High-throughput immunophenotypic characterization of bone marrow- and cord blood-derived mesenchymal stromal cells reveals common and differentially expressed markers: identification of angiotensin-converting enzyme (CD143) as a marker differentially expressed between adult and perinatal tissue sources
Amati et al. Stem Cell Research & Therapy
High-throughput immunophenotypic characterization of bone marrow- and cord blood-derived mesenchymal stromal cells reveals common and differentially expressed markers: identification of angiotensin-converting enzyme (CD143) as a marker differentially expressed between adult and perinatal tissue sources
Eliana Amati 0 1 4
Omar Perbellini 0 1 4
Gianluca Rotta 3
Martina Bernardi 1 2 4
Katia Chieregato 1 2 4
Sabrina Sella 1 4
Francesco Rodeghiero 2
Marco Ruggeri 1 4
Giuseppe Astori 1 4
0 Equal contributors
1 Advanced Cellular Therapy Laboratory, Hematology Unit, S. Bortolo Hospital , ULSS 8 Berica, Contra' San Francesco 41, 36100 Vicenza , Italy
2 Hematology Project Foundation , Vicenza , Italy
3 BD Biosciences Italia , Milano , Italy
4 Advanced Cellular Therapy Laboratory, Hematology Unit, S. Bortolo Hospital , ULSS 8 Berica, Contra' San Francesco 41, 36100 Vicenza , Italy
Background: Mesenchymal stromal cells (MSC) are a heterogeneous population of multipotent progenitors used in the clinic because of their immunomodulatory properties and their ability to differentiate into multiple mesodermal lineages. Although bone marrow (BM) remains the most common MSC source, cord blood (CB) can be collected noninvasively and without major ethical concerns. Comparative studies comprehensively characterizing the MSC phenotype across several tissue sources are still lacking. This study provides a 246-antigen immunophenotypic analysis of BM- and CB-derived MSC aimed at identifying common and strongly expressed MSC markers as well as the existence of discriminating markers between the two sources. Methods: BM-MSC (n = 4) were expanded and analyzed as bulk (n = 6) or single clones isolated from the bulk culture (n = 3). CB-MSC (n = 6) were isolated and expanded as single clones in 5/6 samples. The BM-MSC and CB-MSC phenotype was investigated by flow cytometry using a panel of 246 monoclonal antibodies. To define the markers common to both sources, those showing the smallest variation between samples (coefficient of variation of log2 fold increase ≤ 0.5, n = 59) were selected for unsupervised hierarchical cluster analysis (HCL). Differentially expressed markers were identified by directly comparing the expression of all 246 antigens between BM-MSC and CB-MSC. (Continued on next page)
(Continued from previous page)
Results: Based on HCL, 18 markers clustered as strongly expressed in BM-MSC and CB-MSC, including alpha-smooth
muscle antigen (SMA), beta-2-microglobulin, CD105, CD13, CD140b, CD147, CD151, CD276, CD29, CD44, CD47, CD59,
CD73, CD81, CD90, CD98, HLA-ABC, and vimentin. All except CD140b and alpha-SMA were suitable for the specific
identification of ex-vivo expanded MSC. Notably, only angiotensin-converting enzyme (CD143) was exclusively
expressed on BM-MSC. CD143 expression was tested on 10 additional BM-MSC and CB-MSC and on 10 umbilical
cord- and adipose tissue-derived MSC samples, confirming that its expression is restricted to adult sources.
Conclusions: This is the first study that has comprehensively compared the phenotype of BM-MSC and CB-MSC. We
have identified markers that could complement the minimal panel proposed for the in-vitro MSC definition, being
shared and strongly expressed by BM- and CB-derived MSC. We have also identified CD143 as a marker exclusively
expressed on MSC derived from adult tissue sources. Further studies will elucidate the biological role of CD143 and its
potential association with tissue-specific MSC features.
