RESEARCH Open Access
The apeu ic angiogenesis induced by
human umbilical co d issue-de i ed
mesenchymal s omal cells in a mu ine
model o hindlimb ischemia
Ana Ri a S. Pe ei a
1†
, Te esa F. Mendes
1†
, Augus o Minis o
1,2
, Ma iana Teixei a
3
, Ma iana Filipe
3
, Jo ge M. San os
3
,
Ri a N. Bá cia
3
, J. Goy i-O’Neill
4
, Faus o Pin o
1,5
, Ped o E. C uz
3
, Helde J. C uz
3
and Susana Cons an ino Rosa San os
1,5*
Abs ac
Backg ound: Mesenchymal s em cells de i ed om human umbilical co d issue, e med UCX®, ha e he po en ial
o p omo e a ull ange o e en s leading o issue egene a ion and homeos asis. The main goal o his wo k was
o in es iga e UCX® ac ion in expe imen ally induced hindlimb ischemia (HLI).
Me hods: UCX®, ob ained by using a p op ie a y echnology de eloped by ECBio (Amado a, Po ugal), we e
deli e ed ia in amuscula injec ion o C57BL/6 emales a e unila e al HLI induc ion. Pe usion eco e y, capilla y
and colla e al densi y inc ease we e e alua ed by lase dopple , CD31 immunohis ochemis y and diaphonisa ion,
espec i ely. The ac i a ion s a e o endo helial cells (ECs) was analysed a e EC isola ion by lase cap u e
mic odissec ion mic oscopy ollowed by RNA ex ac ion, cDNA syn hesis and quan i a i e RT-PCR analysis. The
UCX®-condi ioned medium was analysed on Gallios low cy ome e . The capaci y o UCX® in p omo ing
ubulogenesis and EC mig a ion was assessed by ma igel ubule o ma ion and wound-healing assay, espec i ely.
Resul s: We demons a ed ha UCX® enhance angiogenesis in i o ia a pa ac ine e ec . Impo an ly, a e HLI
induc ion, UCX® imp o e blood pe usion by s imula ing angiogenesis and a e iogenesis. This is achie ed h ough
a new mechanism in which du able and simul aneous up egula ion o ans o ming g ow h ac o β2, angiopoie in
2, ib oblas g ow h ac o 2, and hepa ocy e g ow h ac o , in endo helial cells is induced by UCX®.
Conclusions: In conclusion, ou da a demons a e ha UCX® imp o e he angiogenic po ency o endo helial cells
in he mu ine ischemic limb sugges ing he po en ial o UCX® as a new he apeu ic ool o c i ical limb ischemia.
Keywo ds: UCX®, Mesenchymal s em cells, Angiogenesis, A e iogenesis, C i ical limb ischemia, Endo helial cells,
Hindlimb ischemia
Backg ound
C i ical limb ischemia (CLI) is a se e e o m o pe iph-
e al a e y disease (PAD) in which pa ien s wi h occlu-
si e a e ial disease o he legs expe ience ch onic
ischemic es pain, ulce , o gang ene [1]. This synd ome
is associa ed wi h se e e p ognosis, wi h 1-yea mo ali y
exceeding 25 % and abou 30–50 % majo limb ampu a-
ion a 1 yea om diagnosis [1]. The limi a ions o
su gical/endo ascula e ascula iza ion, due he dis ibu-
ion and di useness o a e ial occlusions, a e well
ecognized and ampu a ion, despi e i s associa ed
mo bidi y and mo ali y a es, is o en ecommended
[2]. The goal o limb sal age has s imula ed esea ch in o
al e na i e me hods, including he apeu ic angiogenesis
which can be achie ed ei he by local adminis a ion o
* Co espondence: [email p o ec ed].p
†
Equal con ibu o s
1
Cen o Ca dio ascula da Uni e sidade de Lisboa, A . P o Egas Moniz
1649-028 Lisbon, Po ugal
5
Faculdade de Medicina da Uni e sidade de Lisboa, A . P o Egas Moniz
1649-028 Lisbon, Po ugal
Full lis o au ho in o ma ion is a ailable a he end o he a icle
© 2016 The Au ho (s). Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0
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he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e
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Pe ei a e al. S em Cell Resea ch & The apy (2016) 7:145
DOI 10.1186/s13287-016-0410-4
p o-angiogenic g ow h ac o s and gene- o cell-based
he apies [3].
Mesenchymal s em cells (MSCs) hold g ea p omise as
a he apy o PAD, mainly due o hei pa ac ine ac i i y
and immunosupp essi e capaci y [4]. MSCs a e known
o home speci ically o hypoxic issues ollowing inju y
[5] whe e hey po en ia e ascula g ow h h ough he
elease o p o-angiogenic ac o s [6]. I was p e iously
epo ed ha au ologous, allogeneic and xenogeneic
MSC adminis a ion induced he apeu ic angiogenesis in
animal models o hindlimb ischemia (HLI) [7]. In a
me a-analysis o cell he apy o PAD, i was ound ha
au ologous bone ma ow MSCs deli e y led o imp o ed
indices o ischemia and pain- ee walking [8]. Al hough
i s ha es ed om he bone ma ow, MSCs ha e since
been iden i ied in many o he issues, namely adipose
issue [9] and umbilical co d issue [10]. UCX® in
pa icula a e MSCs ob ained om he human umbilical
co d Wha on’s jelly ha a e isola ed, expanded and c yo-
p ese ed acco ding o a pa en ed me hod (PCT/IB2008/
054067; WO 2009044379) and p oduced acco ding o
ad anced he apy medicinal p oduc (ATMP) guide-
lines [11]. UCX® ul il he MSC c i e ia as de ined by
he In e na ional Socie y o Cellula The apy (ISCT)
[12]. UCX® cells a e ad an ageous in compa ison o
o he sou ces due o he absence o in asi eness in
hei collec ion p ocess, hei as e sel - enewal,
highe cell yield and being mo e po en modula o s
o he immune sys em han bone ma ow MSCs,
making hem mo e a ac i e o allogeneic cellula
he apies.
