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Abs ac
Cell he apy wi h adul s em cells is a new ba le
on o he con ol o aging. Be o e being used
o his pu pose, we need o answe se e al basic
ques ions abou he biochemis y and physiology
o hese cells. This pape p esen s some aspec s
and p elimina y esul s ob ained in ou labo a o y
using s em cells om adipose issue.
In oduc ion
Va ious s a egies a e used o con ol he e ec s
o aging on he no mal unc ions o an o ganism.
An ioxidan s, die , exe cise and e en ac i a ing
enzymes o phase II de oxi ica ion appea o delay
he decay unc ion o a g ea e o lesse ex en .
Howe e , none o hese s a egies p e en biological
aging. Hence, o con ol he e ec s o aging i will
be necessa y o emphasize he need o explo e he
mechanisms o issue epai and egene a ion as a
complemen a y me hodology o o he p e en ion
s a egies. In his sense, Regene a i e Medicine
may ep esen a ascina ing al e na i e o con ol
he aging p ocess.
One o he aspec s o Regene a i e Medicine
is based on he egene a i e capaci y o adul
s em cells a e in mos issues and con ibu e o
his homeos asis and epai . A e issue damage
se e al in acellula and in e cellula pa hways a e
ac i a ed in a coo dina ed a emp o es o e he
issue in eg i y. Wi h aging occu s gene al decline
in he egene a ion po en ial. Besides his loss o
egene a i e capaci y is p oduced by al e a ions o
he s em cells wi h aging, i is hough ha s em
cells ansplan ed in o damaged o aged issues may
ha e he apeu ic and es o a i e capaci y. In ac ,
s em cells ep esen a huge p omise in he he apy
o many aging- ela ed degene a i e diso de s aging
some diseases such diabe es, hea disease, s oke,
Pa kinson, e c [1-4]
Po en ial use o adipose issue s em cells in he con ol
o aging
Ma io F. Muñoz1, F ancisco Zu i a1, Sand o A güelles1, Rocío Vazquez2, José Se es3, Ma ías Guzmán4,
Remedios Guillén4, José An onio Rod íguez5, José Luis Co és6, Jaime Muñoz7, José An onio Pin o 7, Me -
cedes Cano8 and An onio Ayala1.
1 Depa amen o de Bioquímica y Biología Molecula , Facul ad de Fa macia. Uni e sidad de Se illa. Spain. 2 Clínica
Rocío Vazquez. Se illa. 3 Clínica Se es. Se illa. 4 Depa amen o de B oma ología, Toxicología y Medicina Legal. Facul-
ad de Fa macia. Uni e sidad de Se illa. 5Ins i u o de Biomedicina de Se illa. Spain. 6 Cen o de In es igación Biomé-
dica, G anada. Spain. 7 Cen o Andaluz de Biología Molecula y Medicina Regene a i a, Se illa. Spain.8 Depa amen o
de Fisiología. Facul ad de Fa macia. Uni e sidad de Se illa
Co espondence: An onio Ayala ,Dp o. Bioquímica y Biología Molecula . Facul ad de Fa macia. Uni e sidad de
Se illa. C/.T amon ana s/n. 41012 - Se illa
Keywo ds. S em cells; adipose issue; aging.
Acknowledgemen s. This wo k was suppo ed by Spanish Minis e io de Ciencia e Inno ación BFU
2010 20882.
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S em cells
S em cells ha e he abili y o sel - enew
h ough symme ic di isions and he po en ial
o di e en ia ing in o se e al di e en cell ypes
depending on hei deg ee o mul ipo en iali y
h ough asymme ic di ision [1-3,5-8]. I is now
known ha mos o he issues ha e a e y speci ic
popula ion o adul s em cells ha allow hei
egula enewal o egene a ion [6,8]. Thus, i has
been desc ibed he exis ence o hese SC in issues
such as muscle, skin, li e , panc eas, b ain [9],
in es ines, a y issue [10,11].
In highe animals, s em cells ha e been classi ied
in o wo g oups: emb yonic s em cells and o gan-
speci ic s em cells o s em cells om adul issue.
The la e a e able o o igina e cells o a pa icula
o gan in he adul . Ou wo k ocuses on adul s em
cells. The ac i i y o hese s em cells a ies g ea ly
om one o gan o ano he : hose o he bone
ma ow ha o m blood cells a e e y ac i e and
a e con inually di iding, while hose which a e, o
example, in he small in es ine a e mo e inac i e.
Some adul s em cells a e capable o di e en ia ing
in o mo e han one cell ype as mesenchymal s em
cells.
