Cell Transplantation, Vol. 24, pp. 865–877, 2015 0963-6897/15 $90.00 + .

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Printed in the USA. All rights reserved. DOI: http://dx.doi.org/10.3727/096368913X676231
Copyright © 2015 Cognizant Comm. Corp. E-ISSN 1555-3892
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The Human Umbilical Cord Tissue-Derived MSC Population UCX® Promotes

Early Motogenic Effects on Keratinocytes and Fibroblasts and G-CSF-Mediated
Mobilization of BM-MSCs When Transplanted In Vivo
Joana P. Miranda,*1 Elysse Filipe,*1 Ana Sofia Fernandes,*† Joana M. Almeida,‡ José P. Martins,‡
Alexandre De la Fuente,§ Miguel Abal,§ Rita N. Barcia,‡ Pedro Cruz,‡ Helder Cruz,‡
Matilde Castro,* and Jorge M. Santos‡

*Research Institute for Medicines, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal
†Centro de Biociências da Universidade Lusófona (CBios), Lisbon, Portugal
‡ECBio S.A., Amadora, Portugal
§Translational Medical Oncology, Health Research Institute of Santiago (IDIS),
Complexo Hospitalario Universitario de Santiago de Compostela/SERGAS, Santiago de Compostela, Spain

Mesenchymal stromal cells (MSCs) play an important role in tissue regeneration mainly through the secretion of
trophic factors that enhance the repair of damaged tissues. The main goal of this work was to study the paracrine
mechanisms by which an umbilical cord tissue-derived MSC population (UCX®) promotes the migration capacity
of human dermal fibroblasts and keratinocytes, which is highly relevant for skin regeneration. Furthermore, the
differences between paracrine activities of MSCs from the umbilical cord tissue and the bone marrow (BM-MSCs)
were also evaluated. In vitro scratch assays revealed that conditioned media (CM) obtained from both growing
and stationary-phase UCX® cultures induced human dermal fibroblast (HDF) and keratinocyte (HaCaT) migra-
tion. These assays showed that the motogenic activity of UCX® CM to HaCaTs was significantly higher than to
HDFs, in opposition to the effect seen with CM produced by BM-MSCs that preferentially induced HDF migra-
tion. Accordingly, a comparative quantification of key factors with vital importance in the consecutive stages of
wound healing revealed very different secretome profiles between UCX® and BM-MSCs. The relatively higher
UCX® expression of EGF, FGF-2, and KGF strongly supports early induction of keratinocyte migration and
function, whereas the UCX®-specific expression of G-CSF suggested additional roles in mobilization of healing-
related cells including CD34−/CD45− precursors (MSCs) known to be involved in tissue regeneration. Accord-
ingly, in vitro chemotaxis assays and an in vivo transplantation model for chemoattraction confirmed that UCX®
are chemotactic to CD34−/CD45− BM-MSCs via a cell-specific mobilization mechanism mediated by G-CSF.
Overall, the results strongly suggest different paracrine activities between MSCs derived from different tissue
sources, revealing the potential of UCX® to extend the regenerative capacity of the organism by complementing
the role of endogenous BM-MSCs.

Key words: Mesenchymal stromal cells (MSCs); UCX®; Stem cell transplantation; Fibroblast and keratinocyte
migration; BM-MSC mobilization; G-CSF; Skin regeneration

INTRODUCTION complications and increased morbidity. Hence, a number of

The healing of cutaneous wounds, like any other type of
injury, represents a complex process, integrating a number
of sequential molecular events involving clotting, inflam-
mation, cellular migration, proliferation, extracellular matrix
(ECM) deposition, angiogenesis, vasculogenesis, and
remodeling of the mature scar tissue (29,36,50,58). In a
number of medical conditions, such as diabetes, chronic
renal failure, or arterial or venous insufficiency, the success
of this process is jeopardized, resulting in numerous
novel strategies have been attempted, such as the local
application of growth factors and stem cells, to improve the
regeneration process (6,35).
Mesenchymal stromal cells (MSCs) are a subset of stem
cells that can be isolated from a number of adult and neona-
tal tissues (13,16,56). MSCs have been shown to possess
modulatory properties of both immune and inflammatory
reactions, although the specific mechanism by which MSCs
display such behaviors is still under investigation (18,26,57).

Received July 19, 2013; final acceptance December 17, 2013. Online prepub date: December 30, 2013.
1
These authors provided equal contribution to this work.
Address correspondence to Jorge Miguel Santos, ECBio S.A., Rua Henrique Paiva Couceiro, Nº 27, 2700-451 Amadora, Portugal. Tel: +351 214997579;
Fax: +351 214997594; E-mail: miguel.santos@ecbio.com
865
866
In situ engraftment of MSCs to sites of tissue damage
after systemic infusion or localized transplantation has
been demonstrated, yet increasing evidence also shows that
MSCs have the ability to induce tissue regeneration with-
out local engraftment and differentiation. Indeed, several
authors have hypothesized that the MSC-secreted factors
enhance and assist in the repair and regeneration of dam-
aged tissues via paracrine activation of surrounding cells
(7,8,12,24,38). Secreted bioactive factors may induce cell
migration, proliferation, and even differentiation, resulting
in, for example, angiogenesis and vasculogenesis (6,38,44).