Recen ly, i was demons a ed ha UCX® pa ac ine
ac i i y ep esses T cell ac i a ion and p omo es he
expansion o egula o y T cells (T egs) be e han bone
ma ow MSCs [13]. Fu he mo e, by using an acu e
a h i is in i o model i was ound ha UCX® can e-
duce paw edema mo e e icien ly han bone ma ow
MSCs. The use o a ch onic a h i is model showed ha
UCX® induce as e emission o local and sys emic a h-
i ic mani es a ions [13]. The UCX® issue egene a ion
capaci y sugges ed o heuma oid a h i is was la e co -
obo a ed o myoca dial in a c ion. By using a mouse
model o myoca dial in a c ion, i was shown ha UCX®
p ese e ca diac unc ion and a enua e ad e se issue
emodelling a e in amyoca dial ansplan a ion [14].
In e es ingly, i was demons a ed ha his ca diop o ec-
i e e ec was exe ed h ough pa ac ine mechanisms
in ol ing angiogenesis p omo ion [14]. Fu he mo e, in
i o s udies, pe o med wi h condi ioned medium (CM)
p oduced by UCX® g own in classical wo-dimensional
monolaye cul u es, ha e demons a ed he po en ial o
induce ke a inocy e mig a ion in he ea ly s ages o
wound healing [15]. Mo eo e , UCX® we e able o a ac
bone ma ow MSCs in i o wi h po en ial o p omo e
he o ma ion o g anula ion issue, con ac ion by myo-
ib oblas s, angiogenesis, asculogenesis and epi heliza-
ion [15]. These esul s s ongly sugges ha UCX® ha e
he po en ial o p omo e a ull ange o e en s leading o
issue egene a ion and homeos asis. In e es ingly, i
was shown ecen ly ha he la e p oli e a i e and e-
modelling s ages o wound healing p omo ed by he
CM o he wo-dimensional monolaye cul u es we e
imp o ed by using h ee-dimensional cul u e-de i ed
CM [16]. An enhanced sec e ion o healing-inducing
pa ac ine ac o s by UCX® including he ex acellula
cellula ma ix me allop o einase-2 (MMP-2) and
ma ix me allop o einase-9 (MMP-9), collagen I, ib o-
nec in, laminin, collagen IV and angiogenic ac o s
such as ascula endo helial g ow h ac o A (VEGF-A),
g anulocy e-colony s imula ing ac o (G-CSF), ans o m-
ing g ow h ac o β1 (TGF-β1), ib oblas g ow h ac o 2
(FGF-2), hepa ocy e g ow h ac o (HGF) and in e leukin
6 (IL-6) was obse ed in he CM o h ee-dimensional cul-
u es based on sel -assembled sphe oids [16].
In his wo k, ou da a co obo a e ha UCX® enhance
angiogenesis in i o and in i o, by modula ing endo-
helial cells. In i o, we ound ha UCX® p omo e
ubule o ma ion and endo helial cell mig a ion. Impo -
an ly, ou esul s ha e demons a ed ha UCX® in he
se ing o expe imen ally induced unila e al hindlimb
ischemia (HLI) s imula e angiogenesis and colla e al es-
sel de elopmen and he eby imp o e blood pe usion in
he ischemic limb demons a ing he po en ial o UCX®
as new he apeu ic ool o CLI. Fu he mo e, and o he
bes o ou knowledge, his is he i s s udy in an ex-
pe imen al model o hindlimb ischemia showing ha , in
i o and in a sus ained way human MSCs up egula e he
endo helial gene exp ession o se e al p o-angiogenic
playe s (such as ans o ming g ow h ac o β2(Tg β2),
angiopoie in 2 (Ang-2), Fg -2,Hg ), shedding ligh on he
po en ial mechanisms o ac ion o his pa icula ATMP.
Me hods
E hics and egula ions
All animal p ocedu es we e pe o med acco ding o Di ec -
i e 2010/63/EU. The p ocedu es we e app o ed by he in-
s i u ional Animal Wel a e Body and licensed by DGAV,
he Po uguese compe en au ho i y o animal p o ec ion
(license numbe 023861/2013).
Cell cul u e
UCX® we e cul u ed in s a ic monolaye s in α-MEM
wi h 1 g/L glucose and 2 mM glu amine (Sigma-Ald ich,
S . Louis, MO, USA), he ea e designa ed basal medium
(BM), supplemen ed wi h 20 % oe al bo ine se um
(FBS; Gibco®, Mad id, Spain), in a humidi ied incuba o
a 37 °C and 7 % CO
2
.
Pe ei a e al. S em Cell Resea ch & The apy (2016) 7:145 Page 2 o 12
Human umbilical ein endo helial cells (HUVECs)
(Sciencell, Ca lsbad, CA, USA) we e cul u ed in M199
media (Sigma-Ald ich, S . Louis, MO, USA), he ea e
designa ed endo helial basal medium (EBM) supple-
men ed wi h 10 % FBS (Gibco™, The mo Fishe Scien-
i ic, Wal ham, MA, USA), 50 μg/ml endo helial cell
g ow h supplemen (ECGS) (Sigma-Ald ich), 100 μg/ml
hepa in (Sigma-Ald ich) and 1 % penicillin-s ep omycin
(10,000 U/mL, Sigma-Ald ich), he ea e designa ed
endo helial g ow h medium (EGM). Cells we e g own in
lasks coa ed wi h 0.2 % gela in (Sigma-Ald ich) un il
70 % con luence and used up o passage 6.
