Adhesion-Related Selection Of
Circulating Double-Positive CD14ⴙCD34low Cells
To establish whether CD14⫹CD34low EPCs found on day 5 of
culture derived from circulating CD14⫺CD34⫹ hematopoietic SCs (HSCs) or CD14⫹ MNCs, CD14⫹ cells were first
purified from PBMNC suspensions by the immunomagnetic
technique. The few CD14⫺CD34⫹ HSCs (0.012% to 0.2%)
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Figure 1. EPCs obtained by culturing PBMNCs in VEGF are
CD14⫹ cells that exhibit high mRNA levels for, but low surface
protein expression of, CD34. A, FITC-Ulex-lectin (green) binding,
Dil-labeled acLDL (red) uptake, and Topro-3 (blue) nuclear staining in adherent cells found 5 days after culturing PBMNCs in
EGM-MV supplemented with VEGF (Bar⫽20 mm). One representative of 7 experiments is shown. B, Detection by real time
RT-PCR of mRNA for CD14, CD105, CD34, and CD133 in
PBMNCs before culturing (day 0) and on day 5 of culture under
the conditions described in Materials and Methods. Columns
represent mean values (⫾SD) obtained in 13 separate experiments. C, Surface CD34 expression by EPCs, as assessed by
conventional flow cytometry (left) or the ACMFL technique
(right). One representative of 13 separate experiments is shown.
were then isolated from the CD14⫺ fraction by the same
technique, and the degree of purification was assessed by
flow cytometry. CD14⫹ cells were then stained with
CFDA-SE (green) and CD34⫹ HSCs with PKH26 (red), and
both populations were mixed with the unstained
CD14⫺CD34⫺ cells in proportions corresponding to those
present in the starting PBMNC population. On day 5 of
culture, ie, 1 day after the removal of nonadherent cells, the
proportions of green- or red-stained adherent cells were
evaluated. Adherent cells consisted mainly of green (CD14⫹)
cells (⬇90%), whereas the proportions of red (CD34⫹) cells
never exceeded 1% of the adhering population (Figure 2B),
suggesting that, at least at this culture time, CD14⫺CD34⫹
HSCs had not represented a relevant source of EPCs, whereas
the great majority of them originated from CD14⫹ cells. To
exclude the possibility that preselection of CD14⫹ cells
results in a bias for the generation of EPCs from CD34⫹
HSCs, in subsequent experiments CD34⫹ HSCs were first
selected by the immunomagnetic technique and stained with
PKH26 (red), whereas CD14⫹ cells were subsequently
isolated by the same technique from the remaining CD34⫺
fraction and stained with CFDA-SE (green). The 2 stained
populations were mixed together with the unstained CD14CD34⫺ cells, cultured under the conditions described above,
and the proportions of green- or red-stained adherent cells
present on day 5 were evaluated. Again, adherent cells
consisted mainly of green (CD14⫹) cells (⬎90%), whereas
the proportions of red (CD34⫹) cells were never higher than
1% (online Figure I), supporting the concept that independently of which cell fraction was first selected, virtually all
cells giving rise to EPCs were contained in the CD14⫹
population. We therefore asked whether EPCs were derived
from CD14⫹ cells capable of acquiring CD34 after their
culturing or from the selection of a preexisting circulating
subset of CD14⫹CD34low cells. To this end, CD14⫹ cells
were purified from PBMNCs by the immunomagnetic technique and then assessed for both CD14 and CD34 expression.
As expected, by using conventional flow cytometry, virtually
all purified CD14⫹ cells expressed CD14 but not CD34
(Figure 3A). However, when the highly sensitive ACMFL
technique was used, a high proportion of CD14⫹ cells (range
12% to 75%; mean value: 47%⫾16%) were found to coexpress CD34 (Figure 3D). Subsequent isolation by the conventional technique from the remaining PBMNCs of CD34⫹
HSCs (Figure 3B) and their assessment by ACMFL, demonstrated that ⬇100⫻ higher levels of CD34 protein were
present on CD34⫹ HSCs than on CD14⫹CD34low cells
(Figure 3E). By contrast, no CD34 expression was observed
on the remaining CD14⫺CD34⫺ PBMNCs even with the
ACMFL technique (Figure 3C and 3F). Similar results were
obtained by another highly sensitive technique, such as
FASER (Figure 3G, 3H, and 3I). No CD133 expression was
observed on total CD14⫹ or CD14⫺CD34⫺ cells by either
classic flow cytometry, ACMFL, or FASER (data not
shown). The proportions of CD14⫹CD34low cells were then
evaluated by ACMFL in PBMNCs from a total number of 20
healthy subjects, aged between 24 and 40 years. Percentages
of CD14⫹CD34low cells ranged from 0.6% to 8.5% of all PB
leukocytes, the mean value being 4.0%⫾2.7%.
Previous work has shown that EPCs are contained in the
circulating KDR⫹ population.23 To establish whether
CD14⫹CD34low cells are contained in the same population,
KDR-expressing cells were purified from PBMNCs by the
immunomagnetic technique and then assessed for CD14 and
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Figure 2. PBMNC-derived EPCs are
contained in the CD14⫹ cell fraction. A,
CD14⫹, CD14-CD34⫹, or
CD14⫺CD34⫺ cells were purified by the
immunomagnetic technique and then
assessed by classic flow cytometry for
CD14 and CD34 expression. B, Purified
CD14⫹ cells were labeled with CFDA-SE
(green) and purified CD34⫹ HSCs with
PKH26 (red), mixed together with the
unlabeled CD14⫺CD34⫺ cells in proportions corresponding to those found in
the starting PBMNC population, and then
cultured in presence of VEGF. On days 0
and 5 of culture, the percentages of
green- or red-stained cells were evaluated by flow cytometry. One representative of 4 separate experiments is shown.