Adipose issue mesenchymal s em cells (ADSC)
Wi hin he adul s em cell g oup a e he
mesenchymal s em cells (MSC). The MSC belong
o he mesenchyma, which by a di e en ia ion
p ocess will lead o he blood essels, smoo h
muscle, meso helium, lympha ic sys em and
connec i e issue i sel . These cells can be
ob ained om di e en o gans including e al
li e , umbilical co d, and bone ma ow [12,13].
Ano he impo an sou ce is adipose issue, which
con ains p ogeni o cells called adipose issue s em
cells (ADSC) [2,10,14,15].
Ad an age o ADSC
The easy access o he subcu aneous a by
liposuc ion, allows ADSC o be ob ained unde
local anes hesia and wi h minimal discom o o
he pa ien . In addi ion, i s high abundance wi h
no e hical p oblems associa ed wi h i s use, make
adipose issue an impo an sou ce o MSC [16-
18]. Ano he ad an age is ha he p opo ion o
MSC is 500 imes highe [19,20] in adipose issue
han in he bone ma ow, so ha a la ge numbe
o cells can be ob ained wi hou a la ge numbe
o passes, dec easing he isk o ch omosomal
abno mali ies induced senescence in cul u es
[21]. An addi ional ad an age is i s po en ial o
di e en ia e in o bone, ca ilage, endons, skele al
muscle, a , endo helial issue and mac ophages
when g own unde speci ic condi ions o each
lineage [11,13,22]. Su p isingly, he ADSC no
only ha e he po en ial o di e en ia e in o cells o
mesode mal o igin and o gans, bu also hey ha e
he abili y o di e en ia e in o neu ons, endoc ine
cells o he panc eas, hepa ocy es, endo helial cells
and ca diomyocy es [10].
ADSCs can epai and egene a e he issues by
se e al mechanisms: Fi s , he ADSC ansplan ed
in o a damaged o diseased issue can sec e e
cy okines and g ow h ac o s ha s imula e he
eco e y in a pa ac ine manne . The ADSC could
modula e he hos s em cell niche by s imula ing
he ec ui men o endogenous s em cells o a
pa icula si e and p omo e hei di e en ia ion
in o he equi ed lineage. Also, ADSC can p o ide
an ioxidan s so ha oxic subs ances eleased in o
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he local en i onmen a e elimina ed, he eby
p omo ing he eco e y o he su i ing cells.
Ano he mechanism is hough hei in i o
di e en ia ion in o he desi ed line, p io o hei
au ologous ansplan a ion [2]. They can also ac as
immune modula o s [23].
Today i is possible o isola e and cul u e adul
s em cells o he apeu ic use. Once ansplan ed,
hese cells can be used o eju ena e damaged
issue by aging o o he causes. Howe e , no
one knows o su e he consequences o he
s em cells ea men s because he e a e many
basic ques ions abou he biology o hese CM
ha mus be answe ed be o e i s he apeu ic use.
Ob iously, hese cells ha e o su i e and pe o m
hei unc ion in un a ou able condi ions as he
damaged issue mic oen i onmen , whe e many
ac o s needed o s em cells a e lacking. Unde
hese ad e se condi ions, he su i al o he
s em cells will depend on his “molecula heal h”
and i s esponsi eness when implan ed in he
issue. The e o e, s em cells mus ha e a obus
epai mechanisms and esis ance in o de o
pa icipa e in issue egene a ion. I is likely ha
his molecula heal h and s ess esponse depends
on many biochemical and physiological aspec s
ela ed o dono age, li es yle, e c. As hese cells a e
con inuously ecei ing “on-o ” signals o di ide,
i could be ha old cells a e less sensi i e o hese
signals. Consequen ly, he he apeu ic applica ions
o s em cells in adul issue epai equi es a be e
unde s anding o he biology o hese cells, o he
en i onmen o he damaged issue o bo h.
The wo k we a e ca ying ou in ou labo a o y
y o answe se e al o hese basic ques ions abou
ADSC.
Fi s o all, we ocused on he isola ion me hods.
These me hods a e based on magne ic cell so ing
(Mil enyi Bio ech), whe e magne ic mic opa icles
linked o an ibodies ecognize su ace an igen in
he s em cells. The sepa a ion is pe o med on a
column inse ed in an ex emely powe ul magne
o e ain he labeled cells. This me hodology allows
us o build a cell bank o pa ien s o di e en ages
ha mee he c i e ia o “Mesenchymal and Tissue
S em Cell Commi ee o he In e na ional Socie y
o Cellula The apy.”
The i s c i e ia is ha cells mus be adhe en o
he plas ic. The pho og aphs o Figu e 1 show ha
he human ADSC a e adhe en o he cul u e lask
wi hou addi ion o any subs a e.
