Low concen a ions o ni ic oxide delay he
di e en ia ion o emb yonic s em cells and p omo e
hei su i al
JR Tejedo*
,1
, R Tapia-Limonchi
2
, S Mo a-Cas illa
1
, GM Cahuana
1
, A Hmadcha
2
, F Ma in
1
, FJ Bedoya
1
and B So ia
2
Ni ic oxide (NO) is an in acellula messenge in se e al cell sys ems, bu i s con ibu ion o emb yonic s em cell (ESC) biology
has no been cha ac e ized. Exposu e o ESCs o low concen a ions (2–20 lM) o he NO dono die hylene iamine NO adduc
con e s p o ec ion om apop osis elici ed by leukaemia inhibi o y ac o (LIF) wi hd awal. NO blocked caspase 3 ac i a ion,
PARP deg ada ion, down egula ion o he p o-apop o ic genes Casp7,Casp9,Bax and Bak1 and up egula ion o he an i-
apop o ic genes Bcl-2 111,Bcl-2 and Bi c6. These e ec s we e also obse ed in cells o e exp essing eNOS. Exposu e o
LIF-dep i ed mESCs o low NO p e en ed he loss o exp ession o sel - enewal genes (Oc 4,Nanog and Sox2) and he SSEA
ma ke . Mo eo e , NO blocked he di e en ia ion p ocess p omo ed by he absence o LIF and bFGF in mouse and human
ESCs. NO ea men dec eased he exp ession o di e en ia ion ma ke s, such as B achyu y, Ga a6 and Ga a4. Cons i u i e
o e exp ession o eNOS in cells exposed o LIF dep i a ion main ained he exp ession o sel - enewal ma ke s, whe eas he
di e en ia ion genes we e ep essed. These e ec s we e e e sed by addi ion o he NOS inhibi o L-NMMA. Al oge he , he da a
sugges ha low NO has a ole in he egula ion o ESC di e en ia ion by delaying he en y in o di e en ia ion, a es ing he loss
o sel - enewal ma ke s and p omo ing cell su i al by inhibi ing apop osis.
Cell Dea h and Disease (2010) 1, e80; doi:10.1038/cddis.2010.57; published online 7 Oc obe 2010
Subjec Ca ego y: Expe imen al Medicine
Emb yonic s em cells (ESCs) e ain hei abili y o sel - enew
and o di e en ia e in o a g ea a ie y o cell ypes. The genes
Oc 4,Sox2,Nanog and Myc a e key egula o s essen ial o
he main enance o sel - enewal.
1
The di e en ia ion o ESCs
is accompanied by he down egula ion o hese ansc ip ion
ac o s and up egula ion o ansc ip ion ac o s in ol ed in
di e en ia ion.
2
Loss o Oc 4 causes inapp op ia e di e en ia-
ion o ESCs in o ophec ode m, whe eas o e exp ession o
Oc 4 esul s in di e en ia ion in o p imi i e endode m and
mesode m.
3
Nanog egula es plu ipo ency mainly as a
ansc ip ional ep esso o downs eam genes ha a e
impo an o cell di e en ia ion, such as Ga a4 and Ga a6.
4
These ac o s o m a igh ansc ip ional egula o y ci cui ha
main ains ESCs in an undi e en ia ed s a e.
In addi ion, hese ansc ip ion ac o s a e swi ched on and
o by ex e nal signals.
5
Thus, leukaemia inhibi o y ac o (LIF)
main ains he sel - enewal s a e in mESCs h ough ac i a ion
o he JAK/STAT pa hway.
6
The main downs eam a ge o
he LIF is c-myc, an ea ly esponse gene ha p omo es he
exp ession o he egula o y subuni o elome ase.
7
LIF also
ac i a es c-S c y osine kinase, ex acellula signal- egula ed
kinases (ERKs) and he phosphoinosi ide 3-kinase, which
is ele an o he p oli e a ion and su i al o mESCs.
8
Howe e , LIF signalling is no su icien o sus ain sel -
enewal, and addi ional signals a e equi ed o ep ess
di e en ia ion.
9
I has been shown ha IRS-1 exp ession is
necessa y o main aining he sel - enewal s a e in mESCs,
and ha his ac ion is independen o S a 3 phospho yla ion.
10
Also, exp ession o he ac i e o m o Ak is su icien o
main ain he undi e en ia ed pheno ype o hese cells.
11
These epo s sugges ha he phosphoinosi ide 3-kinase/Ak
pa hway is necessa y o he main enance o sel - enewal in
mESCs.
In hESCs, he main enance o undi e en ia ed g ow h is LIF
independen . In his ega d, hESCs g own in eede laye - ee
cul u e equi e a medium condi ioned by ib oblas cul u e.
In he absence o such condi ioned medium, high concen a-
ions o basic ib oblas g ow h ac o (bFGF) and Noggin a e
necessa y o main ain sel - enewal.
12
In ac , ac i a ion o he
FGF pa hway is necessa y o he con ol o su i al and
p oli e a ion in se e al cell ypes.
13
Inc eases in MAP-E k
kinase/ERK and Ak phospho yla ion downs eam o bFGF
ha e been epo ed o occu in hESCs.