CD34 expression by conventional flow cytometry. More than
90% of these cells were CD14⫹, whereas proportions of
CD34⫹ cells were never higher than 1% (Figure 4A).
However, when CD34 expression was assessed by ACMFL,
the great majority of KDR-purified cells were found to be
CD14⫹CD34low cells (Figure 4B).
Finally, the possibility that CD14⫹CD34low cells could
exist also in the BM was investigated. As expected, by using
conventional flow cytometry substantial numbers of both
single positive CD14⫹ or CD34⫹ cells could be detected in
the BM (Figure 4C). Surprisingly, however, when CD14⫹
BM cells were examined for the expression of CD34 by the
ACMFL technique, virtually all them appeared to be
CD14⫹CD34low cells (Figure 4D).
Circulating CD14ⴙCD34low Cells Exhibit
Phenotypic Markers of SCs
We then asked whether circulating CD14⫹CD34low possess
phenotypic markers of SCs. To this end, the expression of
Nanog, a transcription factor that plays key roles in self-renewal
and maintenance of pluripotency in ESCs,12,13 was quantitatively
assessed in several adult human tissues, as well as in freshly
derived circulating cell populations or primary cultures of
different human cell types by real time quantitative RT-PCR
(Figure 5A). High Nanog mRNA expression was detectable in
human teratocarcinoma, whereas normal human BM, heart,
kidney, prostate, skeletal muscle, and spleen exhibited very low
or undetectable Nanog mRNA levels, slightly higher levels
being found only in adult testis (Figure 5B). Very low or
undetectable Nanog mRNA levels were also observed in pri-
mary cultures of keratinocytes, human renal proximal tubular
epithelial cells, human microvascular endothelial cells, human
aortic smooth muscle cells, human dermal fibroblasts, or
PBMNCs. By contrast, substantial amounts of Nanog mRNA
levels were detectable in purified CD133⫹CD34⫹(CD14⫺) or
CD133⫺CD34⫹(CD14⫺) and, even if at lower levels, in
purified CD14⫹ PBMNCs (Figure 5C). However, when purified CD14⫹ cells were sorted into CD14⫹CD34low and
CD14⫹CD34⫺ cells by the ACMFL technique, Nanog expression appeared to be enriched in CD14⫹CD34low cells and was
negligible in the CD14⫹CD34⫺ cell fraction (Figure 5C).
To further support the SC nature of CD14⫹CD34low
PBMNCs and to distinguish them from fully differentiated
CD14⫹CD34⫺ monocytes, CD14⫹CD34low and CD14⫹C34⫺
cells sorted by the ACMFL technique (Figure 6A) were
cultured in EGM-MV supplemented with VEGF. Nanog, as
well as another ESC marker (Oct-4)14 and an adult SC marker
(Bmi-1),15 were assessed before culturing, as well as on day
5 and on day 11 of culture. Low levels of these markers were
found in CD14⫹CD34⫺ cells at all times of culture (Figure
6B), and these cells did not acquire phenotypic markers of
mature ECs (Figure 6C). By contrast, the 3 SC markers were
expressed by fresh derived CD14⫹CD34low PBMNCs and
were maintained at high levels until day 5 of culture, then
declining to became virtually undetectable on day 11, when
the cells acquired the phenotypic markers of ECs (Figure 6B
and 6C). Of note, Nanog protein could also be detected in
both fresh and 5-day-cultured CD14⫹CD34low cells by using
confocal microscopy (online Figure II). Similar results were
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Figure 3. Demonstration of a subset of circulating PBMNCs expressing both CD14 and low CD34 on their surface and its comparison
with CD34⫹ HSCs. Circulating CD14⫹ or CD34⫹ cells were purified by the immunomagnetic technique and then assessed for CD34
expression by classic flow cytometry, as well as by ACMFL or FASER techniques. Detection by classic flow cytometry of CD14 and
CD34 on CD14⫹ (A), CD34⫹ (B), or CD14⫺CD34⫺ (C) cells. Detection by ACMFL of CD34 (black line) or IgG1 isotype control (dotted
line) on CD14⫹ (D), CD34⫹ (E), or CD14⫺CD34⫺ (F) cells. Detection by FASER of CD34 (black line) or IgG1 isotype control (dotted
line) on CD14⫹ (G), CD34⫹ (H), or CD14⫺CD34⫺(I) cells. One representative of 10 separate experiments is shown.
obtained when Oct-4 and Bmi-1 expression was assessed
(data not shown).
It has been shown that Nanog expression in human ESCs is
downregulated when these cells are induced to differentiate
and that, unlike in mouse ESCs, this occurs in a STAT-3–
independent manners.24 We therefore assessed whether
CD14⫹CD34low cells exhibited a similar behavior. Indeed,
although both fresh CD14⫹CD34low cells and EPCs were
found to express gp130 receptors (Figure 7A), and to respond
to leukemia inhibitory factor (LIF) with STAT-3 phosphorylation (Figure 7B), their undifferentiated state was not
maintained, as shown by the downregulation of Nanog
(Figure 7C) and Oct-4 (data not shown) and the upregulation
of ECs markers (Figure 7D).