P epa a ion o condi ioned media
UCX® we e seeded in BM supplemen ed wi h 5 % FBS
and g own un il 90 % con luence. A e washing cells
wi h phospha e-bu e ed saline (PBS), BM (wi hou
se um) was added o 24 hou s. The ea e , BM was e-
placed and condi ioned o 48 hou s a 37 °C and 7 %
CO
2
a e which i was collec ed, cen i uged a 300 g o
10 minu es o emo e cell deb is, il e ed (0.22 μm po e
size, Me ck Millipo e, Bille ica, MA, USA), and concen-
a ed using 5 kDa cu -o spin concen a o s (Agilen
Technologies, San a Cla a, CA, USA) as pe manu ac-
u e ’s ecommenda ions. Con ol condi ioned medium
samples unde wen he same p ocedu e as desc ibed
abo e, bu in he absence o cells.
Quan i ica ion o sec e ed ac o s
The quan i ica ion o HGF, VEGF-A, TGF-β1, IL-8,
pla ele -de i ed g ow h ac o -AA (PDGF-AA) and
FGF-2 in he condi ioned medium was pe o med using
a comme cially a ailable ki (FlowCy omix™; eBioscience,
San Diego, CA, USA) acco ding o he manu ac u e ’s
ins uc ions. Samples we e acqui ed on a Gallios low
cy ome e (Beckman Coul e , B ea, CA, USA) and he
esul s ob ained using FlowCy omix P o 3.0 So wa e
(eBioscience).
Tubule o ma ion assay
The ubule o ma ion assay was pe o med using he hick
gel me hod o p epa a ion. B ie ly, a e hawing o e -
nigh , he ma igel (Co ning, Co ning, NY, USA) was
pla ed in o a p e-cooled 48-well pla e (Nunc™, The mo
Fishe Scien i ic) (180 μl pe well) using a chilled pipe ip.
A e polyme iza ion o ma igel a 37 °C, 5 % CO
2
, o
45 minu es, HUVECs we e inocula ed a a densi y o
4.5 × 10
4
cells/cm
2
on op o he ma igel in 350 μlo
EBM. Cells we e hen incuba ed o 1 hou a 37 °C, 5 %
CO
2
.1×10
6
UCX® we e esuspended in 350 μlBMand
loaded on a 1-μm inse (B and, We heim, Ge many) and
ca e ully placed in a 48-well pla e and incuba ed o
16 hou s a 37 °C, 5 % CO
2
. Con ols wi h no UCX®
included HUVECs in (i) EBM, (ii) EGM and (iii) EBM
supplemen ed wi h FGF-2 (50 ng/ml). A e incuba ion,
inse s we e emo ed and all wells we e pho og aphed
(×10 ampli ica ion) using a Nikon Eclipse Ti-U in e ed
mic oscope wi h a conjuga ed Nikon DS-Qi1Mc came a
(Nikon, Tokyo, Japan). Tubule o ma ion was quan i ied
on ou andom ields pe eplica e, using he Angiogen-
esis analyse om Image J.
Wound-healing assay
HUVECs we e pla ed o con luence in a 24-well pla e
(p e iously coa ed wi h gela ine 0.2 %) wi h EBM
supplemen ed wi h 5 % FBS. A e cul u ing o e nigh ,
wounds we e c ea ed in he monolaye by sc apping he
pla e wi h a s e ile pipe e ip. A e ha , 1 × 10
6
UCX®,
esuspended in EBM supplemen ed wi h 5 % FBS and
loaded on a 1-μm inse (B and, We heim, Ge many),
we e ca e ully placed in a 24-well pla e and incuba ed
o 9 hou s a 37 °C, 5 % CO
2
. In con ols, he same p o-
cedu e was pe o med in he absence o UCX®. Pho o-
g aphs we e aken in P imo e (Ca l Zeiss Mic oscopy,
Jena, Ge many) on a × 4 magni ica ion and h ee inde-
penden wound a eas we e measu ed wi h ImageJ imme-
dia ely a e wounding and 9 hou s la e .
Mice
Twen y- wo-week-old C57BL/6 emale mice, pu chased
om Cha les Ri e Labo a o ies, Ba celona, Spain, we e
used in all expe imen s. The animals we e anaes he ised
wi h a ke amine-mede omidine cock ail (75 mg/kg BW
and 1 mg/kg BW, espec i ely) in ape i oneally o he
su gical p ocedu e as well as o he o he analysis
p ocedu es. The anaes hesia was pa ially e e ed wi h
a ipamezole (5 mg/kg BW). Pos ope a i ely, analgesia
was pe o med (bup eno phine 100 μl/15–30 g BW
e e y 8–12 h) and he animals we e closely moni o ed.
HLI model
A su gical p ocedu e was pe o med o induce unila e al
HLI in he mice. B ie ly, an incision in he skin o e lying
he high o he igh hindlimb o each mouse was made
and he dis al ex e nal iliac a e y and he emo al a e y
and eins we e liga ed and excised. The ein was liga ed
bo h o inc ease he se e i y o he ischemia as well as
o inc ease he echnical ep oducibili y o he model, as
isola ion o he emo al a e y alone o en esul s in ea -
ing o he ein esul ing in haemo hage.