Mesenchymal stromal cells (MSC) are a heterogeneous
population of nonhematopoietic multipotent progenitor
cells capable of self-renewal and differentiation into
mesodermal cell lineages [
]. MSC can be isolated from various
human tissues [
], where they may be recognized as
pericytes and function as a source of cells for tissue repair
and regeneration [
]. Although bone marrow (BM)
remains the most widely recognized source of MSC for
clinical use, the invasive procedure of BM collection has
increased interest for other MSC sources. In this context,
cord blood (CB) has emerged as an alternative to BM,
being collected noninvasively and without major ethical
concerns since it is commonly discarded as a medical waste
]. Given the low frequency of MSC progenitors within
CB, CB-derived MSC have been mostly isolated as single
clones, showing a small spindle-shaped morphology and
unique differentiative and proliferative properties, together
with a normal karyotype after prolonged expansion [
Currently, the widely adopted MSC definition relies on
three minimal criteria according to the International
Society for Cellular Therapy (ISCT), namely: adherence to
plastic under standard culture conditions; expression of
CD105, CD73, and CD90, and lack of expression of
hematopoietic and endothelial surface markers CD14,
CD45, CD34, CD11b, HLA-DR, and CD31; and in-vitro
differentiation potential into osteocytes, chondrocytes, and
adipocytes under appropriate culture conditions [
criteria still represent the accepted standards for the
scientific community to characterize human MSC, despite
functional and phenotypic differences that may exist across
tissue sources, culture conditions, and extent of ex-vivo
]. For instance, an impaired adipogenic potential
of MSC derived from perinatal tissues as CB-MSC is well
documented, likely due to their intermediate state between
adult and embryonic stem cells [
]. Similarly, the
stromal vascular fraction of adipose tissue (AT) meets the
negativity requirements for CD45 and CD31, but not for
CD34 which is expressed at variable levels during the early
stages of culture [
]. Beyond these observations, MSC
characterization remains largely confined to the
abovedescribed criteria, further complicated by the lack of unique
and definitive cell surface markers.
At present, flow cytometry represents the gold standard
clinical tool for studying the immunophenotype of ex-vivo
expanded MSC as part of quality assessment for the release
of MSC produced in compliance with good manufacturing
practice (GMP) standards. Recent advances in
highthroughput flow cytometry allow us to profile hundreds of
human cell surface markers in a single assay, thus
facilitating an efficient and comprehensive analysis of the MSC
surface proteome. However, very few studies have investigated
the in-depth MSC immunophenotype using this approach
] and, to the best of our knowledge, comparative
studies comprehensively characterizing the MSC phenotype
across several tissue sources are still lacking.
The present study provides a comparative and
comprehensive 246-antigen immunophenotypic analysis, performed
with the aim of defining common and differentially
expressed markers between BM- and CB-derived MSC. The
high-throughput screening approach, combined with a
rigorous method of marker selection, allowed us to uncover new
common markers for defining MSC, regardless of the source
and of other variables potentially influencing MSC
phenotype. On the other hand, the exclusive recognition of CD143
as a marker able to segregate adult from perinatal MSC may
facilitate future research towards the identification of
functional differences that may impact MSC efficacy in vivo.
Isolation and ex-vivo expansion of MSC from adult and perinatal sources
MSC were isolated and expanded from BM, CB, AT, and
umbilical cord (UC). For BM, UC, and AT, collection
procedures were approved by the ethics committee of S.
Bortolo Hospital, Vicenza, Italy (Act 40/09 of 16 December
2009). For CB, informed consent was received from the
mothers (IBMDR SCO101, version 2, January 2013 and
reference protocol SIT-VR 13/73).
BM-MSC were generated from washouts of discarded
human BM collection bags and filters (n = 4, median
donor age 31.5 years, range 20–47 years, two males and
two females) after two washing steps with 200 ml saline
and centrifugation at 2000 rpm for 10 min.
Briefly, whole unprocessed total nucleated cells (TNC)
were plated at the concentration of 10 × 104 cells/cm2 in
low-glucose Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% heat-inactivated fetal bovine
serum (FBS) (both from Gibco, Thermo Fisher Scientific,
Waltham, USA), 100 U/ml penicillin, and 100 μg/ml
streptomycin (Sigma Aldrich, Saint Louis, Missouri, USA).