Circulating CD14ⴙCD34low Cells Possess
Clonogenicity and Multidifferentiation Capacity
To provide direct evidence that circulating CD14⫹CD34low cells
also possess the functional features of SCs, CD14⫹CD34low
were separated from CD14⫹CD34⫺ cells, and both populations
assessed for their ability to proliferate in response to stem cell
factor (SCF), fms-like tyrosine kinase 3-ligand (Flt3-L), and
thrombopoietin. Only CD14⫹CD34low cells proliferated in response to SC growth factors, as detected by the CFDA-SE
technique (Figure 8A). Moreover, under the same conditions,
CD14⫹CD34low cells showed the ability to generate clones,
which consisted of at least 30 to 300 cells each (Figure 8B), with
a clonal efficiency equal to 27%⫾6%, although the clonal
efficiency of CD14⫹CD34⫺ was irrelevant (0.8%⫾0.3%).
Figure 4. CD14⫹CD34low cells are the
majority of circulating KDR⫹ cells and
represent the only type of CD14⫹ cells
present in the BM. A, Detection by classic flow cytometry of CD14 (green line),
CD34 (red line), or IgG1 isotype control
(black line) on circulating KDR⫹ cells,
purified by the immunomagnetic technique. B, Detection by ACMFL of CD34
(red line) or IgG1 isotype control (black
line) on KDR⫹CD14⫹ cells. One representative of 4 separate experiments is
shown. C, Detection by classic flow
cytometry of CD14 and CD34 on total
BM-derived MNCs. D, Detection by
ACMFL of CD34 (red line) or IgG1 isotype control (black line) on BM-derived
CD14⫹ cells. One representative of 4
separate experiments is shown.
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Figure 5. Detection by quantitative RT-PCR of Nanog mRNA expression in different adult tissues, freshly derived cells, and primary cell
cultures. A, Amplification plot of Nanog standard curves generated using serial dilutions of a known amount of the plasmid containing
the Nanog amplicon. B, Nanog assessment in pooled mRNA obtained from different human tissues. C, Nanog assessment in pooled
mRNA obtained from primary cultures of keratinocytes, human renal proximal tubular epithelial cells, circulating MNCs, human microvascular endothelial cells, human aortic smooth muscle cells, fibroblasts, and in purified circulating CD133⫹CD34⫹, CD133⫺CD34⫹,
total CD14⫹, CD14⫹CD34low, or CD14⫹CD34⫺, cells.
Finally, sorted CD14⫹CD34low cells cultured under appropriate
conditions were able to differentiate not only into mature ECs
(Figure 6D), but could also originate osteoblasts (Figure 8C),
adipocytes (Figure 8D), or neural cells (Figure 8E). Differentiation into osteoblasts was demonstrated by the ability of almost
every adherent cell to stain for alkaline phosphatase and to form
calcium deposits, which stained with Alizarin Red (Figure 8C).
Moreover, undifferentiated CD14⫹CD34low cells did not express the bone-specific transcription factors Osterix and Runx2,
but their expression was markedly upregulated after the induction treatment (Figure 8C). Differentiation into adipogenic phenotypes was confirmed by characteristic cell morphology and oil
red O staining of lipid vacuoles, that was completely absent from
undifferentiated cells (Figure 8D). Furthermore, real-time RTPCR demonstrated the appearance of very high levels of AP-2
and PPAR␥ mRNA, two adipocyte-specific transcription factors, that were completely absent in undifferentiated
CD14⫹CD34low cells (Figure 8D). Finally, undifferentiated
CD14⫹CD34low cells did not express glial fibrillary acidic
protein (GFAP), neurofilament 200, or the mRNA for neuron
specific enolase, although the expression of all these markers
was markedly upregulated after the induction treatment (Figure
8E). The same multidifferential potential was observed when
differentiation media were added to CD14⫹CD34low-derived
EPCs after 5 days of culture in EGM-MV supplemented with
VEGF (data not shown).
Discussion
Although it has been demonstrated that CD14⫹ cells can
generate EPCs and exhibit revascularizing properties,1–5 they
are considered as terminally differentiated cells and there is
still no proof of their possible “stemness,” or of their
relationships with angioblasts derived from CD34⫹ cells.
Indeed, some authors suggested that the angiogenic effects of
EPCs derived from circulating monocytes might be mediated
by growth factor secretion.9 Recently, however, Kuwana et
al25 described a population of CD14⫹ monocytes that could
differentiate into several distinct mesenchymal cell lineages.
These cells, named as monocyte-derived mesenchymal progenitors (MOMPs), were obtained from circulating MNCs
cultured on fibronectin for 7 days and had a unique molecular
phenotype-CD14⫹CD45⫹CD34⫹.25 MOMPs could be obtained from PB even if deprived of CD34⫹ cells, but their
source, as well as their SC nature, was not clearly defined.
Furthermore, the possible relationship between MOMPs and
EPCs was not investigated.
In this study, we demonstrate that cells which are usually defined
as PBMNC-derived EPCs are CD14⫹ cells characterized by CD34
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Figure 7. Activation of the gp130/STAT3 pathway is not required
for maintaining CD14⫹CD34low cells in their undifferentiated state.
A, Levels of gp130 mRNA were assessed by real time RT-PCR on
freshly-purified CD14⫹CD34low cells and on EPCs derived by culturing PBMNCs. B, Induction by LIF of STAT-3 phosphorylation in
CD14⫹CD34low cells. Cells were left untreated or exposed to LIF
(10 ng/mL) for the indicated time points. Total protein lysates were
analyzed by immunoblotting using antibodies directed against
tyrosine-phosphorylated Stat3 (Y705, upper panel). The membrane
was reprobed for total Stat3 to ensure equal loading (lower panel).