UCX® adminis a ion
A dose o 2 × 10
5
UCX® was injec ed in amuscula ly,
5 hou s pos -ischemia induc ion on he gas ocnemius
muscle ( igh hindlimb) in a olume o 50 μl pe animal,
di ided in o wo injec ions o 25 μl. As a con ol, UCX®
ehicle (PBS) was adminis e ed a he same condi ions.
Pe ei a e al. S em Cell Resea ch & The apy (2016) 7:145 Page 3 o 12
Lase dopple pe usion imaging
The lase dopple pe usion image (Moo LDI V6.0,
Moo Ins umen s L d, Axmins e , UK) was used o as-
sess limb pe usion. Hai was emo ed 1 day be o e lase
dopple analysis using an elec ical sha e ollowed by
depila o y c eam. Blood low was measu ed bo h in he
ischemic leg and he con ala e al one, be o e HLI in-
duc ion (PRE-HLI), immedia ely pos -HLI (POST-HLI),
and a day 7, 14 and 21 pos -HLI (d7 POST-HLI, d14
POST-HLI and d21 POST-HLI, espec i ely). Colou -
coded images o issue pe usion we e eco ded and
poo o no pe usion was displayed as da k blue, and he
highes pe usion le el was displayed as ed. Mean lux
alues we e calcula ed using he Moo LDI V6.0 image
p ocessing so wa e. To accoun o a iables such as
empe a u e and ambien ligh , blood pe usion is
exp essed as he a io o ischemic o non-ischemic limb.
The mice we e placed on a 37 °C hea ing pad o educe
hea loss du ing measu emen s.
Immunohis ochemis y and capilla y densi y analysis
Mice we e sac i iced a day 90 pos -HLI ( o assu e capil-
la y s abiliza ion a e he ischemic inju y). The gas o-
cnemius muscles o bo h legs we e ha es ed, placed in
ans e se o ien a ion on a small co k disc wi h he help
o 10 % agacan h, snap ozen in liquid ni ogen-
cooled isopen ane and s o ed a -80 °C un il sec ioned.
Se en-mic ome e sec ions we e labeled wi h CD31
monoclonal an ibody (Pha mingen, San Diego, CA,
USA). A e ixa ion in ace one o 10 minu es, hyd ogen
pe oxidase (0.3 % dilu ed in me hanol) was added o
30 minu es, a oom empe a u e (RT) and ollowed by
wo washes in PBS o a o al o 10 minu es. Blocking
solu ion (5 % abbi se um in PBS) was applied o
30 minu es, a RT, and he slides we e hen incuba ed
o 1 hou a RT wi h a monoclonal an ibody agains
mouse CD31 a 1:500, dilu ed in 1 % bo ine se um albu-
min (BSA) in PBS. A e h ee washes in PBS o a o al
o 30 minu es, a seconda y bio inyla ed abbi an i- a
IgG an ibody was added a 1:200 in 1 % BSA in PBS and
5 % abbi se um o 30 minu es, a RT. Washes we e
pe o med as be o e and labeled a idin-conjuga ed pe -
oxidase complex (Vec as ain ABC ki ; Vec o Labo a o -
ies, Bu lingame, CA, USA) was used o colo
de elopmen acco ding o he manu ac u e ’s ecom-
menda ions o 30 minu es, a RT. A e insing in PBS
( h ee imes o 5 minu es), DAB pe oxidase subs a e
ki (Dako, Glos up, Denma k) was added o 5 minu es
o localize he immune complexes. The sec ions we e
coun e s ained wi h haema oxylin (Me ck, Kenilwo h,
NJ, USA) o 10 seconds and moun ed wi h en elan
(Me ck). Omission o he i s an ibody was used as a
nega i e con ol. Analysis o issue samples was
conduc ed using a Leica DM2500 up igh b igh ield
mic oscope (Leica Mic osys ems, We zla , Ge many).
Capilla y densi ies, i.e. numbe o capilla ies pe numbe
o myocy es, we e measu ed in wo di e en sec ions o
ou dis inc ana omic a eas o each specimen using he
ImageJ so wa e.
Con as agen pe usion and diaphonisa ion
Nine y days pos -ischemia induc ion ( o assu e ascula
s abiliza ion a e he ischemic inju y), mice we e deeply
anaes he ised and he o so and limbs we e sha ed. A
medial ho aco omy was pe o med o expose he hea
and a needle (26 gauge), a ached o an au oma ic in-
jec o , was in oduced in he le en icle. An incision
was pe o med in he igh a ium o allow enous
d ainage. Mice we e p ima ily pe used wi h hepa inized
se um (3000 IU/L) un il he blood was comple ely e-
mo ed om ci cula ion. A asodila a ion mix u e o ad-
enosine (1 mg/L) and papa e ine (4 mg/L) was
subsequen ly adminis e ed, igh be o e he con as
agen , o 2 minu es. The con as used was a mix u e o
ba ium sul a e (50 %) and gela in (5 %). This solu ion
was kep wa m un il he injec ion ime o a oid hicken-
ing. Con as agen was pe used manually un il he ee
blanched. Righ a e he injec ion he mice we e ans-
e ed o a cold chambe , so ha he con as agen
became solidi ied. All he solu ions we e injec ed wi h a
pe usion a e o 0.7 mL/minu e. Then, he skin o each
mouse was emo ed om he lowe body and diaphoni-
sa ion was pe o med by using a modi ied e sion o he
Spal eholz echnique. B ie ly, he mice we e ixa ed,
decalci ied, whi ened, washed, dehyd a ed by eeze
subs i u ion and placed in o a acuum pump wi h
Spal eholz solu ion (benzyl benzoa e and me hyl salicyl-
a e) un il anspa ency was acqui ed.