Cultures were kept at 37 °C in a humidified 5% CO2
atmosphere. After 72 h, nonadherent cells were removed,
and fresh medium was added (passage (P)0). The resulting
plastic-adherent cells were fed twice a week, harvested at
80–90% confluence using 10× TrypLE™ Select (Gibco,
Thermo Fisher Scientific), and then subcultured at a lower
density (2000 cells/cm2) for 2–3 additional passages.
For the study, nine BM-MSC cultures were generated
from four donors. Cells were expanded and analyzed as
“bulks”—i.e., as a mixture of more colonies isolated as a
whole from the same donor (n = 6, two of which were
analyzed at two different passages)—or as single clones
isolated from the bulk culture (n = 3, Figs. 1 and 3). In
these selected experiments, individual MSC clones
were isolated and picked up from the bulk culture using
10-mm × 10-mm cloning cylinders (Merck Millipore,
Darmstadt, Germany). MSC clones were isolated from the
surrounding cells and then dissociated within the cylinder,
resuspended, transferred to a new vessel as a pure colony,
CB-MSC cultures were established from six donors
(n = 6, four males and two females) as described previously
by our group [
]. Briefly, mononuclear cells (MNC) were
obtained by density gradient centrifugation (Lymphoprep™,
Sentinel Ch. Spa, Milan, Italy) of whole CB diluted 1:1 with
phosphate-buffered saline (PBS; Sigma-Aldrich), and
cultured in low-glucose DMEM supplemented with 20% FBS,
10–7 M dexamethasone (Hospira, Illinois, USA), 100 U/ml
penicillin, and 100 μg/ml streptomycin, at a density
of 1–2 × 106 cells/cm2 and 5–7 × 106 cells/ml. Cells were
incubated at 37 °C in a humidified atmosphere containing
5% CO2. After 1 week from initial plating, nonadherent
cells were removed. The remaining cells were fed once a
week in the absence of dexamethasone and checked for
colony appearance for a maximum of 4 weeks. When reaching
80% confluence, cells were harvested using 10× TrypLE™
Select and subcultured at a density of 4000 cells/cm2. The
medium was replaced twice a week and proliferation
patterns were established by counting cells each week. For the
study, CB-MSC from six independent donors were yielded
and expanded from the starting material as single clones in
five cases and as a combination of a few clones in one case.
AT- and UC-derived MSC were isolated as described
previously by our group [
]. Fresh UC was collected
from the Obstetrics and Gynecology Unit of S. Bortolo
Hospital (Vicenza, Italy) from full-term deliveries after
cesarean section. About 20–30 cm of the whole UC was
rinsed with PBS in presence of 100 U/ml penicillin and
100 μg/ml streptomycin, minced in a Petri dish into small
fragments of about 2–3 mm, transferred to a 75-ml cell
culture flask, and digested with 0.1% collagenase type II
(Gibco, Thermo Fisher Scientific) for 4 h at 37 °C in a
humidified atmosphere containing 5% CO2. An equal
volume of 0.25% trypsin–EDTA (Sigma-Aldrich) was added
for 30 min. The enzyme’s action was blocked with
lowglucose DMEM supplemented with 10% FBS, 100 U/ml
penicillin, and 100 μg/ml streptomycin, and cultures
were kept at 37 °C in a humidified atmosphere
containing 5% CO2. After 4 days, tissue fragments were
removed and the flasks were rinsed twice with PBS,
and then complete fresh medium was added. The
resulting plastic-adherent cells were fed twice a week
and harvested at 80–90% confluence using 10×
TrypLE™ Select, and then subcultured at a density of
1500–2000 cells/cm .