Migration of molecular weight markers is indicated on the left. C,
Strong downregulation of Nanog mRNA levels in CD14⫹CD34low
cells cultured in EGM-MV supplemented with VEGF, which associates with the appearance of EC markers (D), independently of LIF
treatment. Data represent mean values (⫾SD) obtained in 4 separate experiments. In C and D, 1 representative of 4 separate experiments is shown.
Figure 6. CD14⫹CD34low but not CD14⫹CD34⫺ cells express
Nanog, Oct-4, and Bmi-1 mRNA and differentiate into ECs. A,
Total circulating CD14⫹ cells were sorted into CD34⫺ and CD34low
cells by applying the ACMFL technique and assessed by the same
technique for CD34 expression. B, Nanog, Oct-4, and Bmi-1
mRNA expression, as assessed by real time RT-PCR, on the same
populations on days 0, 5, and 11 of culture under the conditions
described in Materials and Methods. C, KDR and vWF mRNA
upregulation in the same samples. Columns represent mean values
(⫾SD) obtained in 6 separate experiments.
surface expression at levels below the detection limits of the classic
flow cytometry. Indeed, by using techniques ⬇100- to 1000-fold
more sensitive, such as ACMFL and FASER, we could demonstrate that virtually all adhering EPCs were double-positive
CD14⫹CD34low cells. These cells originated from the adherencerelated selection of a subset of CD14⫹CD34low cells, representing
proportions between 12% and 75% of circulating CD14⫹ cells,
depending of the subject analyzed. The existence of a circulating
double-positive CD14⫹CD34low population may reconcile apparently contradictory data of the literature, some showing the generation of EPCs from CD14⫹,9,10 and others from CD34⫹6,7 cells,
when recovered by the classic immunomagnetic technique with
anti-CD14 or anti-CD34 antibodies, respectively.1–7,9–10 It can also
explain the variability in the numbers of EPCs isolated in previous
studies from the PB of human adults.1–5 The low levels of CD34
and the higher levels of CD14 expression on these cells is consistent
with the observation that more than 95% of this population is
usually recovered together with CD14⫹ cells, and only an irrelevant percentage can be recovered together with CD34⫹ HSCs, thus
also explaining why PBMNC-derived EPCs appear as CD14⫹
cells if analyzed after their adhesion in culture.
To provide further evidence that CD14⫹CD34low cells are the
major source of PBMNC-derived EPCs, we purified KDR⫹
cells from PB by the immunomagnetic technique and assessed
the expression of CD14 and CD34 by conventional flow
cytometry or ACMFL. Indeed, KDR is expressed by ECs but is
also considered as a critical marker of EPCs,1–5,7,23 and its
expression on CD34⫹ cells has been reported to identify true
SCs and allow to distinguish them from lineage committed
progenitors.26 Only ⬇1% of KDR⫹ cells were represented by
CD34⫹CD14⫺ cells when assessed by conventional flow
cytometry, although ⬇80% of KDR⫹ cells consisted of
CD14⫹CD34low cells. These findings strongly support the concept that CD14⫹CD34low cells are the major source of EPCs
obtainable from PB and probably constitute a subset of pluripotent circulating SCs. Of note, virtually all BM CD14⫹ cells,
when assessed with ACMFL, also appeared to express CD34,
suggesting that circulating CD14⫹CD34low cells probably result
from the migration of a population already present in the BM.
A major difficulty in the identification of the subset that
represents the source of PBMNC-derived EPCs has been represented by the absence of an undoubt stemness marker. The
results of this study demonstrate that purified CD14⫹CD34low
cells express Nanog, presently known as the most important
Romagnani et al
EPCs Derive From Nanogⴙ CD14ⴙCD34low Cells
321
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Figure 8. Growth kinetics, clonogenicity,
and multidifferentiation potential of circulating CD14⫹CD34low cells. A, Proliferation of circulating CD14⫹CD34low (left),
but not of CD14⫹CD34⫺ (right), sorted
cells, cultured in the presence of SC
growth factors (SCF, 50 ng/mL; Flt3-L,
50 ng/mL; thrombopoietin, 50 ng/mL), as
assessed by CFDA-SE labeling. B, Generation of clones from single cells taken
from the CD14⫹CD34low sorted population cultured in the presence of SC
growth factors. C, Induction of osteogenic differentiation in CD14⫹CD34low
cells cultured for 21 days, as described
in Materials and Methods. Left, Cells
were stained for alkaline-phosphatase
and for the presence of mineralized nodules by Alizarin red (red color). Right,
Expression of mRNA for bone-specific
transcription factors, Runx-2, and
osterix, as assessed by real time quantitative RT-PCR. D, Induction of adipocyte
differentiation in CD14⫹CD34low cells,
cultured as described in Materials and
Methods. Left, Lipid vacuoles were
stained with Oil red O. Right, Expression
of mRNA for adipocyte-specific transcription factors, AP2, and PPAR-␥. E,
Induction of neural cell differentiation in
CD14⫹CD34low cells, cultured as
described in Materials and Methods.