14
Mo eo e , bFGF can
unc ion in coope a ion wi h insulin-like g ow h ac o II o
main ain hESC plu ipo ency. Al hough bFGF con ibu es o
cell su i al wi h li le in luence on apop osis, insulin-like
Recei ed 06.5.10; e ised 28.7.10; accep ed 10.8.10; Edi ed by A Finazzi-Ag o0
1
Andalusian Cen e o Molecula Biology and Regene a i e Medicine (CABIMER)-Uni e si y Pablo de Ola ide, CIBERDEM, Se ille, 41092, Spain and
2
Andalusian
Cen e o Molecula Biology and Regene a i e Medicine (CABIMER)-Fundacio
´n P og eso y Salud, Se ille, 41092, Spain
*Co esponding au ho : JR Tejedo, A . Ame
´ ico Vespucio s/n-Edi . CABIMER. Pa que Cien ı
´ ico y Tecnolo
´gico Ca uja 93. Se ille 41092, Spain. Tel: þ34 954 467840;
Fax: þ34 954 461664; E-mail: [email p o ec ed]
Keywo ds: emb yonic s em cell; di e en ia ion; sel - enewal; ni ic oxide
Abb e ia ions: ESCs, emb yonic s em cells; NO, ni ic oxide; LIF, leukaemia inhibi o y ac o ; bFGF, basic ib oblas g ow h ac o ; DETA-NO, die hylene iamine ni ic
oxide adduc ; GFP, g een luo escen p o ein; eNOS, endo helial ni ic oxide syn hase; iNOS, inducible ni ic oxide syn hase; PI3K, phosphoinosi ide 3-kinase;
Ak , p o ein kinase b; GC/PKG, guanyla e cyclase/p o ein kinase G; IGF I, insulin-like g ow h ac o I; IGF II, insulin-like g ow h ac o II; SSEA 1, s age-speci ic an igen 1;
SSEA 4, s age-speci ic an igen 4; AP, alkaline phospha ase
Ci a ion: Cell Dea h and Disease (2010) 1, e80; doi:10.1038/cddis.2010.57
&
2010 Macmillan Publishe s Limi ed All igh s ese ed 2041-4889/10
www.na u e.com/cddis
g ow h ac o -II inc eases p oli e a ion and p e en s apop o-
sis.
15
O he signal ansduc ion pa hways ha ha e a ole in
hESC sel - enewal include he ans o ming g ow h ac o -b
(TGF-b)/Ac i in/Nodal,
16,17
phosphoinosi ide 3-kinase
18
and
Wn
19
pa hways.
Ni ic oxide (NO) is a signalling molecule ha egula es cell
su i al and p oli e a ion in di e se cell ypes.
20,21
I has also
been shown o a ec gene exp ession a he le el o
ansc ip ion and ansla ion.
22
NO is p oduced du ing he
de elopmen o o ganisms by iso o ms o NO Syn hases
(NOSs), and i is easible ha i migh con ibu e o he
egula ion o some ea u es o ESC biology. I has been
epo ed ha a physiological concen a ions, NO unc ions as
a nega i e egula o o s em/p ogeni o cell p oli e a ion, and
i is c i ical o he ini ia ion o di e en ia ion.
23
A dis inc
ea u e NO is ha ac ions inside cells depend hea ily on i s
concen a ion. In he mic omola o nanomola ange, ac i a-
ion o su i al and o he guanyla e cyclase/p o ein kinase G
sys em has been epo ed.
24
Homeos a ic concen a ions o
NO (1–10 mM) can ini ia e signalling pa hways implica ed in
su i al ac ions in RINm5F cells and p omo e he ac i a ion o
c-S c, which a he same ime induces he ac i a ion o IRS-1,
phosphoinosi ide 3-kinase, AKT and eNOS as a pa o he
su i al esponse.
21
Mo eo e , he gene a ion o NO pa ici-
pa es in he ac i a ion o su i al pa hways by insulin-like
g ow h ac o -1 and insulin in b-cells.
25
Thus, many o he
signalling pa hways ac i a ed by NO in o he cell sys ems a e
ope a i e in ESCs. Recen epo s ha e shown ha a
unc ional NO-cGMP signalling sys em migh be in ol ed in
ea ly di e en ia ion e en s o ESCs.
26
Howe e , i has also
been p oposed ha he dual ole o NO in he con ol o ESC
di e en ia ion migh depend on he exp ession o NO-signalling
componen s a he han on he di e en ial exp ession o NOS
iso o ms du ing his pe iod o de elopmen .
24,26
G ea e o is being de o ed o iden i ying s a egies ha
imp o e he sel - enewal and plu ipo ency o ESCs. He e, we
p o ide e idence o a ole o NO in delaying ESC di e en ia-
ion. This e ec is independen o LIF, sugges ing ha his
messenge could be used o imp o e ESC sel - enewal in
cul u e.
Resul s
Di e en ial exp ession o NO syn hases and exp ession
o sel - enewal ma ke s in mESCs. NOS iso o ms gene
exp ession analysis o D3-mESCs ans ec ed wi h a cons uc
con aining he eGFP gene unde con ol o he Oc 4 p omo e
(D3-pOc 4-eGFP-mESCs) and selec ed by so ing e ealed
ha cells cul u ed in he p esence o LIF exp essed he eNOS.
Di e en ia ion o hese cells ollowing LIF wi hd awal o as
emb yoid bodies led o a subs an ial dec ease o eNOS and
an inc ease in he exp ession o he iNOS iso o m. Fu he ,
nNOS exp ession emained unchanged excep o a sligh
inc ease in cells di e en ia ed as emb yoid bodies (Figu e 1a).
A ime cou se s udy e ealed ha eNOS exp ession le els
dec eased signi ican ly om he second day o LIF wi hd awal;
he lowes le els we e obse ed be ween 4 and 7 days
(Figu e 1b uppe ), whe eas iNOS exp ession began on day 5
and peaked on day 7 (Figu e 1b lowe ).
Nex , D3-pOc 4-eGFP-mESCs we e ans ec ed wi h a
ec o ha cons i u i ely o e exp esses he eNOS gene
unde con ol o he CMV p omo e (D3-pOc 4-eGFP-eNOS-
mESC). Bo h g een mESCs we e selec ed by so ing in
con ol condi ions (Figu e 1c and, Supplemen a y Figu e 1).
These cells exp essed Nanog, Oc 4 and Sox2 mo e highly
han did wild- ype D3-mESCs. In addi ion, showed high le els
o Te (Figu e 1c), elome ase ac i i y (Figu e 1d) and eNOS
p o ein (Figu e 1e) ela i e o D3-mESCs. Fu he mo e,
colonies o hese cells emi g een luo escence, as con i med
by luo escence mic oscopy and low cy ome y (Supplemen-
a y Figu e 1a and b).