AT-MSC were obtained from the adipose tissue of
healthy subjects (n = 10) undergoing abdominal plastic
surgery. About 100–150 cm2 of AT was washed with PBS
in the presence of 100 U/ml penicillin and 100 μg/ml
streptomycin to eliminate blood contamination. For the
isolation of the stromal vascular fraction, AT was cut and
digested with 0.1% collagenase type II for 1 h at 37 °C in a
humidified atmosphere containing 5% CO2. The
collagenase activity was then neutralized with 10 ml low-glucose
DMEM. After centrifugation at 140 × g for 10 min, the cell
pellet was resuspended, filtered through a 70-μm nylon
cell strainer (Falcon®, Corning, Corning, NY, USA) and
centrifuged at 270 × g for 4 min. The obtained stromal
vascular fraction was plated into F75 cell culture flasks in
the presence of low-glucose DMEM containing 10% FBS,
100 U/ml penicillin, and 100 μg/ml streptomycin, and
then cultures were kept at 37 °C in a humidified
atmosphere containing 5% CO2. After 3 days, nonadherent cells
were removed and complete fresh medium was added.
The resulting plastic-adherent cells, termed AT-MSC,
were cultured until 80–90% confluent, and then harvested
using 10× TrypLE™ Select and subcultured at a density of
1500–2000 cells/cm .
Cryopreserved MSC from all sources were used for
later experiments. MSC batches with a viability of 90%
or more were considered for flow cytometry analysis.
The BM- and CB-MSC phenotype was investigated using
the BD Lyoplate™ Human Cell Surface Marker Screening
Panel (BD Biosciences, Cat. 560747, San Jose, CA, USA), a
system consisting of three 96-well plates, each well
containing a lyophilized unconjugated antibody specific for a
cell surface protein for a total of 242 monoclonal
antibodies (mAbs). The panel also contains mouse and rat
isotype controls for assessing the isotype-specific background.
Additional purified or fluorochrome-conjugated mouse
anti-human mAbs and related isotype or negative controls
were added to the original panel, including purified
antichondroitin sulfate (9.2.27 clone; BD Pharmingen™),
allophycocyanin (APC)-anti-CD276 (FM276 clone; Miltenyi
Biotec, Bergisch-Gladbach, Germany), Alexa Fluor®
488-anti-vimentin (RV202 clone; BD Pharmingen™), and
APC-anti-α-Smooth Muscle Actin (αSMA, clone #1A4;
R&D Systems, Minneapolis, MN, USA). Isotype controls
included mouse IgG2A (clone #20102; R&D Systems) and
mouse IgG2B (clone IS6-11E5.11; Miltenyi Biotec)
APCconjugated mAbs. BM- and CB-MSC cell staining was
performed according to the manufacturer’s
recommendations with slight modifications. About 40,000 cells in each
well were suspended in 100 μl buffer containing PBS 1%
MACS bovine serum albumin (BSA) Stock Solution
(Miltenyi Biotec) and 5 mM EDTA. Except for
fluorochrome-conjugated mAbs, antibody binding was
detected using Alexa Fluor® 647-conjugated goat anti-mouse
Ig and goat anti-rat Ig secondary antibodies. Intracellular
markers (chondroitin sulfate, vimentin, and αSMA) with
related controls were detected using the BD Intrasure™
Kit, according to the manufacturer’s recommendations.
Samples were acquired (4000 events) using BD
FACSCanto II (BD-Bioscience). Data were analyzed by
FlowJo software (Treestar, Ashland, OR, USA).
Lyoplate data analysis
Fold increase of the median fluorescence intensity with
respect to the isotype control (FI), the percentage of
positive cells (%pos), and robust coefficient of variation
(rCV) were used to describe each marker.
Log2 transformation of FI was performed to improve
the resolution of low FI values.
Subsequently, to define common markers to BM- and
CB-MSC, those showing the smallest variation between
samples (CV of log2 FI ≤ 0.5, n = 59) were selected
(Additional file 1: Figure S1) for unsupervised hierarchical
cluster analysis (HCL). Mean and CV values of log2 FI for
each marker are reported in Additional file 2 (Table S1).