Colla e al essels quan i ica ion
Mice we e kep in Spal eholz solu ion du ing image
acquisi ion, in o de o achie e a homogenous densi y
be ween he issues and he media, wi h minimal abso p-
ion o e lec ion o ligh . Mice en i e limbs we e pho o-
g aphed in a magni ie wi h a ligh sou ce. A e
acquisi ion, images we e aligned and s i ched oge he
using Adobe Pho oshop CS6®, and en i e limb pho o-
g aphs we e ob ained. In o de o exclude he emo al
a e y and all enous s uc u es om his quan i ica ion,
only he colla e al essels we e manually segmen ed by
highligh ing hem using Adobe Pho oshop CS6®. We
conside ed as colla e al essels all he essels wi h a diam-
e e be ween 20 and 300 μm. Fo e e y mouse, an ana-
omically de e minable egion comp ising he liga ion si e
was selec ed and de ined as a egion o in e es (ROI).
Colla e al essels densi y (CVD) was quan i ied in equi a-
len ROIs co esponding o 20 % o he o al limb a ea.
Pe ei a e al. S em Cell Resea ch & The apy (2016) 7:145 Page 4 o 12
The CVD was calcula ed as he a io be ween he ascula
and he ROI a eas.
To exclude a ia ions in he ana omy, pe usion and
diaphoniza ion p ocedu es, he CVD alue o he non-
ischemic limb ob ained o each mouse was assumed o
co espond o 100 %. Acco ding o his assump ion, he
CVD pe cen age in he ischemic limb was calcula ed
ela i ely o he non-ischemic one. The pe cen age o
CVD inc ease was de e mined as he di e ence be ween
he CVD pe cen age among he ischemic and non-
ischemic limbs. Means o CVD pe cen ages and s and-
a d de ia ions we e calcula ed o each expe imen al
g oup. All densi y measu emen s we e pe o med using
ImageJ so wa e.
Lase cap u e mic odissec ion o capilla ies
Mice we e sac i iced a day 70 pos -HLI. Twel e-
mic ome e sec ions o he gas ocnemius muscles we e
labeled wi h CD31 monoclonal an ibody (Pha mingen).
The sec ions we e s o ed a –80 °C un il mic odissec-
ion. The immunohis ochemis y p o ocol desc ibed
abo e was modi ied o imp o e RNA p ese a ion by
using high sal bu e , 2 M NaCl in PBS 1× (a 4 °C) in
all incuba ion and washing s eps [17]. B ie ly, slides we e
placed in ace one, o 5 minu es, ai -d ied, ehyd a ed
wi h 2 M NaCl/PBS (4 °C) and incuba ed, o e nigh , a
4 °C wi h a monoclonal an ibody agains mouse CD31,
a 1:500, in 2 M NaCl/PBS. A e wo washes in 2 M
NaCl/PBS o a o al o 6 minu es, a seconda y bio inyl-
a ed abbi an i- a IgG an ibody was added a 1:200 in
2 M NaCl/PBS and 5 % abbi se um o 30 minu es, a
4 °C. Washes we e pe o med as be o e and labeled
a idin-conjuga ed pe oxidase complex (Vec as ain ABC
ki ; Vec o Labo a o ies) was used o colo de elopmen
acco ding o he manu ac u e ’s ecommenda ions o
30 minu es, a 4 °C. A e insing, DAB pe oxidase
subs a e ki (Vec o Labo a o ies) was added o 5 mi-
nu es o localize he immune complexes. Sec ions we e
dehyd a ed in ice-cold 90 % e hanol ollowed by 100 %
e hanol and allowed o d y. Ten housand capilla ies
we e mic odissec ed using a Zeiss PALM Mic oBeam
Lase Mic odissec ion Sys em (Ca l Zeiss Mic oscopy)
equipped wi h a pulsed solid-s a e 355 nm lase .
Dissec ed capilla ies we e ca apul ed in o a mic o uge
ube adhesi e cap.
RNA ex ac ion, cDNA syn hesis, p e-ampli ica ion and RT-PCR
To al RNA om he mic odissec ed capilla ies was
isola ed using an RNeasy Mic o Ki (QIAGEN, Hilden,
Ge many), including DNase ea men o emo e
po en ial genomic DNA con amina ion. Fo syn hesis
and p eampli ica ion o cDNA, RT
2
P eAMP cDNA
Syn hesis Ki (QIAGEN) was used wi h wo ounds o
p e-ampli ica ion using he ollowing p ime s: Fg -2_F
(5′- ACTCCAGTTGGTATGTGGCACTGA-3′); Fg -2_
R(5′-AACAGTATGGCCTTCTGTCCAGGT-3′); Tg b-
2_F (5′-GCTTTGGATGCGGCCTATTGCTTT-3′); Tg
b-2_R (5′-CTCCAGCACAGAAGTTGGCATTGT-3′);
Ang-2_F (5′-ATCCAACACCGAGAAGATGGCAGT-3′);
Ang-2_R (5′-AACTCATTGCCCAGCCAGTACTCT-
3′); Hg _F (5′-GCATTCAAGGCCAAGGAGAAGGT
T-3′); Hg _R (5′-TCATGCTTGTGAGGGTACTGCG
AA-3′); 18s_F (5′-GCCCTATCAACTTTCGATGGTA
GT-3′); 18s_R (5′- CCGGAATCGAACCCTGATT-3′).