The paracrine secretion of trophic factors has the potential
to induce profound effects on the MSC local environment,
as suggested by the cytokine-mediated control of hemato-
poiesis, which is thought to be regulated by MSCs in the
vascular niche of the bone marrow (8,44,59). The paracrine
action of MSCs has been implicated in cutaneous wound
healing, where it is suggested that these cells secrete a
number of promitogenic and motogenic growth factors
and cytokines, thus leading to enhanced tissue regenera-
tion (10,59,60).
Most of the data so far have been generated with the
prospective autologous application, using bone marrow-
derived MSCs (BM-MSCs) (9,60). BM-MSCs are known
to be endogenously mobilized and to coexist within sites
of injury, actively participating in the healing process
(9,10,60). However, increasing evidence has been sup-
porting neonatal tissues, such as the umbilical cord tissue
and placenta, as better sources of MSCs due to several
reasons. Neonatal tissues confer ease of isolation from
noninvasive and noncontroversial human sources. Further-
more, a relatively higher cell yield is obtained, and the
resulting MSCs have higher expansion capacity and often
higher regenerative potency (2,28,60,61). UCX® in partic-
ular are MSCs obtained from the umbilical cord stromal
tissue using a proprietary method that has recently been
adapted according to advanced therapy medicinal product
(ATMP) certification requirements [EMEA/CAT/486831/
2008/corr (17)] (36a). UCX® comply with the MSC defi-
nition provided by the International Society for Cellular
Therapy (ISCT) (15). Namely, UCX® present fibroblast-
like morphology while adherent to plastic; more than
98% of UCX® within the population express the markers
cluster of differentiation 105 (CD105), CD73, CD90, and
CD44, whereas less than 2% express CD45, CD34,
CD31, CD14, CD19, and human leukocyte antigen
(HLA)-DR. Finally, UCX® can be differentiated into oste-
oblast, chondrocyte, and adipocyte-like cells (46). UCX®
have proven to be nonimmunogenic in vitro and in vivo and
to promote angiogenesis in the scope of an acute myo-
cardial infarction model (38a). Furthermore, UCX® have
recently been demonstrated to have the capacity of
repressing T-cell activation and promoting the expansion
of regulatory T-cells (Tregs) in vitro. Most importantly,
MIRANDA ET AL.
the UCX® capacity for modulating the inflammatory
reaction and for suppressing the immune system was
shown to be higher than that of BM-MSCs (46). These
observations have led us to propose that MSCs from
different tissue sources should not be treated as equal in
terms of their regenerative properties and should there-
fore be discussed and evaluated as separate therapeutic
entities (46).
In this work, the motogenic effect of UCX® to human
dermal fibroblasts (HDFs) and human keratinocytes
(HaCaTs) was demonstrated and shown to be very differ-
ent from that caused by BM-MSCs, further supporting
a distinct, but nevertheless complementary, regenerative
role for UCX® (55). Accordingly, a comparative quantifi-
cation of a selection of paracrine factors with vital impor-
tance during the consecutive phases of wound healing
revealed very different secretome profiles between the
two MSC types. While the UCX® trophic factor profile
suggested a direct impact on the earlier homeostasis and
inflammation stages of wound healing, the BM-MSC tro-
phic profile was better suited to promote later prolifera-
tive and final remodeling activities. Additionally, the
UCX®-specific expression of granulocyte-colony stimulat-
ing factor (G-CSF) was shown to attribute UCX® with a
chemotactic recruitment activity to other circulating or
resident BM-MSCs. Such capacity was confirmed in vivo
through transplantation of UCX® and BM-MSC-seeded
scaffolds in the inner wall of the peritoneal cavity of
severe combined immunodeficient (SCID) mice and by
monitoring interscaffold-specific MSC migration.
Overall, our results are consistent with different regen-
erative activities between MSCs from different tissue
sources and confirm the potential of UCX® as an active
substance for developing ATMPs.
MATERIALS AND METHODS
Ethical and Regulatory Aspects
This study was approved by the Ethics Committee of
the Cascais Hospital Dr. José de Almeida (Portugal), in the
scope of a research protocol with ECBio, S.A. (Amadora,
Portugal). Umbilical cord procurement involving donations,
with written informed consent, was made according to
Directive 2004/23/EC. All the experimental procedures
involving animal models were carried out with the per-
mission of the local laboratory animal committees and in
accordance with internationally accepted guidelines.
UCX® Isolation and Culture
UCX® used in this work were derived from a total of
seven umbilical cords (three females and four males) from
healthy donors, isolated according to Santos et al. (47) and,
when necessary, thawed from a low passage (P3–P5)
working cell bank (WCB). In brief, UCX® were isolated
from human umbilical cord tissue sections that were
UCX® REGENERATIVE ACTIVITY
digested using a precise ratio between tissue mass, tissue
digestion enzyme activity units, digestion solution vol-
ume, and void volume using collagenase. Cells dissoci-
ated from the tissue were recovered by adherence to the
surface of a culture flask (Nunc, Schnelldorf, Germany)
during a static horizontal incubation period and cultured
with a-modified minimum essential medium (a-MEM;
Sigma-Aldrich, Madrid, Spain) supplemented with 20%
fetal bovine serum (FBS; Gibco, Life Technologies, Madrid,
Spain), herein designated as MSC complete medium, at
37°C humidified atmosphere with 7% CO2 in air and cryo-
preserved (47). The WCB was generated by expanding
UCX® in standard MSC culture conditions (46). Cells were
characterized by their capacity to adhere to plastic in stan-
dard MSC culture conditions, specific surface marker
expression, and trilineage differentiation capacity (46). All
cell culture media and supplements were purchased from
Sigma-Aldrich, unless otherwise indicated.