E ec o NO on apop osis induced by LIF wi hd awal in
mESCs. In D3-pOc 4-eGFP-mESCs we s udied he e ec
o low concen a ions o he die hylene iamine ni ic oxide
adduc (DETA-NO) on cell su i al and apop osis. LIF
dep i a ion led o a 6.5- old inc ease in DNA agmen a-
ion (Figu e 2a, shaded ba s); he exposu e o 2–10 mM
DETA-NO con e ed p o ec ion agains his ac ion. DETA-NO
concen a ions o 20 mM o highe induced a signi ican
inc ease in apop osis (3.5- old a 20 mM and eigh - old a
50 mM) in compa ison o LIF con ol cells. Cells g own in
he p esence o LIF, a signi ican e ec o DETA-NO on
DNA agmen a ion was obse ed a 50 mM, he highes
concen a ion es ed (Figu e 2a, open ba s). Nex , we
es ed he e ec o endogenously NO on cell su i al by
using D3-mESC-Oc 4-eGFP-eNOS-mESCs (Figu e 2b).
A signi ican dec ease in DNA agmen a ion was obse ed
in hese cells when compa ed wi h D3 and D3-pOc 4-eGFP-
mESCs cul u ed in he absence o LIF o 7 days.
O e exp ession o eNOS, howe e , did no comple ely
p e en he apop osis induced by LIF wi hd awal. To
iden i y he mechanism in ol ed in he p o ec i e ac ion o
NO, we s udied he exp ession o apop o ic genes in hese
cells (Figu e 2c). D3-pOc 4-eGFP-mESCs g own in he
absence o LIF and in he p esence o 2 mM DETA-NO
showed a dec ease in he exp ession o p o-apop o ic
genes, such as Bax,Bak1,Casp9 and Casp7. The
exp ession le els o he an i-apop o ic genes Bcl2l11,Bcl2
and Bi c6 we e wo- old highe han he le els in cells
cul u ed unde LIF wi hd awal condi ions (Figu e 2c).
Mo eo e , PARP down egula ion and caspase 3 ac i a ion
induced by LIF wi hd awal we e inhibi ed by NO ea men
(Figu e 2d). In addi ion, R1-mESCs cul u ed in he absence
o LIF and in he p esence o 2 mM DETA-NO displayed
a dis inc mo phology when compa ed wi h cells cul u ed
in he absence o LIF, some o hem showing a compac
mo phology wi h well-de ined bo de s ypical o he
‘s emness’ s a e (Supplemen a y Figu e 3).
Impac o NO on he egula ion o plu ipo ency and
di e en ia ion ma ke s in mESCs. A dose-dependency
o DETA-NO was ca ied ou o s udy he e ec o NO on
he plu ipo ency ma ke s Oc 4 and Nanog. Figu e 3a shows
ha Oc 4 exp ession was no a ec ed by exposing cells
o DETA-NO (2–50 mM) in he p esence o LIF, whe eas
Nanog exp ession was enhanced. Oc 4 and Nanog exp es-
sion dec eased when cells we e incuba ed wi h 50 mM
DETA-NO (Figu e 3a). In mESCs cul u ed in he absence
Con ol o di e en ia ion and cell su i al by NO
JR Tejedo e al
2
Cell Dea h and Disease
o LIF, DETA-NO (2–50 mM) p omo ed bo h Oc 4 and Nanog
p o ein exp ession (Figu e 3a).
Using R1-E-mESCs we s udied he e ec o NO in o he
mESC line. R1-E-mESCs cul u ed in he p esence o LIF g ew
in colonies displaying s onge posi i e s aining o alkaline
phospha ase (100%) han cells g own in he absence o LIF
(14%). Supplemen a ion o 2 mM DETA-NO o medium wi hou
LIF pa ially es o ed alkaline phospha ase ac i i y (40%); he
numbe o posi i e colonies dec eased as he concen a ion o
DETA-NO inc eased: 5 mM (36%), 10 mM (25%), 20 mM (20%)
and 50 mM (9.5%) (Figu e 3c). In addi ion, he exp ession o
di e en ia ion ma ke s was analyzed by quan i a i e eal ime
(qRT)-PCR. Ea ly di e en ia ion genes, such as B achyu y,
Ga a4,Ga a6,Fg 5 and Fg 8 a e exp essed in cells cul u ed
in he absence o LIF. DETA-NO supplemen a ion led o
lowe exp ession o hese genes (Figu e 3b). The le els o
exp ession obse ed wi h 2 mM DETA-NO we e compa able
o hose ob ained in cells g own in he p esence o LIF.
Mo eo e , as he concen a ion o NO inc eased, he
exp ession o di e en ia ion ma ke s also inc eased
(Figu e 3b).
NO delays mESC di e en ia ion. We nex in es iga ed
he e ec o exogenous NO on di e en ia ion. In D3-pOc 4-
eGFP-mESCs, he exposu e o LIF led o he exp ession o
GFP in 69.7±8.5% o cells. Howe e , when cells we e
cul u ed in he absence o LIF, he pe cen age o GFP-
posi i e cells dec eased (27.7±5.5%). Supplemen a ion wi h
2mM DETA-NO inc eased he pe cen age o GFP-posi i e
cells (45.4±6.5%) (Figu e 4a).
In addi ion, mESCs cul u ed in he absence o LIF
supplemen ed wi h 2 mM DETA-NO showed signi ican ly
highe exp ession o Oc 4, Sox2 and Nanog han cells
cul u ed in he absence o LIF (Figu e 4b). Wes e n blo
Figu e 1 Di e en ial exp ession o ni ic oxide (NO) syn hases and exp ession o sel - enewal ma ke s in mESCs. (a) NO syn hase exp ession du ing di e en ia ion.