RT-PCR was pe o med acco ding o he manu ac-
u e ’sp o ocolusingPowe SYBR®G een(In i o-
gen, Ca lsbad, CA, USA) and an Applied Biosys ems
(Wal ham, MA, USA) 7500 Fas Real-Time PCR o
he same a ge s desc ibed abo e. The housekeeping
gene used o no malize was 18S. The RT-PCR p o-
g am consis ed o an ini ial dena u a ion s ep, a 95 °
C, o 10 minu es ollowed by 50 cycles, a 95 °C, o
15 seconds and a 60 °C, o 1 minu e. The ela i e
quan i ica ion was pe o med acco ding o he com-
pa a i e me hod (2
-ΔΔC
; Applied Biosys ems Use
Bulle in no. 2P/N 4303859), wi h he non-ischemic
muscle as an in e nal calib a o . The o mula used is
2
-ΔΔC
=2
-[ΔC (sample) - ΔC (calib a o )]
,whe eΔC
(sample) = C (sample) –C ( e e ence gene). Fo he
in e nal calib a o ΔΔC = 0 and 2
0
=1. Fo he
emaining samples he alue o 2
-ΔΔC
indica es he
old change in gene exp ession ela i e o he calib a-
o . The ΔC alue o each sample is he a e age o
iplica es.
S a is ical analysis
Fo he wound-healing assay, he alues assume no mal
dis ibu ion and unequal a iances and he e o e an inde-
penden wo- ailed es was pe o med. Fo dopple ana-
lysis, an independen wo- ailed es was also pe o med
a days 7, 14 and 21. The alues assume no mal dis ibu-
ion. Only a day 7, we canno assume equal a iances.
Fo capilla y and colla e al analysis, he alues also assume
no mal dis ibu ion and equal a iances and he e o e an
independen wo- ailed es was also pe o med. Fo he
mas e junc ions and segmen leng h, one-way independ-
en analysis o a iance (ANOVA) was de eloped as da a
ollowed a no mal dis ibu ion, hough equali y o
a iances could no be assumed o he i s . The e o e, a
Games-Howell co ec ed pos hoc es was used o iden-
i y di e ences be ween g oups. Fo he meshes a ea, as
no mali y could no be assumed and so a K uskal-Wallis
non-pa ame ic es was de eloped ollowed by i s pai ed
analysis o iden i y di e ences be ween g oups. The e ec
size and powe was de e mined by using he G-Powe
so wa e in all ype o analyses. p< 0.05 was in e p e ed o
deno e s a is ical signi icance.
Pe ei a e al. S em Cell Resea ch & The apy (2016) 7:145 Page 5 o 12

Resul s
UCX® p omo e ubulogenesis and endo helial cell
mig a ion
We i s e alua ed he e ec o UCX® on he capilla y
s uc u e o ma ion. A ma igel ubule o ma ion assay
was used as an in i o model. HUVECs we e seeded
on o ma igel in endo helial basal medium (EBM) and
co-cul u ed wi h UCX®. As a con ol HUVECs we e
seeded in EBM, whe e we do no expec o isualize
capilla y-like s uc u es o med by HUVECS. Fo ha
eason, wo posi i e con ols we e added and HUVECs
we e seeded bo h in endo helial g ow h medium (EGM)
ha con ains se e al p o-angiogenic ac o s such as
VEGF and FGF-2 and in EBM supplemen ed wi h FGF-2
(EBM-FGF-2). As shown in Fig. 1a and quan i ied in
Fig. 1b, UCX® induce ubule o ma ion by HUVECs and
hese s uc u es consis en ly showed a signi ican in-
c ease in he numbe o mas e junc ions, o al mesh
a ea and o al segmen leng h when compa ed o hose
om HUVECs cul u ed in EBM o EGM. Wi h he ex-
cep ion o he o al mesh a ea, all he measu ed pa ame-
e s we e also signi ican ly di e en be ween HUVECs
cul u ed wi h UCX® in EBM and HUVECs cul u ed in
EBM supplemen ed wi h FGF-2. Nex , we assessed he
mig a o y capaci y o HUVECs seeded in EBM supple-
men ed wi h 5 % o se um co-cul u ed wi h UCX® wi h-
ou cell o cell con ac . Wi h his objec i e, a wound-
healing assay was pe o med and as a con ol HUVECs
we e seeded in he same cul u e condi ions bu in he
absence o UCX®. As shown in Fig. 2a and b, he wound
a ea dec eased mo e apidly in he p esence o UCX®
han in he con ol condi ion, sugges ing ha UCX®
p omo e he mig a ion o HUVECs ia a pa ac ine
sec e ion. Taken oge he , hese esul s clea ly show ha ,
in i o, UCX® induce angiogenic p ocesses in endo he-
lial cells.