Cell Characterization by Flow Cytometry
For surface marker expression analyses (n = 7), cells
were incubated for 1 h at 4°C with the antibodies in 2%
(w/v) bovine serum albumin (Sigma-Aldrich) solution and
analyzed by flow cytometry using a Gallios imaging flow
cytometer (Beckman Coulter, Inc., Pasadena, CA, USA).
The antibodies used (anti-human) and their conjugates
were CD105-phycoerythrin (PE), CD73-allophycocyanine
(APC), CD90-PE, CD44-APC, CD14-Peridinin-chlorophyll
protein/cyanine 5.5 (PerCp/Cy5.5), CD45-PerCp/Cy5.5,
CD34-fluorescein isothiocyanate (FITC), CD31-FITC,
CD19-Pacific blue, and HLA-DR-Pacific blue. The isotype
controls used were Pacific blue IgG1, Pacific blue IgG2a,
IgG1k PerCp/Cy5.5, IgG2a PerCp/Cy5.5, IgG1k PE,
IgG1k APC, and IgG1k FITC. The antibodies’ final dilu-
tion was 1:100, with the exception of the antibodies con-
jugated with PerCp/Cy5.5, where a dilution of 1:400
was used. All antibodies were purchased from Biolegend
(San Diego, CA, USA), except for CD105-PE, which was
acquired from eBioscience (San Diego, CA, USA).
Cell Characterization by Trilineage Differentiation
To induce adipogenic differentiation (n = 7), cultured
cells were incubated in MSC complete medium, supple-
mented with 10 µg/ml insulin, 200 µM indomethacin,
0.5 mM isobutylmetylxantine, and 1 µM dexamethasone,
for 3 days, and subsequently 1 day in adipogenic main-
tenance medium, consisting of MSC complete medium sup-
plemented with 10 µg/ml insulin. Medium replacement
cycles were repeated during 21 days. For chondrogenic
differentiation, cells were grown in suspension as pellets,
incubated in Dulbecco’s modified Eagle’s medium (DMEM),
1% FBS, 2 mM L-glutamine, 6.25 µg/ml insulin, 10 ng/
ml transforming growth factor-b (TGF-b1) (Tebu-bio,
Le-Perray-en-Yvelines, France), and 50 µM ascorbate-2-
867
phosphate. The medium was replaced every 3 days dur-
ing 21 days. To induce osteogenic differentiation (n = 4),
cells were incubated in a-MEM supplemented with 10%
FBS, 2 mM glutamine, 10 mM b-glycerol phosphate, 50 µg/
ml ascorbate-2-phosphate, and 100 nM dexamethasone.
The medium was replaced every 3 days during 21 days.
For cytochemical staining, cells were fixed with 4% (w/v)
paraformaldehyde (Sigma-Aldrich) for 20 min. In adipo-
genic and osteogenic differentiation protocols, cells were
stained with oil red O (Sigma-Aldrich) and alkaline phos-
phatase (Sigma-Aldrich), respectively. For chondrogenic
differentiation (n = 3), spheroids were cut into sections
and stained with alcian blue (Sigma-Aldrich). The pre-
sence of stained cells was confirmed by use of an inverted
microscope with phase contrast (DMIL HC; Leica,
Wetzlar, Germany).
Production of UCX® and BM-MSC Conditioned
Media (CM)
For conditioned media (CM) production, UCX® and
BM-MSCs (Cat. P10576, batch 2095; Innoprot, Bizkaia,
Spain) were first sequentially adapted to low serum con-
centrations by a reduction of the amount of FBS in the
cell culture medium from 20% to 2% (Fig. 1a). When at
2% FBS, cultures were maintained until 80% confluence
after which (i) either UCX® or BM-MSCs were trypsinized
(Gibco, Life Technologies, Madrid, Spain) and seeded at
a 1 × 104 cells/cm2 density in MSC medium supplemented
with 2% FBS and cultured for a conditioning period of 7
and 4 days, respectively (growing UCX®-CM and grow-
ing BM-MSC-CM), or (ii) UCX® medium was simply
replaced with MSC medium supplemented with 2% FBS
and maintained in culture for a conditioning period of
3 days (confluent UCX®-CM). The control sample con-
sisted of MSC complete medium containing 2% FBS that
was never in contact with cells. CM and control were con-
centrated with 5 kDa cutoff spin concentrators (Agilent
Technologies, Santa Clara, CA, USA) as per manufactur-
er’s recommendations. CM used for comparative studies
based on scratch assays were normalized for total protein
concentration. Protein content was quantified using the
bicinchoninic acid protein assay kit (Thermo Scientific,
Waltham, MA, USA) according to the manufactur-
er’s instructions.
In Vitro Scratch Assays
The in vitro scratch assays were performed on primary
HDFs (from a 38-year-old Caucasian female, Cat. DF-F;
Zen-Bio, Research Triangle Park, NC, USA) and on
HaCaTs (from a 62-year-old Caucasian male, Cat. 300-493;
CLS Cell Lines Service GmbH, Eppelheim, Germany)
essentially as described by Liang et al. (33). Primary HDF
and HaCaT cells were seeded into 24-well plates (Nunc,
Schnelldorf, Germany) at a density of 2.5 × 104 cells/cm2
 868

Figure 1. Motogenic effect of UCX®-CM and BM-MSC-CM to HDF and HaCaT cells. (a) Scheme of CM production as described in Materials and Methods. (b) Images of the
scratch area on HDF and HaCaT cell cultures at time 0 h and 20 h or 40 h, respectively, after CM treatment. (c) Graphical representation of the integrated area migrated by
HDF and HaCaT cells after contact with different CMs. *p < 0.05; **p < 0.01. Scale bar: 500 µm. UCX®, umbilical cord tissue-derived mesenchymal stromal cell (MSC) popu-
lation; BM, bone marrow; FBS, fetal bovine serum.