So ed D3-pOc 4-eGFP mESCs we e cul u ed o 7 days in he p esence o absence o LIF a ached o a su ace and o 14 days by hanging d op p o ocol (EB p epa a ion).
eNOS, iNOS and nNOS exp ession we e de ec ed by RT-PCR. The images a e ep esen a i e o h ee expe imen s. (b) Time cou se o eNOS and iNOS exp ession. R1-E
mESCs we e cul u ed in he absence o LIF o 7 days. eNOS and iNOS exp ession we e de ec ed by qRT-PCR, b-ac in was used as an endogenous con ol and cycle
h eshold (C ) alues we e no malized wi h espec o hose o cells cul u ed in he p esence o LIF (day 0). Da a shown a e om h ee independen expe imen s. *P 0.005
wi h espec o day 0. (c) qRT-PCR o undi e en ia ion ma ke s. D3 mESCs, D3-pOc 4-eGFP mESCs and D3-pOc 4-eGFP-eNOS mESCs we e cul u ed in he p esence o LIF
and gene exp ession was de e mined by qRT-PCR. Da a a e he mean ±S.E.M. o h ee expe imen s; *P 0.005 when compa ed wi h D3 cells and **P 0.005 when
compa ed wi h D3-pOc 4-eGFP cells. (d) Telome ase ac i i y in D3 mESCs, D3-pOc 4-eGFP mESCs and D3-pOc 4-eGFP-eNOS mESCs. Each da a poin ep esen he
mean ±S.E.M. o h ee expe imen s; *P 0.005 when compa ed wi h o he cell ypes. (e) Wes e n blo analysis o eNOS in D3 mESCs, D3-pOc 4-eGFP mESCs and D3-
pOc 4-eGFP-eNOS mESCs. Rep esen a i e image o h ee independen expe imen s
Con ol o di e en ia ion and cell su i al by NO
JR Tejedo e al
3
Cell Dea h and Disease
analysis also showed highe le els o hese p o eins, bu wi h
Sox2 and Nanog in a di e en pa e n han he gene
exp ession. I is o no e ha he le els o he p o eins shown
we e simila o hose ound in undi e en ia ed cells
(Figu e 4c). In non- ans ec ed D3-mESCs, he exp ession
o B achyu y (Figu e 4d) inc eased by LIF-wi hd awal was
cancelled by 2 mM DETA-NO. In addi ion, he co-exp ession o
SSEA-1 and Oc 4 p o ein (Figu e 4e and Supplemen a y
Figu e 2A) dec eased in he absence o LIF, whe eas
simul aneous ea men wi h DETA-NO main ained he Oc
4 and SSEA-1 signals. To add ess he e ec o NO on
apop osis and plu ipo ency/di e en ia ion, we moni o ed
caspase 3 ac i a ion and Nanog and B achyu y exp ession
in he p esence o DETA-NO and s au ospo ine, an induce o
apop osis (Supplemen a y Figu e 2C). S au ospo ine cancels
he p o ec i e e ec o NO on apop osis induced by LIF
wi hd awal wi hou a ec ing he e ec o NO on Nanog o
B achyu y exp ession.
Endogenous eNOS o e exp ession main ains mESCs in
an undi e en ia ed s a e. A second app oach was o s udy
he e ec o endogenous NO p oduc ion by eNOS o e -
exp ession using D3-pOc 4-eGFP-eNOS-mESCs. G ow h in
he p esence o LIF esul ed in 70.9±3.1% GFP-posi i e
cells. In he absence o LIF, he p opo ion o GFP-posi i e
cells dec eased o 51.8±9.1%. The p opo ion o GFP-
posi i e cells d opped o 16.4±5.8% when NOS ac i i y was
inhibi ed wi h 400 mM L-NMMA (Figu e 5a).
qRT-PCR e ealed ha he exp ession o plu ipo ency
ma ke s was lowe in D3-pOc 4-eGFP-eNOS-mESCs cul-
u ed in he absence o LIF in compa ison wi h hose g own
in i s p esence, bu hei exp ession dec eased when
NOS ac i i y was inhibi ed wi h 400 mM L-NMMA (Figu e 5b).
A simila e ec was obse ed in wes e n blo analysis
(Figu e 5c). The p o ein le els o Oc 4 dec eased signi-
ican ly, whe eas Sox2 also dec eased, bu his change
was no signi ican . I is o no e ha he le els o Nanog
did no change signi ican ly be ween he wo condi ions
(Figu e 5c). In con as , when NOS ac i i y was inhibi ed,
he exp ession o he B achyu y gene inc eased wi h espec
o i s exp ession in cells cul u ed wi h LIF (Figu e 5d). The
co-exp ession o SSEA-1 and Oc 4 p o ein dec eased in
he absence o LIF, and i was comple ely inhibi ed when
NO p oduc ion was blocked wi h 400 mM L-NMMA (Figu e 5e
and Supplemen a y Figu e 2B). In he p esence o LIF,
L-NMMA pa ially educed Oc 4 exp ession bo h in wild- ype
mESCs and mESCs o e exp essing eNOS (Supplemen a y
Figu e 2D).
Figu e 2 E ec o di e en NO concen a ions on apop osis and cell su i al. (a) E ec o DETA-NO on DNA agmen a ion. D3-pOc 4-eGFP mESCs we e cul u ed o
7 days in he p esence o absence o LIF ea ed wi h di e en concen a ions (2–50 mM) o DETA-NO. DNA agmen a ion was measu ed. Da a a e he mean ±S.E.M. o i e
independen expe imen s. *P 0.005 e sus. D3 mESCs cul u ed in he p esence o LIF and in he absence o DETA-NO (b) D3, D3-pOc 4-eGFP and D3-pOc 4-eGFP-eNOS
mESCs we e cul u ed in he p esence o absence o LIF o 7 days and DNA agmen a ion was measu ed. Da a a e he mean ±S.E.M. o i e independen expe imen s.
*P 0.005 e sus. D3 and D3-pOc 4-eGFP mESCs cul u ed in he absence o LIF. **P 0.005 e sus. mESCs cul u ed in he p esence o LIF. (c) Analysis o p o-apop o ic
and an i-apop o ic gene exp ession in D3-pOc 4-eGFP mESCs. Cells we e cul u ed in he absence o LIF and ea ed wi h 2 mM DETA-NO gene exp ession was de e mined
by qRT-PCR. b-ac in was used as an endogenous con ol and C alues we e no malized wi h espec o he C o cells cul u ed in absence o LIF. Da a a e he
mean ±S.E.M. o h ee expe imen s. (d) E ec o DETA-NO on PARP and caspase 3 clea age. D3-pOc 4-eGFP mESCs we e cul u ed in he p esence o absence o LIF
o 7 days and supplemen ed wi h 2 mM DETA-NO. PARP and clea ed caspase 3 we e de ec ed by wes e n blo . The posi i e con ol o apop osis was 1 mM S au ospo ine.