Left, Cells were stained for GFAP and
NF200. Right, Expression of mRNA for
neural enolase. Columns represent mean
values (⫾SD) obtained in 4 separate
experiments. In A and B, as well as in
the left part of C, D, and E, 1 representative experiment is shown.
marker of stemness in mouse and human ESCs,12,13,27 at both
mRNA and protein level. By contrast, Nanog was virtually
absent from differentiated human adult cells and strongly downregulated when EPCs differentiated into ECs. The stemness
phenotype of circulating CD14⫹CD34low cells was then confirmed by the detection of Oct-4, another marker of ESCs14 and
of Bmi-1, an adult SC marker which plays a central role in
self-renewal.15 These findings not only provide the first demonstration that Nanog can be a useful marker for the identification
of human adult SCs, but also suggest the possibility that it plays
a crucial role in maintaining their undifferentiated state. This
possibility is only apparently in contrast with the expression by
EPCs of acLDL and Ulex-lectin, which are considered as
markers of committed endothelial progenitors. Indeed, acLDL
and Ulex-lectin expression has also been detected in different
BM cell types, including SCs.3 It has been shown that Nanog
expression in mouse ESCs is maintained through a signal
transduction pathway involving the gp130 receptors and
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August, 19, 2005
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STAT-3 activation.24 By contrast, stimulation of human ESCs
by gp130 cytokines was not sufficient to maintain these cells in
an undifferentiated state.24 Likewise, despite the expression of
gp130 receptors, the addition of LIF to VEGF-containing cultures induced STAT-3 phosphorylation, but it was unable to
maintain their undifferentiated state, as shown by the downregulation of both Nanog and Oct-4 and the appearance of markers
of ECs. These findings demonstrate that signaling through the
gp130/STAT3 pathway is insufficient to prevent the onset of
differentiation at least in this type of adult human SCs. Very
recently, the ability of activin A to maintain high Nanog and
Oct-4 levels, as well as pluripotency, in human ESCs has been
reported.28 Whether activin A is able to play the same role in
double-positive CD14⫹CD34low cells remains to be established.
The expression of Nanog and Oct-4 strongly suggested that
circulating CD14⫹CD34low cells might represent multipotent
SCs. Accordingly, these cells exhibited proliferative response
to SC growth factors, clonogenicity, and multidifferentiation
potential, as shown by their ability to give rise not only to
ECs, but also to osteoblasts, adipocytes, or neural cells. By
contrast, CD14⫹CD34⫺ cells neither exhibited clonogenicity nor multidifferentiation capacity, suggesting their nature
of fully differentiated monocytes. Taken together, these data
provide the first evidence that a relevant, even if variable,
percentage of CD14⫹ cells consist of double-positive
CD14⫹CD34low cells showing phenotypic and functional
features of multipotent SCs. They also suggest the possibility
that the purification of these cells may provide a useful tool
to get high numbers of purified EPCs for possible practical
use in neovascularization and for the repair of tissue damages.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Acknowledgments
This work was supported by the Ministery of Health of
Tuscany (Italy).
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CD14+CD34low Cells With Stem Cell Phenotypic and Functional Features Are the Major
Source of Circulating Endothelial Progenitors
Paola Romagnani, Francesco Annunziato, Francesco Liotta, Elena Lazzeri, Benedetta
Mazzinghi, Francesca Frosali, Lorenzo Cosmi, Laura Maggi, Laura Lasagni, Alexander
Scheffold, Manuela Kruger, Stefanie Dimmeler, Fabio Marra, Gianfranco Gensini, Enrico
Maggi and Sergio Romagnani
Circ Res. 2005;97:314-322; originally published online July 14, 2005;
doi: 10.1161/01.RES.0000177670.72216.9b
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2005 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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http://circres.ahajournals.org/content/suppl/2005/07/14/01.RES.0000177670.72216.9b.DC1.html
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CIRCRESAHA/2005/108050/R1
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“Online Data Supplements”
(a) Expanded Materials and Methods
Reagents. Ficoll-Hypaque was purchased from Pharmacia (Uppsala SW). Unconjugated and
FITC, PE-, allophycocyanin (APC)-, or peridin chlorophyll protein (PerCP)-conjugated antiCD1a (HI149 mouse IgG1), anti-CD3 (SK7, mouse IgG1), anti-CD4 (SK3, mouse IgG1), antiCD8 (SK1, mouse IgG1), anti-CD11c (S-HCL-3, mouse IgG2b), anti-CD14 (MΦ9, mouse
IgG2b), anti-CD16 (3G8, mouse IgG1), anti-CD19 (4G7, mouse IgG1), anti-CD31 (L133,1,
mouse IgG1), anti-CD34 (My10, mouse IgG1), anti-CD45 (2D1, mouse IgG1), anti-CD80
(L307.4, mouse IgG1), anti-CD86 (2331, mouse IgG1), anti-HLA-DR (L243, mouse IgG2a)
mAbs were purchased from Becton Dickinson (Mountain View; CA). Unconjugated antiVEGFR2 (KDR) mAb (KDR-2, mouse IgG1) was purchased from Sigma-Aldrich Co. (St
Louis; MS), unconjugated anti-Tie-2 mAb (mouse IgG1) was purchased from R&D Systems
(Minneapolis; Mn). Anti-CD1c (AD58E7, mouse IgG2a), anti-CD133 (AC133, mouse IgG1)
mAbs were purchased from Miltenyi Biotec (Bisley; Germany). Anti-CD105 mAb (SN6, mouse
IgG1) was purchased from Ancell (Bayport, MN). Goat anti-mouse IgG1 Abs, labelled with PE
or Alexa Fluor 633, were purchased from Molecular Probes (Eugene; OR). Anti-CD14 and antiCD34 mAbs conjugated with magnetic beads were obtained from Miltenyi Biotec. The 5-(and
6-)carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) was purchased from Molecular
Probes. The PKH26 red fluorescent cell linker dye was purchased from Sigma-Aldrich. To-pro3 was purchased from Molecular Probes, Eugene, OR, USA.