MIRANDA ET AL.
UCX® REGENERATIVE ACTIVITY
and 4.0 × 104 cells/cm2, respectively, with DMEM-High
Glucose (DMEM basal medium) supplemented with 10%
FBS, herein designated as DMEM complete medium.
Once cultures reached 80% confluence, culture medium
was replaced with DMEM basal medium without FBS for
24 h to guarantee the inhibition of cell proliferation, while
enabling cell migration. Scratches of approximately 0.5 mm
in width were then performed on the monolayer with a
sterile P200 pipette tip (Orange, Braine-L’Alleud, Belgium).
After performing the scratch, the culture medium was
replaced with 200 µl of DMEM basal medium supple-
mented with the different groups of CM and control
diluted to a final concentration of 500 µg/ml total protein.
Additional controls consisting of CM generated from
HDF or HaCaT cultures were also performed in order to
monitor for self-chemotactic effects (data not shown). The
area of the scratch was measured at 0 h for both cell
types and at 20 h and 40 h postscratch for HDF and
HaCaT cells, respectively. UCX®-CMs were derived from
a total of n = 3 umbilical cord samples, and a total of
n = 6 independent experiments was performed. Photographs
were taken at an amplification of 40× on a Nikon Eclipse
Ti-U inverted microscope with a conjugated Nikon DS-
Qi1Mc camera (Nikon, Tokyo, Japan). Cellular migra-
tion was analyzed in the NIS-Elements BR 3.10 program
by integrating the final cell-free area within the scratch,
calculated in relation to the total initial area of the scratch
at 0 h.
Trophic Factor Quantification
A commercially available kit (FlowCytomix™;
eBioscience) was used to determine the concentrations of
epidermal growth factor (EGF), fibroblast growth factor-2
(FGF-2), G-CSF, hepatocyte growth factor (HGF), inter-
leukin-6 (IL-6), TGF-b1, and vascular endothelial growth
factor-A (VEGF-A) in CM from growing UCX®, grow-
ing BM-MSCs, and control samples. Samples were
acquired on a Gallios imaging flow cytometer, and the
results were obtained using FlowCytomix Pro 3.0 Soft-
ware. Quantification of keratinocyte growth factor (KGF)
in these samples was performed using an enzyme-linked
immunosorbent assay kit (Quantikine; R&D Systems,
Minneapolis, MN, USA). The quantification of all growth
factors was obtained from a total of n = 2 umbilical cord
samples (300 events acquired/growth factor) and was
expressed as pg/106 cells/day and normalized against the
control background.
In Vitro Chemotaxis Assay
BM-MSCs were labeled with octadecyl indocarbo-
cyanine (DiD, 1 µM; Life Technologies, Madrid, Spain)
for 30 min at 37°C. Fifty thousand labeled BM-MSCs
were seeded in serum-free media in the upper compartment
of a Boyden chamber (Corning, Amsterdam, Netherlands)
869
and were allowed to migrate through the porous mem-
brane (8 mm) for 48 h in response to the following stimuli
from the lower compartment: (i) serum-free media (nega-
tive control), (ii) serum-free media supplemented with the
universal chemoattractant EGF at 10 ng/ml (positive con-
trol), (iii) 200,000 nonlabeled BM-MSCs in serum-free
media seeded on the bottom of the well (control for self-
mediated chemotaxis), (iv) 200,000 nonlabeled UCX® in
serum-free media seeded on the bottom of the well, and
(v) 200,000 UCX® in serum-free media supplemented with
anti-G-CSF as an inhibitor (hG-CSF, Anti-G-CSF Human,
500-P43-B; Tebu-Bio). Upon incubation, migratory BM-
MSCs were removed from the bottom of the membrane
using trypsin and quantified using a FLUOstar OPTIMA
fluorimeter (BMG Labtech, Ortengerg, Germany) oper-
ated at 549 nm excitation/565 nm emission. Results from
three independent experiments are represented as ratios
compared to negative controls.
In Vivo Scaffold-Based Transplantation Model
for Chemoattraction
Either BM-MSCs or UCX® labeled with 1 µM DiD
was suspended in culture medium at an appropriate con-
centration (50,000 cells/20 ml) and seeded into 3D Insert-
polystyrene/polycaprolactone (PS/PCL) with nanomesh
scaffolds (3D-Biotek, Hillsborough, NJ, USA) 24 h before
implantation in the inner wall of the peritoneal cavity of
8- to 9-week SCID mice (female SCID beige mice; Charles
River, Barcelona, Spain). For this, mice were anesthetized
with Ketolar® (75 mg/kg; Parke-Davis, Pfizer, Madrid,
Spain) and metomidine (1 mg/kg; Orion Pharma, Barcelona,
Spain) and an incision performed in the peritoneum to intro-
duce and fix the scaffolds with BM-MSCs and UCX® in
the left upper part and lower part of the peritoneal cavity
at 1 cm of distance approximately, with the help of surgi-
cal glue (Vetbond®; 3M, St. Paul, MN, USA). Atipamezol
(1 mg/kg; Orion Pharma), a metomidine antagonist, was
supplied after operation. One week after implantation, mice
were sacrificed, and MSC migration was evaluated using an
in vivo imaging system (IVIS; Perkin-Elmer, Santa Clara,
CA, USA) operated at 644 nm excitation/665 nm emis-
sion. Figures are representative images of two independent
experiments using a minimum of four animals per group.