Figu es a e ep esen a i e o h ee independen expe imen s
Con ol o di e en ia ion and cell su i al by NO
JR Tejedo e al
4
Cell Dea h and Disease
Low amoun s o NO main ain hESC plu ipo ency. In he
hESC HS181 line, he p esence o 8 ng/ml bFGF main ained
he undi e en ia ed ea u es o colonies o up o i e
passages (Figu e 6a, uppe ow, le pic u e). A e i e
passages in he absence o bFGF, he colonies displayed
whi e a eas, which a e ypical o he di e en ia ed s a e
(Figu e 5a, uppe ow, middle pic u e). When cells we e
cul u ed in bFGF-dep i ed condi ions supplemen ed wi h
2mM DETA-NO o up o i e passages, undi e en ia ed
cha ac e is ics we e es o ed and sub-cul u ing was possible
o 10 passages (Figu e 5a, uppe ow, igh pic u e). In
hESC colonies cul u ed o i e passages, DETA-NO was
obse ed o inc ease Oc 4 exp ession, which was much
g ea e han ha no ed unde bFGF condi ions (Figu e 5a,
bo om ow). Quan i a i e gene exp ession alues we e
ob ained o plu ipo ency ma ke s and di e en ia ion genes
in hESCs. The exp ession o Nanog, Oc 4 and Sox2
conside ably declined in cells cul u ed in he absence o
bFGF; in con as , cells ea ed wi h DETA-NO main ained
highe le els o hese mRNAs, ye s ill lowe han cells
cul u ed wi h bFGF (Figu e 6b). These esul s we e
con i med by wes e n blo ing (Figu e 6c). By con as ,
di e en ia ion genes, such as B achyu y and Ga a-4
showed a signi ican inc ease in exp ession in he absence
o bFGF ha was cancelled by addi ion o DETA-NO
(Figu e 6d). Nex , we examined he exp ession o he cell-
su ace an igen SSEA-4. The esul s showed ha SSEA-4
exp ession disappea ed a e i e passages o bFGF
wi hd awal, bu was p esen when he cul u e medium was
supplemen ed wi h 2 mM DETA-NO (Figu e 6e).
Discussion
E idence ga he ed in ecen yea s indica es ha NO
pa icipa es in he egula ion o cell di e en ia ion and issue
mo phogenesis,
23,27
bu i s con ibu ion o he con ol o ESC
Figu e 3 Dose-dependen in luence o DETA-NO on plu ipo ency and di e en ia ion in mESCs. (a) Wes e n blo analysis o Nanog and Oc 4 in non- ans ec ed D3
mESCs cul u ed in he absence o p esence o LIF and supplemen ed wi h di e en DETA-NO concen a ions (2–50 mM) o e 7 days. The image is ep esen a i e o i e
expe imen s. (b) Dose-dependen e ec o DETA-NO on he exp ession o di e en ia ion ma ke s in R1-E mESCs cul u ed o 5 days; b-ac in was used as he endogenous
con ol and C alues we e no malized wi h espec o he C o cells cul u ed in absence o LIF. Da a a e he mean ±S.E.M. o i e independen expe imen s. *P 0.005
e sus. cells cul u ed in he absence o LIF. **P 0.005 e sus. cells cul u ed in he absence o LIF supplemen ed wi h 2 mM DETA-NO. (c) Alkaline phospha ase ac i i y
e ained by cells ea ed wi h di e en concen a ions o DETA-NO. Pe cen ages we e ob ained wi h espec o he numbe o colonies in he cul u e in p esence o LIF. Scale
ba s, 20 mM. Figu es a e ep esen a i e o h ee independen expe imen s. Pe cen age da a a e he mean ±S.E.M. o h ee independen expe imen s. *P 0.005 when
compa ed wi h cells cul u ed in he absence o LIF
Con ol o di e en ia ion and cell su i al by NO
JR Tejedo e al
5
Cell Dea h and Disease

biology emained o be subs an ia ed. He e, we show ha
low amoun s o NO gene a ed ei he om chemical dono s
o ollowing eNOS o e exp ession egula e apop osis, sel -
enewal and di e en ia ion o ESC. When mESCs we e
cul u ed unde op imal condi ions in he p esence o LIF, high
exp ession le els o eNOS and sligh ly lowe le els o nNOS
we e de ec ed. A speci ic dec ease in he eNOS iso o m was
appa en when cells we e exposed o one o wo di e en ia-
ion p o ocols, oge he wi h high exp ession o iNOS. The
high exp ession o iNOS indica es ha high le els o NO
du ing sequen ial di e en ia ion complemen he e ec s
induced by LIF wi hd awal. High NO was p e iously shown
o inc ease ESC di e en ia ion owa ds de ini i e endode m,
ca diomyocy es and neu ons.