Tissues and cells. Pooled mRNAs from the following human tissues were obtained from
Clontech (New Jersey; NJ): bone marrow (n=10 donors), heart (n=10), kidneys (n=6), prostate
(n=32), skeletal muscle (n=7), spleen (n=14), testis (n=45) and teratocarcinoma (n=6). Primary
cultures of human keratinocytes, human renal proximal tubular epithalial cells (HRPTEC),
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CIRCRESAHA/2005/108050/R1
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human microvascular endothelial cells (HMVEC), human aortic smooth muscle cells
(HASMC), human dermal fibroblasts (HDF) were obtained from Clonetics (New Jersey; NJ).
Isolation of CD14+ monocytes or CD34+ hemopoietic stem cells (HSC). Briefly, peripheral
blood MNCs (PBMNCs) suspensions were obtained from healthy donors and then incubated in
phosphate buffer pH 7.2 plus 0.5% FCS (Hyclone, Logan; UT), plus 2 mM EDTA for 20 min
at 4° C in the presence of anti-CD14 mAb, conjugated with colloidal super-paramagnetic
microbeads. The positive selection of CD14 cells was performed on LS+ columns (Milteny
Biotec). CD34+ HSCs were recovered from the CD14 negative fraction by using the same
procedure.
Cell Cultures. PBMNCs (8 x 106 cells/well), CD14+ cells, CD14+CD34- cells, CD14+CD34low
cells were plated on six-well culture dishes, coated with human fibronectin (Sigma) and
maintained in endothelial basal medium (EBM-MV) (Clonetics), supplemented with EGM-MV
SingleQuotes, 100 ng/ml of human recombinant VEGF (Peprotech; London, UK), and 20%
FCS. The human pluripotent embryonal carcinoma cell line NTERA-2 clone D1 was purchased
from European Collection of Cell Cultures (ECACC), Salisbury, Wiltshire, UK.
Flow cytometry analysis of cell surface molecules. After staining with appropriately
conjugated Abs and washings, cells were analyzed on a BDLSRII cytofluorimeter, using the
DIVA software (BD Biosciences). The area of positivity was determined using an isotypematched control Ab. 104 events for each sample were acquired.
ACMFL. Briefly, liposomes were prepared by the ‘extrusion method’, using 45%
phophatidylcholine, 10% phosphatidylglycerol, 5% phosphatidylethanolamine, and 45%
cholesterol. The liposomes contained 10 mM Cy5 dye and superparamagnetic microparticles
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CIRCRESAHA/2005/108050/R1
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(diameter, 50–100 nm; absorbance at 450 nm, 500). Small, non-magnetic, liposomes were
excluded by filtration on magnetic cell separation columns, which results in uniformly sized
magnetofluorescent liposomes approximately 200–300 nm in size. Liposomes were then
conjugated with biotin. Cells were incubated in cold PBS pH 7.2, plus 0.5% BSA and 0.02%
NaN3 in the presence of rabbit Ig (10 mg/ml). After 10 min, cells were incubated for 15 min
with a FITC-conjugated anti-CD34 mAb (or a FITC-conjugated isotype control) and with a PEconjugated anti-CD14 mAb. After washing, cells were incubated for additional 15 min with a
digoxigenin-conjugated anti-FITC mAb. Cells were then washed and incubated for 15 min. with
an anti-digoxigenin/anti-biotin construct, washed twice and incubated for 30 min in the presence
of biotin-conjugated liposome. After extensive washings, cells were analyzed on a BDLSR II
cytofluorimeter, using the Diva software (Becton Dickinson). The area of positivity was
determined using an isotype-matched mAb, a total of 105 events for each sample were acquired.
FASER technique. The FASER tecnique was performed as indicated by the manufacter (Faser
kit-PE, Miltenyi Biotec). Briefly, cells were labeled with a PE-conjugated anti-CD34 or CD133
mAb and then incubated with FcR blocking reagent and PE-Activator reagent for 10 minutes in
the dark at 4−8 °C. After incubation cells were washed and then incubated with FcR blocking
reagent and PE-Enhancer reagent for 10 minutes in the dark at 4−8 °C. After extensive
washing the amplification of the PE fluorescence was performed for additional two rounds, cells
were then analyzed on a BDLSR II cytofluorimeter, using the Diva software (Becton
Dickinson). The area of positivity was determined using an isotype-matched mAb, a total of 105
events for each sample were acquired.
Western blotting. CD14+CD34low cells were either left untreated or exposed to (10 ng/ml) LIF
for 5 to 60 min. At the end of incubation, cells were quickly placed on ice and washed with icecold PBS. The monolayer was lysed in RIPA buffer (20mM Tris-HCl, pH 7.4, 150mM NaCl,
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CIRCRESAHA/2005/108050/R1
4
5mM EDTA, 1% nonidet P-40, 1mM Na3VO4, 1mM phenyl methyl sulfonyl fluoride, 0.05%
[w/v] aprotinin) and transferred to microcentrifuge tubes. Insoluble proteins were discarded by
centrifugation at 12,000 rpm, at 4ºC. Protein concentration in the supernatant was measured in
triplicate using a commercially available assay (Pierce, Rockford, IL). Equal amounts of total
cellular proteins were separated by SDS-PAGE, and analyzed by Western blotting using the
specified antibodies.