Statistical Analysis
All statistical analyses were done with SPSS 21.0
(Chicago, IL, USA). Results are presented as mean and with
standard errors of the mean (SEM) where possible. To esti-
mate the significance of the differences between groups, the
parametric Student’s t-test was used. As a result of multi-
ple subgroup assessments, a Bonferroni correction was
used to adjust the p value of the in vitro scratch assays
(a/4) and in vitro chemotaxis assays (a/3). Differences
were considered to be significant for values of p < 0.05.
870
RESULTS
In this work, UCX® were freshly isolated from a total
of seven umbilical cord samples, and when necessary for
experimentation, cells were thawed from a low passage
WCB (P3–P5). UCX® and BM-MSCs were expanded in
all cases and consistently presented fibroblast-like mor-
phology while adherent to plastic; more than 98% of the
cells within the corresponding populations consistently
expressed CD105, CD73, CD90, and CD44, whereas less
than 2% expressed CD45, CD34, CD31, CD14, CD19,
and HLA-DR. Finally, both types of MSCs could consis-
tently be differentiated into osteoblast, chondrocyte, and
adipocyte-like cells following the MSC criteria as defined
by the ISCT (15) (results not shown).
In Vitro Motogenic Effects of UCX® and BM-MSC to
HDFs and HaCaTs
In order to evaluate the motogenic potential of UCX® to
HDFs and HaCaTs, CM was derived from UCX® and its
activity compared to that derived from BM-MSCs (Fig. 1a).
Both CMs were supplied to HDF and HaCaT cell cultures
and specific cell migration evaluated using in vitro scratch
assays. Prior to CM production, both types of MSCs were
progressively adapted to growth conditions containing
sequentially lower FBS concentrations in order to reduce the
amount of serum components in the CM that could poten-
tially mask their trophic activity (Fig. 1a). CM was finally
produced by cultures growing in 2% FBS, the lowest con-
centration that still allowed for MSC growth and expansion.
Also, in an attempt to elucidate for the importance of meta-
bolic rate in the quality of the secreted trophic factors, CM
were produced by UCX® cultures in exponential phase (grow-
ing UCX®-CM) and stationary phase (confluent UCX®-
CM). Both HDF and HaCaT cells in contact with either
growing UCX®-CM or confluent UCX®-CM exhibited a
significantly increased migration capacity, with a slightly
more prominent effect being observed for CM produced by
more metabolically active, exponentially growing UCX®
(Fig. 1b, c). Interestingly, the motogenic effect of growing
UCX®-CM was considerably higher with keratinocytes
(more than threefold higher than control) than fibroblasts
(1.8-fold higher than control), contrarily to the cell migra-
tion promoted by CM produced by exponential phase BM-
MSC cultures (growing BM-MSC-CM) that showed a
clear selectivity for fibroblasts (twofold higher than control)
(Fig. 1c). The results demonstrated that the two different
types of MSCs have diverse paracrine activities, resulting in
differential motogenic effects to HDFs and HaCaTs.
Trophic Factor Quantification in UCX® and BM-MSC
Conditioned Media
In order to establish a correlation between the differen-
tial motogenic effects caused by the paracrine activity of
MIRANDA ET AL.
UCX® and BM-MSCs to HDFs and HaCaTs, respec-
tively, a number of essential trophic factors known to be
representative of the several phases of wound healing
were quantified. Thus, the concentrations of HGF, G-
CSF, TGF-b1, KGF, EGF, FGF-2, VEGF-A, and IL-6
were determined in growing UCX®-CM, growing BM-
MSC-CM, and CM control samples (Fig. 2a). The results
clearly show different secretome profiles between UCX®
and BM-MSCs under our growth conditions (Fig. 2a). In
turn, a pie chart representation of trophic factor expression
distribution between growing UCX®-CM and growing
BM-MSC-CM revealed that i) factors secreted preferen-
tially by UCX® (such as G-CSF, EGF, FGF-2, and KGF)
have been commonly implicated in earlier stages of
wound healing, namely, homeostasis and inflammation
(1,5,27,30,40–42,48,53,62), while ii) factors secreted pref-
erentially by BM-MSCs (such as TGF-b1, IL-6, and
VEGF-A) have been more closely associated with the later
proliferation and remodeling phases of wound resolution
(4,19,20,22,32,34,39) (Fig. 2b). Moreover, given what is
known about G-CSF-mediated mobilization of circulating
BM-MSCs to sites of injury (14,21,43,45,54), a signifi-
cant UCX®-specific expression of G-CSF led us to pro-
pose a mechanism where UCX® could further promote
chemotactic recruitment of BM-MSCs (Fig. 2b).
In Vitro Evaluation of G-CSF-Mediated UCX®
Chemotactic Mobilization of BM-MSCs
The hypothesis that UCX® could promote chemotactic
mobilization of BM-MSCs was first evaluated in vitro.