Figu e 1. Human ADSC (42 y s) in cul u e lask. 10x
magni ica ion.
A p e equisi e o conside ing he cul u ed
ADSC as MSC, is o demons a e ha hey ha e
he po en ial o di e en ia e in o a leas wo
di e en cell ypes [11,13,22,24]. Fo his, cells
we e cul u ed in speci ic di e en ia ing media.
Figu e 2 shows pho og aphs aken be o e and a e
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di e en ia ion in o adipocy es, whe e i can be
obse ed usi o m cells con aining lipid acuoles.
The ed s aining o hese acuoles (Fig. 2 and B)
shows he di e en ia ion in o adipocy es ADSCs.
Fu he mo e, he p esence o black-pu ple
shown by he p esence o alkaline phospha ase
in os eoblas s gene a ed by ADSC di e en ia ion
(Fig. 2C)
In addi ion o he abili y o adhe ence o plas ic
and di e en ia ion, he esul s p o ided by low
cy ome y se e o con i m he na u e o he cells
ob ained. Figu e 3 show he esul s ob ained om
a cell popula ion o human ADSC. These g aphs
ep esen s side sca e (Side Sca e , SS), which
gi es in o ma ion abou he exis ence o di e en
cell popula ions. The o wa d sca e ( o wa d
sca e , FS), gi es an insigh on cell size. As can be
seen, he sample con ains a unique cell popula ion,
e lec ed in he g aph as a single cloud.
To e i y ha he ob ained cells mee he
minimum s anda ds equi ed by he commi ee
o mesenchymal s em cells and issues o he
in e na ional cell he apy, cells mus ha e he
su ace ma ke s CD73, CD90 and CD105. On he
con a y, hey should no con ain CD14, CD34,
CD45, CD19, CD79α and human leukocy e
an igen (HLA)-DR.
Figu e 2. Di e en ia ion o human ADSC in o adipocy es
and os eoblas s. A) Pic u e o human adipocy es ob ained
om ADSC (pa ien o 37 yea s) p io o s aining, 10X
magni ica ion. B) Pho og aph a e ixa ion and s aining
p ocess. C) Pho og aph o os eoblas s a e ixing and s ai-
ning 20X magni ica ion.
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The his og ams ep esen he numbe o e en s
coun ed on each exposed channel. In ou case,
he channel FL1, FL2 and FL4 a e se o exci e
he luo opho es FITC, PE, Pe CP, espec i ely.
Conside ing he an ibodies used, (CD14-Pe CP,
CD20-Pe CP, CD34-Pe CP, CD45-Pe CP,
CD105-FITC and CD90-PE), FL1 channel gi es
us in o ma ion on he numbe o cells exp essing
CD90 ma ke (Fig. 3B); FL2 channel gi es
in o ma ion abou he p opo ion o CD105-
exp essing cells (Fig. 5C) and FL4 channel
indica es he pe cen age o posi i e cells is any
o he ollowing ma ke s: CD14, CD20, CD34
and CD45 (Fig. 3D). The i s his og am (Fig.
3B) indica es ha 94.53% o he sample is posi i e
o he CD90 ma ke . The second his og am
(Fig. 3C) shows ha almos 100% o he sample
exp essed he ma ke CD105. Only 9.5% o he
cells ha e one o he ollowing ma ke s: CD14,
CD20, CD34, CD45.
Finally, we ha e used a me hod o labeling and
acking ha allow us o moni o s em cells once
hey a e injec ed in i o. This me hod is based in
ans ec ing he ADSC wi h he luci e ase gene
by using len i i us. A e in ape i oneal injec ion
o luci e in, cells ca ying he luci e ase gen can
be obse ed by using an in i o imaging sys em.
Figu e 4A shows a cul u e o ans ec ed ADSC in
a 25 cm 2 lask p io o adminis a ion o 3 luci e in
used as a con ol o bioluminescence. Figu e 4B
shows he same cul u e a e he adminis a ion o
D-luci e in.
Figu e 3. S udy o he deg ee o cellula homogenei y
and pheno yping o human ADSC. Da a we e ob ained
om he low cy ome e FC-500 (Beckman-Coul e ). The
esul s o posi i e e en s o he luo opho e a e in %.

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Conclusion
In summa y, we can say ha cell he apy open a
new ba le on in he con ol and ea men o
aging ela ed diseases. Howe e , he e a e many
basic ques ions abou he biology o hese s em cells
ha should be answe ed o p edic he he apeu ic
po en ial o hese cells, some o which a e going o
be known a e he comple ion o he he p esen
s udy because. The main objec i e o his wo k is
o iden i y he ac o s a ec ing he “ obus ness” o
he biochemis y and physiology o ADSC.
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