The log2 FI values of markers with low variation among
samples were analyzed using MeV software (TM4
Software Development). Unsupervised HCL was performed
using Euclidean distances as the distance metric and the
complete linkage as linkage method of analysis [
Flow cytometry analysis of angiotensin-converting enzyme (ACE) (CD143) expression
The expression of ACE was analyzed on 10 additional
samples for each source (BM, CB, UC, AT) using
phycoerythrin (PE) mouse anti-human angiotensin converting
enzyme (CD143) (clone BB9) and PE mouse IgG1
isotype control (clone MOPC-21), both purchased from BD
Pharmingen™. One hundred thousand cells were stained
for 15 min at room temperature in the dark with the
specific antibody or isotype control. At least 5000 events
in the morphological MSC gate were acquired on a BD
FACSCanto II (BD Biosciences). Data were analyzed by
FlowJo software in terms of FI and %pos.
The expression of all 246 markers was compared using
Mann-Whitney U test. Each marker was considered
differentially expressed between BM- and CB-MSC if the
Mann-Whitney p value was ≤ 0.01. In addition, the
differences in ACE (CD143) expression between BM-, UC-, CB-,
and AT-MSC were computed by Mann-Whitney U test.
Identification of markers commonly expressed on MSC
Starting from 246 antigens and proceeding to an
unsupervised hierarchical classification of those
showing the lowest variation across samples in terms of
CV (n = 59), we identified four groups of markers
according to their relative expression intensity (Fig. 1a
and Additional file 3: Table S2). A total of 18 markers
clustered as strongly expressed and showed log2 FI values
higher than 5.6, meaning that they had a median
fluorescence intensity at least 48.5 times higher when compared
to the isotype controls or the negative markers (Fig. 1b).
Beyond CD90, CD105, and CD73, this included CD44,
CD13, CD29, HLA-ABC, vimentin, beta-2-microglobulin,
CD147, CD151, CD276, CD47, CD59, CD81, CD98,
CD140b, and alpha-SMA. These markers were found
consistently expressed by both BM- and CB-MSC (n = 9 and
n = 6, respectively), regardless of sample heterogeneity
ascribable to tissue source or passage history.
Furthermore, none of them was found to be differentially
expressed when comparing bulk cultures with the derived
clones (n = 3), differently to other antigens (CD57, CD35,
CD6, CD21, CD25, CD34, CD62E, CD62L, CD43, CD33,
CD56, and CD229) which, however, did not fall in the group
of common MSC markers (Additional file 4: Figure S2).
Analysis was further refined on the markers having the
highest percentage of positivity and the narrowest
distribution for each sample. Overall, all the identified markers
apart from CD140b and alpha-SMA showed a % of
positivity close to 100% and a very low % rCV in most
samples, thus being suitable for the identification of in-vitro
expanded MSC (Fig. 2). Conversely, the expression of
alpha-SMA was characterized by a high donor-to-donor
heterogeneity and CD140b showed high positivity in all
samples except one (Fig. 2).
Identification of markers differentially expressed on BM-MSC and CB-MSC
In order to uncover determinants able to segregate MSC
according to origin, the expression of the starting 246
antigens was compared between BM- and CB-MSC.
CD130, CD141, and CD143 showed significant
expression differences between the sources (U Mann-Whitney
cut-off p value ≤ 0.01, Fig. 3a and b). Among them,
CD141 was expressed only on CB-MSC, but at variable
levels. CD130 was expressed by BM-MSC, although also
at low levels by CB-MSC. Importantly, ACE (CD143) was
constantly expressed solely by BM-MSC. Therefore, this
latter marker only clearly distinguished between the two
MSC sources. The exclusive surface localization of CD143
on BM-MSC was further verified on 10 additional
MSC samples for each source, further confirming
previous data (p < 0.0001, Fig. 4).
Evidence of CD143 as a discriminating marker between adult and perinatal MSC
The analysis of CD143 expression on AT- and UC-MSC
(n = 10 for each MSC source) confirmed the presence of
CD143 only on the surface of AT-MSC (p < 0.0001,
Fig. 4), endorsing the hypothesis of its exclusive
expression on MSC derived from adult sources. These data
therefore support a role for CD143 as a discriminating
marker between adult and perinatal MSC sources.