28–30
LIF dep i a ion induces also di e en ial exp ession o
NOS iso o ms. eNOS exp ession signi ican ly dec eased
a e LIF wi hd awal un il day 6, a e which i main ained a
low exp ession le el, whe eas iNOS exp ession began o
inc ease a e day 5 and was highes a day 7. The le el o
exp ession o eNOS is associa ed o plu ipo ency as wild- ype
Figu e 4 Exogenous NO a es s he di e en ia ion induced by LIF wi hd awal in mESCs. (a) D3-pOc 4-eGFP mESCs om passage 10 a e so ing we e cul u ed o
7 days in he p esence (le panel) o absence (medium panel) o LIF and in he absence o LIF plus 2 mM DETA-NO ( igh panel). GFP in ensi y was measu ed by low
cy ome y. Cells wi h luo escence in ensi y simila o highe han ha obse ed o mESCs ans ec ed wi h he pOc 4-eGFP cons uc and cul u ed o 7 days in he p esence
o LIF we e conside ed posi i e o eGFP (M1 popula ion). Each g aph is ep esen a i e o i e independen expe imen s (n¼5). (b) qRT-PCR analysis o Oc 4, Sox-2 and
Nanog in D3-pOc 4-eGFP mESCs cul u ed o 7 days unde he indica ed condi ions. b-ac in was used as he endogenous con ol and C alues we e no malized wi h espec
o he C o cells cul u ed in he absence o LIF. Da a a e he mean ±S.E.M. o h ee expe imen s. *P 0.005 e sus. cells cul u ed in he absence o LIF. (c) Wes e n blo
analysis o Oc 4, Sox-2 and Nanog in D3-pOc 4-eGFP mESCs cul u ed o 7 days unde he indica ed condi ions. Rep esen a i e images and densi ome y quan i ica ion a e
om i e independen expe imen s. (d) qRT-PCR analysis o B achyu y exp ession in D3-pOc 4-eGFP mESCs. b-ac in was used as he endogenous con ol and C alues
we e no malized wi h espec o he C o cells cul u ed in he absence o LIF. Da a a e he mean ±S.E.M. o h ee expe imen s. *P 0.005 e sus. cells cul u ed in he
absence o LIF. (e) Con ocal images o cells posi i e o SSEA-1 in non- ans ec ed D3 mESCs cul u ed o 7 days in he p esence (le panel) o absence (middle panel) o LIF
o in he absence o LIF plus 2 mM DETA-NO ( igh panel). Nuclei a e coun e -s ained wi h DAPI. Images a e om h ee independen expe imen s. Scale ba s a e 20 mm
Con ol o di e en ia ion and cell su i al by NO
JR Tejedo e al
6
Cell Dea h and Disease
D3-mESCs om passage 30 exp essed lowed le els o
eNOS, and low exp ession o Oc 4, Nanog, Sox2, Te and
low elome ase ac i i y. On he o he hand so ed D3-pOc 4-
eGFP-mESCs had inc eased le els o eNOS when compa ed
wi h D3 wild- ype cells and also had inc eased le els o Oc 4,
Nanog, Sox2, Te and elome ase ac i i y and eNOS o e -
exp ession signi ican ly inc eased he exp ession o Oc 4,
Sox2, Te and elome ase ac i i y ela i e o D3-pOc 4-
eGFP-mESCs. Fu he mo e, when eNOS ac i i y was
blocked, undi e en ia ion ma ke s dec eased no ably. These
indings suppo he no ion ha gene a ion o low and
sus ained le els o NO migh be ins umen al o he
main enance o plu ipo ency. The hypo hesis ha p ecise
cellula esponses a e di e en ially egula ed by speci ic NO
concen a ions was p e iously p oposed.
31
Concen a ions in he ange 1–10 mM o DETA-NO and
endogenous NO gene a ion by eNOS o e exp ession a e
also capable o hal ing apop osis induced by LIF s a a ion.
Figu e 5 Endogenous p oduc ion o NO by eNOS o e exp ession main ains mESC sel - enewal a e LIF wi hd awal. (a) D3-pOc 4-eGFP-eNOS mESCs om passage
10 a e so ing we e cul u ed o 7 days in he p esence (le panel) o absence (medium panel) o LIF o in he absence o LIF plus 400mM L-NMMA ( igh panel). GFP in ensi y
was measu ed by low cy ome y. Cells wi h luo escence in ensi y simila o highe han ha obse ed o mESCs ans ec ed wi h he pOc 4-eGFP cons uc and cul u ed o
7 days in he p esence o LIF we e conside ed posi i e o eGFP (M1 popula ion). Each g aph is ep esen a i e o i e independen expe imen s (n¼5). (b) qRT-PCR analysis
o Oc 4, Sox-2 and Nanog in D3-pOc 4-eGFP-eNOS mESCs cul u ed o 7 days unde he indica ed condi ions. b-ac in was used as he endogenous con ol and C alues
we e no malized wi h espec o he C o cells cul u ed in he p esence o LIF. Da a a e he mean ±S.E.M. o h ee expe imen s. *P 0.005 e sus. cells cul u ed in he
absence o LIF. (c) Wes e n blo analysis o Oc 4, Sox-2 and Nanog in D3-pOc 4-eGFP mESCs cul u ed o 7 days unde he indica ed condi ions. Rep esen a i e images and
densi ome y quan i ica ion a e om i e independen expe imen s (d) qRT-PCR analysis o B achyu y exp ession in D3-pOc 4-eGFP-eNOS mESCs. b-ac in was used as he
endogenous con ol and C alues we e no malized wi h espec o he C o cells cul u ed in he absence o LIF. Da a a e he mean ±S.E.M. o h ee expe imen s. *P 0.005
e sus cells cul u ed in he p esence o LIF. (e) Immuno luo escence images o cells posi i e o SSEA-1 in D3-pOc 4-eGFP-eNOS mESCs cul u ed o 7 days in he p esence
(le panel) o absence (medium panel) o LIF o in he absence o LIF plus 400 mM L-NMMA ( igh panel). Nuclei we e coun e -s ained wi h DAPI. Images a e ep esen a i e o
h ee independen expe imen s. Scale ba s a e 20 mm
Con ol o di e en ia ion and cell su i al by NO
JR Tejedo e al
7
Cell Dea h and Disease
Fu he mo e, ea men o mESCs wi h NO nega i ely
egula es he p o-apop o ic esponse and no ably inc eases
he an i-apop o ic esponse. These esul s a e consis en wi h
p e ious epo s in o he cell models in which i was shown
ha NO p e en ed apop osis by egula ing Bcl2 amily
p o eins.
32
In addi ion, mESCs o e exp essing Bcl2 main ain
he cha ac e is ics o undi e en ia ed, plu ipo en ESCs
du ing long- e m cul u ing and main ain hei po en ial o
di e en ia e in o ma u e cell ypes.
33
All hese da a a e in
ag eemen wi h o he esul s desc ibing he an i-apop o ic and
su i al e ec s o NO.