Real-Time Quantitative RT-PCR (TaqManTM). Total RNA was extracted (RNeasy Micro
kit; Qiagen; Hilden, Germany) and treated with DNase I (Qiagen) in order to eliminate possible
genomic DNA contamination. Quantitation of CD14, CD34, CD105, CD133, Bmi-1, gp130,
Runx2, osterix, neuronal enolase, PPAR-γ, AP2 and KDR was performed using Taq-Man gene
expression assays (Applied Biosystems; Warrington, UK). The vWf primers and probes were:
VIC
probe,
5'-CCCCAGAGCTGCGAGGAGAGGAAT-3’;
forward
5’-
CTGGAGGACGGCCACATT-3’; reverse 5’-ACTCATACCCGTTCTCCCGG-3’. The Oct-4
primers and probes were: VIC probe, 5'-CAGCCACATCGCCCAGCAGC-3’; forward 5’GAAACCCACACTGCAGCAGA-3’;
reverse
5’-GGACCACATCCTTCTCGAGC-3’.
Standard curves were generated using serial dilutions of mRNA obtained from positive samples,
expressing high levels of mRNA for each gene. To specifically detect NANOG mRNA
expression in a quantitative manner in different human tissues and cultures, several pairs of
oligos were prepared to recognize the human orthologue of the mouse Nanog gene. The human
NANOG gene displays ten processed pseudogenes, arised by retrotransposition of mRNA and
designated NANOGP2 to NANOGP11, and a duplication pseudogene, designated NANOGP1.
Since only NANOGP1, NANOGP5 and NANOGP11 are expressed, we choosed a pair of oligos
unable to recognize the mRNA sequences possibly encoded by these pseudogenes. Furthermore,
the oligos were choosen to span an exon-intron boundary and the DNA presence was checked in
every sample. Only samples containing mRNA, but no contaminating DNA, were used for all
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CIRCRESAHA/2005/108050/R1
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analyses, to avoid artifacts related to the amplification of the unusually high number of
NANOG-related non-functional pseudogenes. The pair of oligos choosen for their high
specificity were used to set up a Taq-Man quantitative RT-PCR, together with a VIC-labelled
fluorescent probe. Primers and probes were the followings: NANOG: VIC probe, 5'TCCATCCTTGCAAATGTCTTCTGCTGAGAT-3';
forward
5'-
GATTTGTGGGCCTGAAGAAAACT-3'; reverse 5'-AGGAGAGACAGTCTCCGTGTGAG3'. This set of primers displayed selective specificity, high sensitivity, and optimal amplification
efficiency, as tested on a plasmid encoding the NANOG cDNA sequence. mRNA levels were
quantitated by comparing experimental levels to standard curves, generated using serial
dilutions of the same amount of the plasmid (Fig. 5A). Numbers of cell analyzed were counted
by three independent observer and each sample was assayed in triplicate.
In vitro multidifferentiation. For in vitro differentiation into osteoblasts, adipocytes or neural
cells, sorted circulating CD14+CD34low cells were cultured under conditions known to induce
differentiation into various cell types. For osteogenic induction adherent cells were cultured in
α-MEM, 10% horse serum, containing 100 nM dexamethasone, 50 µM ascorbic acid and 2 mM
β-glycero-posphate (all from Sigma). The medium was changed twice a week for 3 weeks. At
the end of the treatment, cells were stained with alkaline phosphatase magenta
immunohistochemical solution (Sigma) according to the manifacture instructions. To detect
intracellular calcium deposits, cells were fixed with 10% formalin and stained with 2% alizarin
red S (Sigma) for 3 min, followed by extensive washing and staining with hematoxylin. The
expression of bone-specific transcription factors, Runx-2 and osterix, was assessed at mRNA
level by using real time quantitative RT-PCR. For adipogenic differentiation, cells were
incubated in DMEM high glucose (hg) containing 10% FBS (Hyclone), 1 µM dexamethasone,
0.5 µM 1-methyl-3-isobutylxanthine (IBMX), 10 µg/ml insulin, and 100 µM indomethacin, (all
from Sigma). After 72 hours, the medium was changed to DMEM hg, 10% FBS and 10 µg/ml
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CIRCRESAHA/2005/108050/R1
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insulin for 24 h. These treatments were repeated three times. The cells were then maintained in
DMEM hg, 10% FBS and 10 µg/ml insulin for one additional week. At the end of the treatment
the lipid vacuoles were stained with Oil red-O (Sigma). Briefly, cells were washed with PBS,
fixed with 10% formalin for 30 min, rinsed with 60% isopropanol and incubated with 0.1% Oil
red-O for 5 min. After rinsing in water, the cells were counterstained with hematoxylin. The
expression of PPAR-γ mRNA was assessed by real time quantitative RT-PCR.
For neurogenic differentiation cells were plated in DMEM hg, 10% FBS. After 24 h medium
was replaced with DMEM hg, 10% FBS containing B27 (Invitrogen), 10 ng/ml EGF
(Peprotech) and 20 ng/ml bFGF (Peprotech). After 5 days, cells were washed and incubated
with DMEM containing 5 µg/ml insulin, 200 µM indomethacin and 0.5 mM IBMX in the
absence of FBS for 5 hours. At the end of the treatment, cells were fixed with methanol for 10
min, washed and immunofluorescence performed as described elsewhere with a pAb antineurofilament 200 (NF200) and a mAb anti-glial acidic fibrillary protein (GFAP). The
expression of neuronal specific enolase was assessed at mRNA level by real time quantitative
RT-PCR.