Bone marrow remains the primary MSC source for
clinical applications even if its collection is characterized by
an invasive procedure. Therefore, the search for
alternative sources has increased over the past years. In this
regard, a comprehensive characterization of MSC isolated
and expanded ex vivo from alternative sources is
essential to unravel potential functional differences that may
impact the in-vivo efficacy of MSC [
]. To that
purpose, MSC heterogeneity within and between the
several tissue sources needs to be properly investigated
and its assessment not limited to measuring the level of
expression of the classical surface markers.
In the present study we used a high-throughput flow
cytometry-based screening approach to investigate the
immunophenotype of both BM- and CB-derived MSC.
The first endpoint was to find new common markers
able to complement the existing minimal panel for MSC
Starting from 246 antigens and proceeding to an
unsupervised hierarchical classification of the markers with
the lowest variation across the samples, we came to
select just 18 (7.3%) strongly expressed markers. These
markers were shared and consistently expressed by all
MSC samples regardless of the source, the frequency of
MSC clones in the starting material, or the extent of
exvivo expansion, thus being independent of all the
variables that could influence MSC phenotype. In this
regard, the peculiarity of our analysis resided in the use of a
stringent CV cut-off of log2 FI as a screening method to
define a common set of markers uniformly expressed by
MSC cultures differently isolated and expanded.
Beyond CD90, CD105, and CD73 and other markers
known to be associated with MSC phenotype, such as
CD29, CD44 CD13, HLA-ABC, and vimentin, several
nonclassical markers were found constantly expressed at
high levels by both BM- and CB-MSC. These markers,
including CD59, CD81, CD47, CD276, CD151, CD147,
CD98, and beta-2-microglobulin, are involved in various
nonspecific biological processes such as immune
activation or inhibition and epithelial-to-mesenchymal
]. Their similarity with the expression
patterns of classical MSC markers, resulting from the
analysis of % of positive cells and rCV, particularly
supports their suitability as additional reference markers
for the specific identification of MSC. The consistent
expression of nonclassical markers on the surface of
ATMSC is also supported in the literature [
] using a
242-antigen high-throughput screening analysis. It
should be mentioned that the other authors detected a
wealth of other molecules not distinguished by us; this
discrepancy may be partially due to the restrictive
marker selection applied in the present study.
Okolicsanyi and co-authors demonstrated that
commercially available human MSC co-express MSC and
neural markers during extended culture [
neural markers, reported to influence neural
differentiation, were CD304, CD271, CD200, CD146, CD73,
CD56, and CD24. Except for CD304, not included in our
246-antigen screening panel, only CD73 and CD146
clustered as strong and intermediate markers,
respectively, while the others did not fall into the group of the
59 antigens selected in the present study as the less
variable markers for BM- and CB-MSC. Therefore, this
neural panel did not allow us to identify subgroups of
MSC samples (Additional file 5: Figure S3).
The pericyte markers CD140b and alpha-SMA appeared
to be not consistently expressed across all donors, despite
being shared at high levels by both MSC sources. Hence,
their role in MSC characterization is currently under
investigation. It is conceivable that CD140b represents a
relevant marker for MSC, being identified together with
CD276 by Camilleri and co-authors [
]. This latter
marker was found by the authors constitutively expressed
at robust levels on AT-MSC.
Besides the identification of common and strongly
expressed markers for MSC, an important outcome of the
present study was to find the existence of markers
differentially expressed among sources. Increasing evidence in
this regard suggests that MSC from different origins may
have differences in marker expression and inherent
]. Our comparative analysis unraveled
only 3/246 (1.2%) markers differentially expressed
between BM- and CB-MSC, specifically CD130, CD141, and
CD143. CD130 was more represented on BM-MSC,
although expressed at low levels also by CB-MSC, while
CD141 was expressed only by CB-MSC, but not
consistently across samples. Conversely, the expression of CD143
was exclusively traceable at intermediate levels on
BMMSC in all the investigated samples. The observed high
variability in CD130 and CD141 expression, corroborated
by literature data on cultured AT-MSC [
16, 17, 29
], led us
to consider these markers as being not eligible for further
investigation. On the other hand, the consistent surface
localization of CD143 only on BM-MSC encouraged the
hypothesis, further verified on an independent set of 10
samples for each source, of a role for this marker in
discriminating BM-MSC from CB-MSC. Furthermore, its
exclusive recognition on AT-MSC but not on UC-MSC
allowed us to extend ACE/CD143 as a marker able to
segregate adult from perinatal MSC.