A
B
C
Fig. 1 UCX® p omo e ubulogenesis. Ma igel assay was pe o med by seeding human umbilical ein endo helial cells (HUVECs) in endo helial basal
medium (EBM), endo helial g ow h medium (EGM), EBM supplemen ed wi h ib oblas g ow h ac o 2 (FGF-2) (EBM + FGF-2) o EBM co-cul u ed wi h
UCX®. (a) Rep esen a i e images om he di e en expe imen al condi ions a e shown. (b) Quan i a i e e alua ion demons a ed signi ican ly
enhanced mas e junc ions, o al mesh a ea and o al segmen leng h in capilla y-like s uc u es o med in HUVECs co-cul u ed wi h UCX® when
compa ed o he o he expe imen al condi ions. Fo mas e junc ions, ollowing Games-Howell pos hoc es he mean di e ences be ween UCX® and
he o he expe imen al condi ions we e: EBM (mean di . =24.27; p= 0.01); EGM (mean di . = 19.67; p= 0.03) and EBM + FGF-2 (mean di . = 13.8;
p= 0.034). The e ec size o basal was 2.08 and powe 0.98. Fo he meshes a ea, ollowing pai wise compa isons, he equi alen o a pos hoc
K uskal-Wallis es , signi ican di e ences be ween UCX® and EBM o UCX® and EGM we e obse ed wi h KW = 22.66, p< 0.01 o KW = 18.77, p=0.01,
espec i ely. Fo segmen leng h, ollowing Games-Howell pos hoc es , UCX® show signi ican di e ence when compa ed o he o he g oups: EBM
(mean di . = 4920; p< 0.001); EGM (mean di . = 3346; p= 0.01) and EBM + FGF-2 (mean di . = 2223; p= 0.016). E ec size o basal was 3.85 and powe
1. Scale ba , 500 μm
Pe ei a e al. S em Cell Resea ch & The apy (2016) 7:145 Page 6 o 12
UCX® sec e e p o-angiogenic ac o s
Since i was al eady demons a ed ha he bene icial
e ec s p omo ed by UCX® esul om a pa ac ine sec e-
ion, we decided o e alua e he sec e ion o six ep e-
sen a i e ophic ac o s c i ical o angiogenesis in he
condi ioned medium o an UCX® monolaye seeded in
basal medium (BM) o 48 hou s. As a con ol, he same
ophic ac o s we e measu ed in BM ha had no been
in con ac wi h cells. The exp ession o HGF, VEGF-A,
TGF-β1, in e leukin 8 (IL-8), pla ele -de i ed g ow h
ac o -AA (PDGF-AA) and FGF-2 we e analysed. Ou
esul s show a signi ican inc ease o HGF, TGF-β1, IL-8
and PDGF-AA in he condi ioned medium o UCX®
when compa ed o he con ol (Table 1), demons a ing
ha UCX® sec e e p o-angiogenic ac o s o he ex acel-
lula medium.
UCX® inc ease pe usion eco e y a e HLI
In o de o e alua e he he apeu ic po en ial o UCX® in
a HLI mouse model, he pe usion eco e y was assessed
o e ime a e ischemia induc ion and UCX® adminis-
a ion. Fi e hou s a e su gical induc ion o unila e al
HLI, UCX® o hei ehicle (as a con ol) we e adminis-
e ed in he ischemic muscle and pe usion was mea-
su ed o e ime. As shown in Fig. 3a and quan i ied in
Fig. 3b, a d ama ic educ ion in blood low was obse ed
in he ischemic limb immedia ely a e su ge y, in com-
pa ison o he con ala e al limb, ollowed by a g adual
no maliza ion o ischemic blood low o e ime. Impo -
an ly, UCX® ma kedly imp o ed blood low eco e y a
7, 14 o 21 days pos -HLI, compa ing wi h con ol mice,
demons a ing ha UCX® adminis a ion con ibu es o-
wa d he unc ional eco e y o ischemic issues.
A
B
Fig. 2 UCX® s imula e endo helial cell mig a ion. Human umbilical ein endo helial cells (HUVECs) we e seeded o con luence in endo helial basal
medium (EBM) supplemen ed wi h 5 % FBS and cul u ed o 16 hou s a e wounding. Immedia ely a e wound, HUVECs we e co-cul u ed wi h
UCX® o no (con ol) o 9 hou s. (a) Rep esen a i e images a e shown. The le and igh panels conce n HUVEC monolaye immedia ely a e o
9 hou s pos -wounding, espec i ely. (b) The wound a ea was measu ed immedia ely a e and 9 hou s pos -wounding by using ImageJ so wa e
( h ee independen measu emen s we e pe o med o each expe imen al g oup; (2.03) = 4.66; *p< 0.05); e ec size was 3.82 and powe 0.93)
Pe ei a e al. S em Cell Resea ch & The apy (2016) 7:145 Page 7 o 12
UCX® inc ease capilla y and colla e al essel densi y a e
HLI
As blood low eco e y depends on bo h angiogenesis
and a e iogenesis we examined whe he UCX® would
a ec capilla y and colla e al essel densi ies in hindlimb
muscles. Fi e hou s a e su gical induc ion o unila e al
HLI, UCX® o hei ehicle (as a con ol) we e adminis-
e ed in he ischemic muscle and mice we e sac i iced
90 days pos -HLI induc ion. Capilla y densi y was
assessed h ough quan i ica ion o CD31-posi i e capil-
la ies on his ological sec ions o gas ocnemius muscle.