20,21,25
A ole o su i al p ocesses in
he main enance o he undi e en ia ed s a e in ESCs is also
suppo ed by se e al o he s udies.
8,11,14,15,18
In addi ion,
using s au ospo ine, an induce o apop osis, oge he wi h
Figu e 6 Exogenous NO a es s he di e en ia ion induced by bFGF wi hd awal in hESCs cul u ed in a eede - ee sys em. (a) Uppe panel: Rep esen a i e phase
con as images showing he mo phology o hESCs cul u ed o i e passages in he p esence (le ) o he absence (middle) o bFGF and in he absence o bFGF plus 2 mM
DETA-NO ( igh ). Images a e ep esen a i e o 10 expe imen s. Lowe panel: immuno luo escence images o cells posi i e o Oc 4 in hESCs cul u ed o i e passages in he
p esence (le ) o absence (middle) o bFGF o in he absence o bFGF plus 2 mM DETA-NO ( igh ). Images a e ep esen a i e o h ee expe imen s. Scale ba s a e 20 mm.
(b) qRT-PCR analyses o Oc 4, Sox-2 and Nanog in H181 hESCs cul u ed o 10 days unde he indica ed condi ions. b-ac in was used as he endogenous con ol and
C alues we e no malized wi h espec o he C o cells cul u ed in he p esence o bFGF. Da a a e he mean ±S.E.M. o h ee expe imen s. *P 0.005 e sus cells cul u ed
in he absence o bFGF. **P 0.005 e sus cells cul u ed in he p esence o LIF. (c) Wes e n blo analysis o Oc 4, Sox-2 and Nanog in H181 hESCs cul u ed o 10 days
unde he indica ed condi ions. Rep esen a i e images and densi ome y quan i ica ions a e om i e independen expe imen s. (d) qRT-PCR analysis o B achyu y and Ga a4
exp ession in H181 hESCs . b-ac in was used as he endogenous con ol and C alues we e no malized wi h espec o he C o cells cul u ed in he absence o bFGF. Da a
a e he mean ±S.E.M. o h ee expe imen s. *P 0.005 e sus cells cul u ed in he absence o bFGF. (e) Con ocal images o cells posi i e o SSEA-4 in hESCs cul u ed o
i e passages in he p esence (le panel) o absence (middle panel) o bFGF o in he absence o bFGF plus 2 mM DETA-NO ( igh panel). Nuclei we e coun e -s ained wi h
DAPI. Images a e ep esen a i e o h ee expe imen s. Scale ba s a e 20 mm
Con ol o di e en ia ion and cell su i al by NO
JR Tejedo e al
8
Cell Dea h and Disease
DETA-NO, we showed ha s au ospo ine cancels NO e ec
on caspase 3 ac i a ion induced by LIF wi hd awal. In con as ,
he e ec o NO on Nanog and B achyu y le els was no
al e ed. These esul s sugges ha NO p o ec i e ac ion is
independen o i s e ec on plu ipo ency and di e en ia ion.
The da a p esen ed in his epo also p o ide e idence o a
ole o NO in ESC plu ipo ency. In he absence o LIF, a low
le el o DETA-NO main ains he sel - enewal p og am in
some colonies by enhancing he exp ession o sel - enewal
ma ke s and inc easing alkaline phospha ase ac i i y. In
addi i on, bo h mESCs and hESCs cul u ed in he absence
o LIF o bFGF and in he p esence o NO, a e capable o
o ming e a omas in immunosupp essed mice (Supple-
men a y Figu e 4). P e ious epo s ha e indica ed a
equi emen o Nanog up- egula ion o he main enance o
sel - enewal.
1,4,9,34
Masui e al.
35
ound ha Sox2 con ols he
ansc ip ional ac i a ion o Oc 4. In ac , i has been ecen ly
demons a ed ha Oc 4 exp ession is di ec ly ela ed o Sox2
exp ession.
35,36
In acco dance wi h hese obse a ions, ou
s udy showed ha Oc 4, Nanog and Sox2 exp ession le els
a e ma kedly inc eased by he addi ion o NO. Mo eo e ,
when eNOS ac i i y is blocked, Sox2 exp ession dec eases.
One possibili y is ha NO could be con olling sel - enewal
and plu ipo ency by modula ing Oc 4 exp ession by inc eas-
ing Sox2. Fu he mo e, in his con ex he ela ionship
be ween eNOS exp ession and he main enance o he
undi e en ia ed s a e is ele an . In his ega d, was obse ed
ha a ound 50% o D3-eNOS cells cul u ed in he absence o
LIF ailed o di e en ia e, whe eas NOS inhibi ion by L-NMMA
main ained only 16% o cells exp essing GFP unde con ol o
he Oc 4 p omo e . Analysis o he gene exp ession and
p o ein immunocy ochemis y co obo a ed hese esul s. I is
in e es ing ha he p o ein le els did no co ela e well wi h
mRNA le els, sugges ing ha plu ipo ency ma ke egula ion
in ol es he egula ion o p o ein le els. In his espec , oles
o caspase ac i a ion and pos - ansla ional modi ica ion o
sel - enewal p o eins ha e been ecen ly pu o wa d.
5
I is
impo an o no e ha he e ec s o NO on di e en ia ion and
he main enance o plu ipo ency a e dose dependen , wi h
mo e bene icial e ec s a lowe concen a ions. Ea ly di e -
en ia ion ma ke s, such as B achyu y, Ga a 4, Ga a6, Fg 5
and Fg 8 a e conside ably ep essed.
The SSEA-1 su ace ma ke o undi e en ia ed cells and
colonies posi i e o alkaline phospha ase s aining also
showed ha a e i e days o cul u e wi hou LIF, se e al
cells emained wi h ma kedly undi e en ia ed ac i i y and
mo phology. Mos o he cul u ed cells showed e idence o an
ini ial di e en ia ion p ocess, sugges ing ha NO could be
ac ing in a selec i e way on he o al ESC popula ion. This is in
acco dance wi h he no ion ha ESCs exhibi a high deg ee o
he e ogenei y e lec ed in hei a iega ed exp ession o some
o he plu ipo ency genes, p omiscuous ac i a ion o lineage-
speci ic genes and he luc ua ing low o di e en ia ing cells in
cul u ed popula ions.