ACE/CD143, also known as hematopoietic progenitor
cell marker, is a zinc-dependent carboxydipeptidase
member of the renin-angiotensin system, with broad
substrate specificity and a well-recognized role in blood
pressure regulation and vascular remodeling [
Moreover, ACE/CD143 is involved in the development and
regulation of hematopoiesis [
]. Zambidis et al. first
demonstrated that the enzymatic activity of ACE,
producing angiotensin II, is required for hemangioblast
expansion and differentiation into either blood or endothelial
cells, through the modulation of angiotensin II-binding
]. The monoclonal antibody BB9, raised initially
to human BM stromal cells [
], recognizes the somatic
form of ACE as a marker of human hematopoietic stem
cells in human embryonic, fetal, and adult hematopoietic
tissues at all stages of hematopoietic ontogeny [
was found to react with a subpopulation of CD34+ACE+
cells capable of sustaining multilineage hematopoietic cell
engraftment in adult BM, and mobilized peripheral blood,
fetal liver, and umbilical CB after transplantation in NOD/
SCID mice [
]. Therefore, the expression of ACE
accompanies the emergence of hematopoiesis in all
bloodforming tissues, starting from the human embryo where
the marker identifies pre-hematopoietic mesodermal
precursors ACE+CD34–CD45– responsible for definitive
Our observation that CD143 is constitutively expressed
by BM-MSC is in accord with the reactivity of BB9 with
BM stromal cells [
], and therefore corroborates the
hypothesis of a locally active renin-angiotensin system
within the BM. The presence of CD143 on the surface of
adult AT-derived MSC agrees with the observations of
Matsushita et al. [
] describing that endogenous
angiotensin II production is increased in MSC
undergoing adipocyte differentiation via increased local
renin expression, suggesting that endogenous
angiotensin II secreted by MSC and differentiated
adipocytes contributes to the modulation of adipogenesis.
This observation is further supported by the scarce
propensity to generate adipose tissue exhibited by
The absence or presence of ACE/CD143 on different
MSC sources is intriguing and could unravel different
MSC properties. Together with a suggested role of
CD143 in hematopoiesis and in MSC capacity to drive
adipogenesis, Silva and co-authors reported that low
expression of ACE can be used as a biomarker to identify
an endothelial cell subpopulation that is more capable of
driving neovascularization [
]. Further studies are
required to better understand the significance of CD143
expression and its potential association with
tissuespecific MSC features. Certainly, since no marker able to
predict the in-vivo efficacy of MSC has been identified,
the demonstration of a functional role of CD143 may be
of considerable clinical impact and would allow a more
careful selection of MSC-based products according to
the clinical goals.
To the best of our knowledge, this is the first study that
comprehensively compared the phenotype of BM-MSC
and CB-MSC. What emerges is a shared and strong
expression of 18 markers; eight of them are
nonclassically associated with the MSC phenotype but
consistently expressed regardless of tissue harvest, clonal
isolation, or extent of ex-vivo expansion, and
therefore could be considered as additional reference
markers for MSC.
Besides an in-depth characterization involving 246
different markers, this study also provides a rigorous
method of analysis that allowed us to select only the
markers with the smallest variation across heterogeneous
samples, thus overcoming any bias potentially
influencing MSC phenotype.