Generation of clones Generation of clones from sorted circulating CD14+CD34low cells was
achieved by limiting dilution in 96 well plates. Cells were initially plated in IMDM (Invitrogen)
15% FBS (Hyclone, Logan, Utha), containing 50 ng/ml stem cell factor (SCF, R&D system), 50
ng/ml fms-like tyrosine kinase 3-ligand (Flt3-L, R&D system), 50 ng/ml thrombopoietin (TPO,
R&D system) and 25% conditioned medium, obtained from CD14+ cells cultured for 5 days in
the same medium. After two weeks of culture, the medium was replaced with IMDM w/o FBS,
50 ng/ml (SCF), 50 ng/ml Flt3-L, 50 ng/ml TPO, 25% conditioned medium.
Confocal microscopy Adherent cells were incubated at 37°C with Dil-labeled acLDL
(Molecular probes) for 1 hr and, after fixation (2% formaldehyde), they were incubated for 1 hr
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with FITC-labeled Ulex europaeus agglutinin I (Ulex-lectin; Sigma). Staining of nuclei with
Topro-3 was used to verify whether almost all adherent cells were acLDL+ Ulex-lectin+. Slides
were mounted in anti-fading mounting media (Vectashield; Vector Laboratories, Burlingame,
CA), and examined by conventional confocal microscopy on a Zeiss LSM 510 META
microscope system (Carl Zeiss; Jena, Germany). For NF200 and GFAP immunofluorescence,
cells were fixed with methanol for 10 min, washed with PBS and incubated for 30 min with
normal goat serum. Cells were subsequently incubated for 30 min with pAb anti NF200
(1:1000, Sigma) or mAb anti GFAP (1:500, Chemicon, Chandlers Ford, UK). For Nanog
detection cells were fixed with acetone for 5 minutes. For Oct-4 and Bmi-1 cells were fixed
with 4% paraformaldehyde for 20 minutes. Cells were subsequently incubated for 15 min at
37°C followed by 1 hour at 4°C with the respective Ab. The anti-human Nanog rabbit pAb
(1:75) was obtained from Abcam, Cambridge, UK; the anti-human Oct-4 rabbit pAb (1:100)
was obtained from Santa Cruz Biotechnology Santa Cruz, CA, USA; the anti-human Bmi-1
(1.50) mAb was obtained from Upstate, NY, USA. After washing with PBS, cells were
incubated for 30 min with goat anti-rabbit AlexaFluor 488 (1:1000, Invitrogen) or goat antimouse AlexaFluor 488 (1:1000, Invitrogen). For Nanog, Oct-4 and Bmi-1, all the reagents were
prepared in 0.5% saponin-containing PBS to permeabilize cell membranes. Nuclei were
counterstained with To-pro-3. Slides, mounted in anti-fading mounting media, were examined
by confocal microscopy on a Zeiss LSM 510 META microscope system.
Statistical analysis Mean group values were compared by using one-way analysis of variance
(ANOVA) and Post-hoc comparisons were carried out using the Bonferroni correction. Due to a
non parametric distribution, comparisons of Nanog mRNA levels among different groups were
performed by Mann Whitney U-test. Data are expressed as mean ± SD unless otherwise stated.
A p value <0.05 was considered statistically significant.
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(b) Online Figures
Online Fig. 1: PBMNC-derived EPCs are contained in the CD14+ cell fraction.
(A) CD14+, CD14-CD34+ or CD14-CD34- cells were purified by the immunomagnetic technique
and then assessed by classic flow cytometry for CD14 and CD34 expression. (B) Purified CD14+
cells were labeled with CFDA-SE (green) and purified CD34+ HSCs with PKH26 (red), mixed
together with the unlabeled CD14-CD34- cells, in proportions corresponding to those found in the
starting PBMNC population, and then cultured in presence of VEGF. On days 0 and 5 of culture,
the percentages of green- or red-stained cells were evaluated by flow cytometry. One representative
of 4 separate experiments is shown.
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Online Fig. 2: Contemporaneous detection of Nanog mRNA by quantitative RT-PCR and
Nanog protein by confocal microscopy in CD14+CD34low cells before and after 5 days of
culture.
(A) Nanog mRNA (left) and protein (right) expression in freshly derived circulating
CD14+CD34low cells purified as described in Materials and Methods. (B) Nanog mRNA (left) and
protein (right) expression in the same cells after 5 days of culture in presence of VEGF. (C) Nanog
mRNA (left) and protein (right) expression in the human pluripotent embryonal carcinoma cell line
NTERA-2 clone D1. (Bar=100 µm).
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(c) Online Table
Online Table 1: Surface marker expression by fresh PBMNCs and after their
culturing to obtain EPCs.
Surface markerA
(%)
CD1a
CD1c
CD3
CD11c
CD14
CD16
CD19
CD31
CD34
CD45
CD80
CD86
CD105
CD133
HLA-DR
A
Total
PBMNCs
Adherent cells-5 days(EPCs)
0.05±0.05
1.75±0.2
58.1±0.1
32.9±0.4
18.7±1.5
17.4±1.3
6.8±1.0
60.5±0.8
0.15±0.05
95.4±0.2
0.7±0.2
19.2±1.0
8.2±1.2
0.2±0.05
38.3±0.2
2.3±0.7
0.5±0.1
7.9±0.9*
86.0±1.9*
84.9±2.0*
81.8±2.8*
2.7±0.6*
84.0±4.8*
0.7±0.2
98.2±0.4
3.1±0.1
74.3±6.6*
83.1±1.8*
0.6±0.3
86.5±3.2*
Surface markers expression was evaluated by flow cytometry in freshly derived
PBMNCs and in adherent cells found on day 5 of culture with EGM-MV supplemented
with VEGF.
* Asterisk expresses significant differences in marker expression between fresh and
cultured cells. Results represent mean values (+ SE) from 4 separate experiments (p <
0.05).
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