As expec ed, he capilla y densi y was g ea e in he
ischemic e sus he non-ischemic hindlimb in bo h ex-
pe imen al g oups (Fig. 4b). No ably, he le el o capil-
la y densi y in he ischemic hindlimb ea ed wi h UCX®
was signi ican ly highe han he one obse ed in he
con ol ischemic hindlimb, as shown in Fig. 4a and
quan i ied in Fig. 4b. In o de o e alua e he colla e al
essel densi y (CVD), mice we e diaphonised and an
equi alen egion o in e es (ROI), co esponding o
20 % o he limb a ea, was selec ed o CVD quan i ica-
ion (Fig. 4c). A signi ican ly highe CVD inc ease was
obse ed in UCX®- ea ed mice when compa ed o he
con ol (Fig. 4d). Taken oge he , ou esul s show ha
A
B
Fig. 3 UCX® inc ease pe usion eco e y. UCX® o hei ehicle (as a con ol) we e adminis e ed in he ischemic gas ocnemius muscle 5 hou s a e HLI
induc ion. (a) Rep esen a i e lase dopple low images be o e (PRE-HLI), immedia ely a e (d0 POST-HLI) and a 7, 14 and 21 days pos -HLI induc ion
(d7 POST-HLI, d14 POST-HLI, d21 POST-HLI). (b) Quan i a i e e alua ion o blood low exp essed as a a io o ISC o NISC limb demons a ed signi ican ly
enhanced limb blood pe usion in UCX®- ea ed mice a 7, 14 and 21 days pos -HLI. (n = 16 o each expe imen al g oup; D7: (22.69) = 4.26; ***p< 0.001;
e ec size was 1.51 and powe 0.98; D14: (30) = 4.7; ***p< 0.001; e ec size was 1.66 and powe 0.99; D21: (30) = 7.22; ***p< 0.001; e ec size was 2.56
and powe 0.99). HLI hindlimb ischemia, ISC ischemic, NISC non-ischemic
Table 1 P o-angiogenic ac o s p esen in he condi ioned medium om UCX® o basal medium
UCX® CM (pg/ml)
Mean±SEM
Basal CM (pg/ml)
Mean±SEM
- es
HGF 2,64 ± 0,718 0,001 ± 0 0,0144
VEGF-A 2,025 ± 1,043 0,001 ± 0 0,1099
TGF-β1 0,983 ± 0,122 0,001 ± 0 0,0005
IL-8 0,765 ± 0,069 0,001 ± 0 0,0001
PDGF-AA 0,123 ± 0,045 0,001 ± 0 0,0412
FGF-2 0,0317 ± 0,009 0,02 ± 0 0,2874
CM condi ioned medium
Pe ei a e al. S em Cell Resea ch & The apy (2016) 7:145 Page 8 o 12
UCX® signi ican ly augmen capilla y densi y and CVD
inc ease a e ischemic inju y.
UCX® up egula e he endo helial Tg -β2, Ang-2, Fg -2 and
Hg exp ession in esponse o HLI
To elucida e he mechanism o ac ion o UCX® in ou
HLI model we in es iga ed i UCX® could change he
endo helial gene exp ession. Fo his pu pose, 5 hou s
a e su gical induc ion o unila e al HLI, UCX® o hei
ehicle (as a con ol) we e adminis e ed in he ischemic
muscle and mice we e sac i iced a day 70 pos -HLI in-
duc ion. The gas ocnemius muscle sec ions we e
s ained o CD31 and isualized using a Lase Cap u e
Mic odissec ion mic oscope. CD31-posi i e cells we e
dissec ed, isola ed and analysed by quan i a i e RT-PCR
o he exp ession o se e al p o-angiogenic ac o s.Ou
esul s show ha ansc ip s o Tg -β2, Ang-2, Fg -2 and
Hg we e clea ly up egula ed in endo helial cells isola ed
om muscle o he ischemic limb, compa ing wi h hose
ound in endo helial cells om he con ala e al limb.
This is obse ed exclusi ely in mice ea ed wi h UCX®.
Con ol mice show he opposi e end, down egula ing
he exp ession o he angiogenic genes in endo helium
om he ischemic limb, when compa ed wi h he
con ala e al limb (Fig. 5). These esul s sugges ha he
mechanism o ac ion o UCX® in he HLI mouse model
in ol es he up egula ion o se e al angiogenic genes in
he endo helial cells p esen in he ischemic muscle.
Discussion
CLI is a mani es a ion o pe iphe al a e ial disease ha
desc ibes pa ien s wi h ch onic ischemic es pain, ulce s
o gang ene [1]. A ailable he apies a e limi ed and pa-
ien s may equi e ampu a ion. The goal o limb sal age
has s imula ed esea ch in o al e na i e me hods, includ-
ing he apeu ic angiogenesis. S em cell he apy holds
g ea po en ial o he apeu ic angiogenesis, bu i s clin-
ical ansla ion has been slow due o (i) leng hy p oce-
du es pe o med ex i o in which s em cells a e
manipula ed and o en lose iabili y, di e en ia e o
AB
CD
Fig. 4 UCX® inc ease capilla y and colla e al densi ies. UCX® o hei ehicle (as a con ol) we e adminis e ed in he ischemic gas ocnemius muscle
5 hou s a e HLI induc ion. (a) Rep esen a i e sec ions om con ol and UCX®- ea ed ischemic gas ocnemius muscles a 90 days pos -HLI. Capilla ies
and myocy es we e iden i ied by CD31 immunohis ochemis y and haema oxylin, espec i ely. Scale ba , 125 μm. (b) Quan i a i e analysis, a 90 days
pos -HLI, e ealed inc eased capilla y densi y (capilla ies/myocy e) in UCX®- ea ed ischemic gas ocnemius muscles compa ed o con ol ischemic
ones. (n = 3 and n = 7 o con ol and UCX®, espec i ely; (8) = 4.44; **p= 0.0057; e ec size was 3.064 and powe 0.97). (c) Illus a i e images o se-
lec ed egions o in e es (ROI) o con ol and UCX®- ea ed mice. ISC and NISC limbs a e shown. Scale ba , 1 mm (d) Da a a e ep esen ed as he pe -
cen age o colla e al essel densi y (CVD) inc ease o he ISC limb ela i ely o he NISC one. A 90 days pos -HLI, UCX®- ea ed mice p esen ed
signi ican ly highe CVD inc ease (%) when compa ed o con ol mice (n = 5 o each expe imen al g oup; (8) = 7.63; ***P=0.000062; e ec size was
4.82 and powe 0.99). ISC ischemic, NISC non-ischemic
Pe ei a e al. S em Cell Resea ch & The apy (2016) 7:145 Page 9 o 12