The main result of the work is the exclusive
recognition of ACE/CD143 as a marker able to segregate
adult from perinatal MSC, being CD143 unequivocally
expressed on BM- and AT-MSC but not on UC- and
CB-MSC. This finding may facilitate future research
towards the identification of functional differences
between the MSC sources that may impact their in-vivo
Additional file 1: Figure S1. Transformation and selection. a) The log2
transformation of FI was performed to improve the resolution of low FI
values. b) The markers with the smallest variability across samples
independently from the source were selected according to a CV of log2
FI ≤ 0.5. Accordingly, 59 markers represented below the dotted line were
subsequently considered for unsupervised hierarchical cluster analysis.
(PDF 439 kb)
Additional file 2: Table S1. Mean and CV values of log2 FI for each of
246 investigated markers. (DOCX 22 kb)
Additional file 3: Table S2. List of the lowest variable markers (n = 59,
CV cut-off of log2 FI ≤ 0.5) sorted by function and cluster expression.
Markers with no expression were assigned to cluster 1, low-expressed
markers to cluster 2, and intermediate markers to cluster 3, while a total
of 18 markers clustering as very strong were assigned to cluster 4.
(PPTX 50 kb)
Additional file 4: Figure S2. Differentially expressed markers between
bulk cultures and single derived clones. The expression of each marker
(n = 246) was compared by paired t test (n = 3; p < 0.01). (TIFF 1629 kb)
Additional file 5: Figure S3. Neural marker classification according to
unsupervised HCL. Heat-map expression of the markers identified by
]: CD24, CD200, CD271, CD56, CD146, and CD73. Antigen
expression is color coded from white (no expression) to red (strong
expression). Data are presented as log2 FI (median fluorescence intensity
on the isotype control). (TIFF 1568 kb)
ACE: Angiotensin-converting enzyme; APC: Allophycocyanin; AT: Adipose
tissue; BM: Bone marrow; BSA: Bovine serum albumin; CB: Cord blood;
DMEM: Dulbecco’s modified Eagle’s medium; FBS: Fetal bovine serum;
FI: Fold increase; HCL: Hierarchical cluster analysis; ISCT: International
Society for Cellular Therapy; mAb: Monoclonal antibody; MNC: Mononuclear
cells; MSC: Mesenchymal stromal cells; PBS: Phosphate-buffered saline;
PE: Phycoerythrin; rCV: Robust coefficient of variation; TNC: Total nucleated
cells; UC: Umbilical cord; SMA: Smooth muscle antigen
We are indebted to all the mothers and donors who gave their consent to
donation. We are grateful to Alberta Alghisi, Chiara Lievore, Cinzia Tagliaferri,
Silvia Fortuna, and all the technicians of the Transfusion Medicine
Department of San Bortolo Hospital (Vicenza, Italy) for the contribution to
cord blood collection. Thanks to Ilaria Giaretta for her assistance during flow
cytometry acquisition, and to Marta Crocco for her contribution to sample
processing. Finally, we are indebted to Rosaria Giordano, Lorenza Lazzari, and
all the staff of Cell Factory Franco Calori (Milan, Italy) for the continuous
exchange of views and ideas.
This work was partially funded by the Associazione Vicentina per le Leucemie
ed i Linfomi and Associazione Italiana Contro le Leucemie-Linfomi e Mieloma
Availability of data and materials
All data generated and/or analyzed during this study are included in this
article and its supplementary information files. All data on which our
conclusions depend are available on request.
EA and OP designed the research, analyzed/interpreted results, and wrote
the manuscript. GR was involved in the conception, design, and critical
revision of the manuscript. MB, KC, and SS provided technical assistance and
participated in sample processing. FR and MR critically reviewed the
manuscript. GA was involved in the conception, design, coordination of the
study, critical revisions for important intellectual content and manuscript final
approval. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The present study was approved (reference protocol SIT-VR 13/73) and informed
consent for cord blood collection and donation for research was received from
the mothers (specific section in the form IBMDR SCO101, version 2, January
2013). All collection procedures were approved by the ethics committee of S.
Bortolo Hospital, Vicenza, Italy (Act 40/09 of 16 December 2009).
Consent for publication
Informed consent included the consent for publication.
GR has supported the present work in his function as a Becton Dickinson
Italia associate. All remaining authors declare that they have no
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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