For this, DiD-labeled BM-MSCs were placed in the upper
compartment of Boyden chambers and allowed to migrate
toward the lower compartment in response to different
chemotactic stimuli. As shown in Figure 3, BM-MSCs
migrate in the presence of EGF (universal chemoattract-
ant positive control) or BM-MSCs (self-mediated chemo-
taxis positive control). In turn, UCX® seeded on the
bottom of the well were clearly chemotactic to BM-MSCs,
resulting in a 1.7-fold migration increase when compared
to self-mediated chemotaxis. Moreover, when the chemo-
tactic assay was performed in the presence of hG-CSF
neutralizing antibodies (a–G-CSF), the UCX®-mediated
recruitment was significantly hindered (Fig. 3). These results
indicate that (i) UCX® are chemotactic to BM-MSCs, and
(ii) cell-specific expression of G-CSF by UCX® is an
important factor determining the paracrine chemotactic
activity to BM-MSCs in vitro.
In Vivo Evaluation of UCX® Potential for Chemotactic
Mobilization of BM-MSCs
In order to confirm whether the UCX® chemotactic
effect to BM-MSCs was kept intact in vivo, two 3D-
Biotek Insert-PS/PCL scaffolds were transplanted into the
peritoneum of SCID mice, one seeded with BM-MSCs and
 UCX® REGENERATIVE ACTIVITY

Figure 2. Trophic factor quantification in growing UCX®-CM and growing BM-MSC-CM. (a) Quantification of HGF, G-CSF, TGF-b1, KGF, EGF, FGF-2, VEGF-A, and IL-6
in growing UCX®-CM and growing BM-MSC-CM normalized against control background and expressed in pg of growth factor/cell number in culture/number of conditioning
days (pg/106 cells/day). *p < 0.05; **p < 0.01. (b) Pie chart representation of the relative trophic factor concentrations in growing UCX ®-CM and growing BM-MSC-CM revealed
that factors secreted preferentially by UCX® have been commonly implicated in earlier stages of wound healing, while factors secreted preferentially by BM-MSCs have been
more closely associated with the later phases of wound resolution. A proposed mechanism where UCX ® can further promote a G-CSF-mediated chemotactic recruitment of BM-
MSCs is represented by the arrow line on top.
871
872 MIRANDA ET AL.

Figure 3. G-CSF-mediated chemotactic activity of UCX® cells to BM-MSCs in vitro. Graphical representation of the relative BM-
MSC migration through the Boyden chamber pores (8 mm) and schematic representation of experimental setup. UCX® seeded on
the bottom of the well were chemotactic to BM-MSCs. Moreover, when the chemotactic assay was performed in the presence of
a-G-CSF neutralizing antibodies, the UCX®-mediated recruitment was significantly hindered. Relative migration was determined with
respect to the negative control. *p < 0.05.

the other with UCX®. MSC-specific chemotactic mobiliza-
tion was assessed by coupling either fluorescently labeled
UCX® or BM-MSCs with their nonlabeled counterparts
(Fig. 4). The left panel on Figure 4 shows fluorescence
emission by MSCs at time 0, right after scaffold implan-
tation. In turn, the center panels show the location of both
scaffolds in the internal wall of the peritoneum 1 week past
cell transplantation, at sacrifice day 7. Panels on the right
show the corresponding fluorescent image at day 7, and the
smaller insets, on the bottom right-hand corner of these
panels, are fluorescent images of the corresponding scaffolds
ex vivo. The experiment with DiD-labeled BM-MSCs can
be followed in the upper panels of Figure 4 (A, B). DiD-
labeled BM-MSCs seeded in the upper scaffold migrated
toward the lower scaffold containing unlabeled UCX® as
demonstrated by a strong fluorescent signal emanating from
the bottom scaffold, as well from a migration path con-
necting both scaffolds (Fig. 4A, B). In turn, when labeled
UCX® were located in the upper compartment, a mobili-
zation of much lower significance was detected toward the
lower scaffold seeded with unlabeled BM-MSCs (Fig. 4C,
D). The column histogram in Figure 4 shows the significant
difference between the quantified fluorescence intensity,
in arbitrary units, in the UCX® scaffold, resulting from
the mobilization of DiD-labeled BM-MSCs (Fig. 4B),
compared to fluorescence intensity in the BM-MSC scaf-
fold, resulting from a much lower mobilization of DiD-
labeled UCX® (p < 0.001) (Fig. 4D). Lastly, in a control
experiment, DiD-labeled BM-MSCs were seeded in the
upper scaffold and coupled with an empty scaffold on
the bottom. The result was that no signal was detected
from the bottom scaffold at day 7, discarding any migra-
tion due to chemotactic inflammatory signals caused by
the scaffold implantation procedure alone (Fig. 4E, F).
The signal detected around the incision scar in experi-
ments 1 and 2 was most likely due to nonspecific MSC
homing to the inflammation site caused by the incision
(Fig. 4B, D, white arrows). Overall, our observations clearly
demonstrate the existence of a UCX®-specific chemo-
tactic activity to BM-MSCs in vivo.
DISCUSSION
In a still widely accepted classical model of wound
healing, four consecutive and overlapping phases can be
considered: homeostasis, inflammation, proliferation, and
remodeling, although many authors congregate homeosta-
sis and inflammation in a single inflammatory stage (37).
Figure 2b depicts the wound healing process attempting to
divide the four healing phases within a temporal sequence
of events. Nevertheless, in vivo, there are overlapping
sequences occurring simultaneously within the wound site.