37
The mechanisms by which NO may modula e he di e -
en ia ion o ESCs a e a ma e o deba e. S udies on signal
ansduc ion pa hways ac i a ed by NO could shed ligh on
his issue. In ac , exposu e o NO does no induce S a 3
phospho yla ion in mouse o human ESCs (Supplemen a y
Figu e 5). This inding indica es ha he mechanism o ac ion
o NO is independen o his pa hway. This obse a ion is
consis en wi h epo s showing egula ion o Nanog ha is
independen om he LIF/S a 3 pa hway
4
and wi h he ac
ha Nanog o e -exp ession in condi ions o LIF dep i a ion is
able o main ain sel - enewal and inc ease he p oli e a ion o
ESCs. Mo eo e , he NO-induced ac i a ion o c-S c and Ak
obse ed in ESCs (Supplemen a y Figu e 5) is simila o ou
p e ious indings in o he cell ypes.
21
These esul s a e also
consis en wi h da a epo ed by Anne en e al.
38
and Rubbin
e al.,
39
whe e hey show ha IRS-1 p o eins, he PI-3K/Ak
sys em and c-S c amily p o eins a e impo an o mESC sel -
enewal. Howe e , he mechanisms in ol ed in he egula ion
o hese signalling pa hways by NO need o be s udied in
g ea e de ail.
Cu en ly, di e se mechanisms a e known ha con ol he
ansi ion o ESC om sel - enewal and he plu ipo en s a e o
di e en ia ion. All o hem in ol e he dynamic and in ica e
ne wo k o plu ipo ency and di e en ia ion p o eins, he i s o
which mus be swi ched o o go ahead wi h di e en ia ion.
High amoun s o NO con ol he mobiliza ion and di e en ia-
ion o a ious o gan-de i ed p ecu so cells, p obably by
con olling he ac i a ion o p omo e egions o some genes
and by en o cing epigene ic changes in ol ed in cell-cycle
a es and di e en ia ion.
22,40
We p o ide e idence o a ole
o low NO in con olling s em cell sel - enewal and di e en i-
ion. The na u e o he e en s igge ed in his no el p ocess a
he gene egula ion le el dese es u he s udy.
Ma e ials and Me hods
Cell cul u e and gene a ion o ans ec an s. Th ee emb yonic s em-
cell lines we e used: (i) mESC lines D3 and R1-E, ob ained om he ATCC and
(ii) hESC line HS181, kindly p o ided by he Ka olinska Ins i u e.
The D3 and R1-E mESCs we e cul u ed a low densi y in Dulbecco’s modi ied
Eagle’s Medium (Gibco, Ca lsbad, CA, USA) supplemen ed wi h 15% e al bo ine
se um (Gibco), nonessen ial amino acids (1%), 2-me cap oe hanol (0.1 mM), L-glu amine
(4 mM), sodium py u a e (1 mM), penicillin (100 IU/ml) and s ep omycin (0.1 mg/ml).
The undi e en ia ed s a e was main ained by adding 1000 U/ml LIF (Gibco) o he
cul u e medium. Fo he gene a ion o ans ec an s, mESCs we e elec opo a ed a
800 mV/50 mF wi h 30 ng o linea ized p-Oc 4-eGFP-pgk hyg o plasmid o ob ain
D3-pOc 4-eGFP mESCs, and wi h 30 ng o ci cula pCDNA3.1-eNOS (p o ided by
D DJ S ewa , Depa men o Medicine, Uni e si y o To on o, To on o, On a io,
Canada) o ob ain D3-eNOS mESCs. Selec ion was ini ia ed 48 h ollowing
ans ec ion wi h 500 mg/ml hyg omicin o 400 mg/ml G418, espec i ely. Double
ans ec an s (D3-pOC 4-eGFP-eNOS mESCs) we e selec ed by he addi ion o he
wo an ibio ics oge he . Fo expe imen s wi h mESCs, cells we e cul u ed in
colonies in he absence o p esence o LIF. Fo ea men s, inc easing
concen a ions o DETA-NO we e added o he cul u e medium o cells g own in
he absence o LIF. The eNOS inhibi o L-NMMA was used a 400 mg/ml.
hESCs we e cul u ed on mi omycin C-inac i a ed human o eskin ib oblas
eede cells (CRL-2429, ATCC) a 371C, 5% CO
2
in Dulbecco’s modi ied Eagle’s
Medium/KO supplemen ed wi h 20% knock-ou se um eplacemen (Gibco In i ogen
Co po a ion, Ca lsbad, CA, USA), 2 mM L-glu amine (Gibco In i ogen co po a ion),
0.1 mM 2-me cap oe hanol (Gibco In i ogen Co po a ion), 1% nonessen ial amino
acids (Gibco In i ogen Co po a ion) and 100 U/ml pe 100 mg/ml penicillin/s ep omycin
(Gibco In i ogen Co po a ion). Cells we e subcul u ed e e y 10 days by mechanical
spli ing. Fo he expe imen al condi ions desc ibed he e, hESCs we e cul u ed on
Ma igel-coa ed issue cul u e pla es (BD Biosciences, San Jose, CA, USA) in he
p e ious medium in he absence o p esence o 8 ng/ml bFGF and 2 mM DETA-NO.
RNA isola ion and cDNA syn hesis. Collec ed ESCs we e washed wi h
PBS 1 and hen esuspended in Easy Blue eagen (In on Bio echnology, Seoul,
Ko ea) o RNA isola ion. Following phenol/chlo o o m ex ac ion, o al RNA was p eci-
pi a ed in 2-p opanol, washed wi h e hanol, suspended in nuclease- ee wa e and s o ed
a 201C un il being used o cDNA syn hesis. The abso bance o he o al RNA o
Con ol o di e en ia ion and cell su i al by NO
JR Tejedo e al
9
Cell Dea h and Disease