Accumulated evidence in this field has drawn up an
extensive list of growth factors and cytokines secreted by
MSCs that are involved in the various processes leading
to tissue regeneration (4,20,32,58). Within the cutaneous
wound milieu, inflammation, cellular migration, prolifera-
tion, differentiation, ECM deposition, angiogenesis, vascu-
logenesis, maturation, and remodeling are all necessary
responses where MSC-secreted trophic factors may play a
role (9,10,24,25,38,49–51,55,59).
In line with the previously demonstrated differential capa-
city between UCX® and BM-MSCs for immune suppression
 UCX® REGENERATIVE ACTIVITY

Figure 4. Chemotactic activity of UCX® cells to BM-MSCs in vivo using a SCID mouse intraperitoneal, two-scaffold, implant model. (Left panels): Photos of mice right after
small-incision scaffold implantation, at day 0. (Right panels) Postmortem images of mice at day 7. (A, C, E) Photos showing scaffold location. (B, D, F) Images of mice with
superimposed in vivo imaging system (IVIS) detection, representing the density (color gradient) of octadecyl indocarbocyanine (DiD)-labeled cells. The bottom right-hand panels
are ex vivo IVIS images of scaffolds, after excision from animals. The column histogram consists of quantified fluorescence intensity, in arbitrary units, in the UCX ® scaffold,
resulting from the mobilization of DiD-labeled BM-MSCs (B), compared to fluorescence intensity in the BM-MSC scaffold, resulting from a much lower mobilization of DiD-
labeled UCX® (D) (p < 0.001). Data were obtained from two independent experiments using a minimum of four animals per group; ***p < 0.001. SCID, severe com-
bined immunodeficient.
873
874
(46), we herein report a clear, distinct, but nonetheless com-
plementary effect of their paracrine motogenic activities to
HDFs and HaCaTs. In agreement with what had been
previously reported for BM-MSCs (55), we demonstrate
a preferential trophism of BM-MSCs to HDFs when
compared to HaCaTs. Conversely, our results showed
that the effect of UCX® on inducing HaCaT migration
was significantly higher when compared to the corre-
sponding effect on HDFs. Given the relatively higher
importance of keratinocyte migration in the early stages of
wound healing, we hypothesized that the paracrine healing
potential of UCX® could therefore be more relevant dur-
ing the initial stages leading to skin regeneration, where
early keratinocyte migration plays a major role (9,55).
Accordingly, UCX® and BM-MSCs presented clearly
distinct secretome profiles. Furthermore, within our spe-
cific pool, those factors either exclusively or majorly
expressed by UCX® could in fact be directly correlated to
events occurring during the earlier phases of the healing
process. The relatively secluded expression of EGF, FGF-2,
and KGF by UCX®, contrasting with the relatively clois-
tered expression of VEGF-A (and to some extent IL-6)
by BM-MSCs, could alone explain the differential effects
observed on HDF and HaCaT migration. EGF, for exam-
ple, has been shown not only to stimulate HaCaT migration,
but also to induce the autocrine secretion of all members
of the EGF family. Some of these EGF family members
possess even higher promitogenic and motogenic-inducing
capacities upon HaCaTs so important for hemostasis.
In addition, increasing evidence suggests that the EGF
receptor pathway has a major impact on the early inflam-
matory/immune reactions necessary for proper skin regen-
eration early after physical injury (31,40,48,53). In turn,
FGF-2 is essential for the organization and morphogenesis
of suprabasal HaCaTs acting through a chemotactic mech-
anism (5,27,42). Additionally, FGF-2 has been shown to
induce leukocyte recruitment on endothelium, implicating
this growth factor on early inflammatory stages of the
healing process (62). Finally, although KGF has preferen-
tially been correlated with epithelialization events (1), its
early action on wound healing has been recently shown
in vivo in a mouse model (41). Experiments demonstrated
that KGF is essential to the proliferation of keratinocytes
and that the early activity of KGF on keratinocyte function
may also play a role in the regulation of VEGF gene
expression and consequently on angiogenesis during fol-
lowing stages of the healing process (41). On the other
hand, in our culture conditions, factors such as the domi-
nant VEGF isoform VEGF-A (VEGF165) and IL-6 were
secreted preferentially by BM-MSCs. VEGF-A, in partic-
ular, has been shown to be expressed in a specific tempo-
ral pattern in wounds, namely, in the later proliferation
phase occurring 3 to 7 days postwound, when capillary
growth and differentiation are at a maximum (Fig. 2b) (3).
MIRANDA ET AL.
Similarly, VEGF-A-inducible matrix metalloproteinase 1
(MMP-1), MMP-2, and tissue inhibitor of metalloproteinase
1, all peaking at 2 to 5 days after excisional wounding (22),
support a role of VEGF-A during fibroblast proliferation and
migration, ECM synthesis leading to granulation tissue for-
mation, angiogenesis, and vasculogenesis (20). In accordance
with our results, VEGF-A has been targeted primarily at
fibroblasts (and macrophages) in deeper wounds (39), thus
correlating the BM-MSC-CM fibroblast-specific motogenic
activity seen by us with its VEGF-A contents. Like VEGF-
A, IL-6 was also mainly expressed by BM-MSCs in our
assays. Pursuant to what is known about VEGF-A, IL-6 has
also been shown to be crucial to epithelialization and to
influence granulation tissue formation during the later stages
of tissue regeneration (34).
In summary, the functional analysis of the factors
included in our test pool does substantiate our hypothesis
that, contrasting to BM-MSCs, the direct impact of
UCX® paracrine activity could be centered on the earlier
phases of the regenerative process. Nevertheless, equiva-
lent expression of other important factors, such as HGF
and TGF-b1, further suggest overlapping roles between
UCX® and BM-MSCs. HGF is a cytokine known to play
multiple roles during the various stages of wound healing,
from neutrophil, monocyte, and mast cell recruitment to
the secretion of matrix metalloproteinases, ECM proteins,
integrins, and collagenases necessary for keratinocyte
migration across the fibronectin-rich provisional wound
matrix (4,19,20,23,32,58). In turn, TGF-b1 is highly
expressed by keratinocytes and has been shown, along
with elements from the VEGF family, to be a key factor
for the formation of granulation tissue due to its pro-
mitogenic effect upon dermal fibroblasts, while also
inducing the expression of ECM proteins and matrix
deposition by these same cells (32). Furthermore, TGF-b1
aids in the angiogenic process and has been reported as
necessary for fibroblast differentiation into myofibroblasts
that are responsible for wound contraction (4,20,32).
Finally, our secretome analysis has revealed a quite
remarkable UCX®-specific expression of G-CSF in our
growth conditions. In the context of tissue regeneration, it
has come to our understanding that circulating factors and
systemic-derived cells play an important role in numerous
aspects of homeostasis and repair. The capacity of G-CSF
to mobilize platelets, infiltrating inflammatory cells and cir-
culating hematopoietic stem cells has been well documented
and currently widely used for clinical transplantation (11).
However, a putative circulating population of CD45−/
CD34− nucleated cells has also been found in the periph-
eral blood of human adults that could be mobilized to the
injured tissue through a mechanism involving G-CSF (14,21,
43,45,54). Studies using parabiotic models have demon-
strated the participation of circulating MSCs in wound
repair (52). Autologous BM-MSCs multiply in the bone
UCX® REGENERATIVE ACTIVITY
Figure 5. UCX®-mediated therapeutic pathways. Representation of the different pathways by which UCX® can actively impact the
wound healing process. Gray arrows highlight G-CSF-mediated mechanisms, namely, early impact recruitment of platelets, immune infil-
trate cells, and CD34+ hematopoietic precursors, and later-impact recruitment of CD34−/CD45− BM-MSCs. ECM, extracellular matrix.
marrow and migrate to the injured sites immediately after
burn injuries, contributing to the inflammatory response,
autolysis, and neovascularization along with hematopoi-
etic and endothelial progenitor cells (6). More recently,
the relative contribution of grafted human MSCs and host
stem/progenitor cells in promoting wound healing has been
evaluated by using a novel asymmetric wound model in
normal and impaired healing diabetic (db/db) mice (49).
MSCs significantly improved healing in both normal and
impaired animals by inducing wingless-related mouse
mammary tumor virus integration site 3a (Wnt3a), VEGF,
and platelet-derived growth factor receptor-a and increas-
ing the number of preexisting host MSCs recruited to the
wound bed. Improvement was seen in both the grafted
and nongrafted sides, suggesting a systemic response to
MSC engraftment. Most importantly, healing was enhanced
despite the rapid loss of grafted human MSCs, suggesting
that mobilizing the host response was the major outcome
of grafting MSCs to tissue repair (49).
As demonstrated in vitro, a G-CSF-dependent pathway
is possibly implicated in the motogenic potential of
UCX® in vivo. Such a discovery, together with distinct
motogenic activities to skin cells, could constitute impor-
tant distinctive factors between UCX® and BM-MSCs in
terms of their future applications. During the early phases
of healing, the paracrine activity of UCX® can directly
benefit the process in two ways: (i) by mitogenic and
motogenic activity to keratinocytes and (ii) by promoting
G-CSF-mediated direct recruitment of platelets, infiltrate
cells, such as neutrophils, macrophages, mast cells, and
CD34+ hematopoietic precursor cells (Fig. 5). In turn, the
relatively lower trophism of UCX® to fibroblasts, so
875
important during the later phases of healing, would be
compensated by inducing the chemotactic mobilization of
either resident or circulating BM-MSCs with potential to
promote the formation of granulation tissue, contraction
by myofibroblasts, angiogenesis, vasculogenesis, and epi-
thelialization (Fig. 5).
Overall, the results presented in this work validate the
motogenic potential of UCX® to dermal fibroblasts and
keratinocytes and consolidate distinct paracrine activities
of MSCs from different tissue sources. The G-CSF-
mediated BM-MSC recruiting capacity of UCX® extends
its potential to a full range of events leading to tissue
regeneration and homeostasis.
ACKNOWLEDGMENTS: The authors thank Prof. Filomena
Nunes, Director of the Hospital of Cascais Dr. José Almeida,
for the umbilical cord sample collection. This work was cofunded
by FCT (EXPL/DTP-FTO/0308/2013, Pest-OE/SAU/UI4013/2011,
and Ciência2008 to J.P.M.), QREN 2008/1467 - TERACEL to
ECBio, and ECBio’s own research funds. H. Cruz and P. Cruz
are shareholders of ECBio; J. M. Santos, M. Filipe, M. Teixeira,
and R. N. Barcia are employees of ECBio; J. P. Miranda is the
spouse of J. M. Santos. No other competing financial interests exist.
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