1
2Mesenchymal s em cell like (MSCl) cells gene a ed om human emb yonic
3s em cells suppo plu ipo en cell g ow h
4Nó a Va ga
a
Q1 , Zol án Ve éb
b
, É a Rajna ölgyi
b
, Ka alin Néme
a
, Fe enc Uhe
a
, Balázs Sa kadi
a
,
5Ágo a Apá i
a,
⇑
6
a
Memb ane Resea ch G oup o he Hunga ian Academy o Sciences, Semmelweis Uni e si y, Budapes , Hunga y
7
b
Depa men o Immunology, Medical and Heal h Science Cen e, Uni e si y o Deb ecen, Deb ecen, Hunga y
8
9
11
a icle in o
12 A icle his o y:
13 Recei ed 12 Sep embe 2011
14 A ailable online xxxx
15 Keywo ds:
16 Human emb yonic s em cells
17 Mesenchymal s em cell like (MSCl) cells
18 Feede cell
19 Immunosupp essi e ea u e
20
21
abs ac
22
Mesenchymal s em cell like (MSCl) cells we e gene a ed om human emb yonic s em cells (hESC)
23
h ough emb yoid body o ma ion, and isola ed by adhe ence o plas ic su ace. MSCl cell lines could
24
be p opaga ed wi hou changes in mo phological o unc ional cha ac e is ics o mo e han 15 passages.
25
These cells, as well as hei fluo escen p o ein exp essing s able de i a i es, e ficien ly suppo ed he
26
g ow h o undi e en ia ed human emb yonic s em cells as eede cells. The MSCl cells did no exp ess
27
he emb yonic (Oc 4, Nanog, ABCG2, PODXL, o SSEA4), o hema opoie ic (CD34, CD45, CD14, CD133,
28
HLA-DR) s em cell ma ke s, while we e posi i e o he cha ac e is ic cell su ace ma ke s o MSCs
29
(CD44, CD73, CD90, CD105). MSCl cells could be di e en ia ed owa d os eogenic, chond ogenic o adi-
30
pogenic di ec ions and exhibi ed significan inhibi ion o mi ogen-ac i a ed lymphocy e p oli e a ion,
31
and hus p esen ed immunosupp essi e ea u es. We sugges ha cul u ed MSCl cells can p ope ly
32
model human MSCs and be applied as e ficien eede s in hESC cul u es.
33
Ó2011 Published by Else ie Inc.
34
35
36
37
1. In oduc ion
38
Mesenchymal s em (s omal) cells (MSCs), o iginally isola ed
39
om bone ma ow [1], p o ide a suppo i e mic oen i onmen o
40
hema opoie ic s em cells. Mo eo e , MSCs show mul ipo en s em
41
cell cha ac e is ics [2] and special immunological ea u es [3]. MSCs
42
canalsobeob ained om a ious issuesincludingpe iphe alblood,
43
umbilical co d, placen a, ein wall, muscle, adipose and connec i e
44
issues [4]. MSCs can be di e en ia ed in o canonical mesode mal
45
issues,e.g.bone, a and ca ilage,bu mayalso e ainawide di e -
46
en ia ion capaci y [5]. MSCs play a ole in issue epai due o hei
47
di e en ia ion po en ial and immunosupp essi e ac i i ies [6],as
48
well as by ophic e ec s media ed by g ow h ac o s and cy okines
49
hey p oduce [7].
50
These ea u es ini ia ed he use o MSCs in a ious cell-based
51
he apies and by now mo e han 50 clinical ials ela ed o his cell
52
ype ha e been epo ed (see h p://www.clinical ials.go )[8].
53
Howe e , he e a e se e al limi a ions o la ge scale, ep oducible,
54
and well cha ac e ized p oduc ion o human MSCs. The aspi a ion
55
o MSCs om dono s is an in asi e p ocedu e and se e al epo s
56
ha e demons a ed dono - and issue-dependen a iabili y o hu-
57
man bone-ma ow-de i ed MSCs [4,9–13]. Isola ion and en ich-
58
men o issue-de i ed MSCs usually esul s in he e ogeneous
59
cell popula ions, and a long e m ex i o expansion o human MSCs
60
has been shown o educe eplica i e capaci y, impai di e en ia-
61
ion po en ial, al e gene exp ession p ofiles, and lead o ka yo ype
62
ins abili y [9,10].
63
In con as o MSCs, plu ipo en human emb yonic s em cell
64
(hESCs) lines ha e he capaci y o unlimi ed g ow h and sel - e-
65
newal [14] and can di e en ia e in o all cell ypes o he human
66
body [15]. The e a e nume ous s udies in es iga ing he di e en i-
67
a ion o hESCs, especially o yield he clinically mos ele an cell
68
ypes, including ca diomyocy es, hemopoie ic cells, neu onal o
69
panc ea ic cells ( o a ecen e iew see [16]).
70
Recen publica ionsha ealso epo ed hede i a iono MSC-like
71
cells om hESC lines [17–20], by using a ious p o ocols and p o-
72
ducing MSCs wi h di e en cha ac e is ics. In he p esen s udy
73
we ha e es ablished a me hod o de i e MSCl cell lines om hESCs
74
wi h well defined gene and p o ein exp ession pa e ns and immu-
75
nological ea u es, examined hei di e en ia ion po en ial, sup-
76
po i e ole o cul u ing hESCs, and p oduced gene ically
77
modified MSCl cell clones s ably exp essing fluo escen ma ke p o-
78
eins. This wo k may significan ly help ou unde s anding o human
79
MSC cha ac e is ics and he use o hese cells in bio echnology
80
applica ions.
0006-291X/$ - see on ma e Ó2011 Published by Else ie Inc.
doi:10.1016/j.bb c.2011.09.089
Abb e ia ions: hESC, human emb yonic s em cell; MSC, mesenchymal s em
(s omal) cells; bmMSC, bone ma ow de i ed MSC.
⇑
Co esponding au ho . Add ess: Memb ane Resea ch G oup o he Hunga ian
Academy o Sciences, Semmelweis Uni e si y, Dioszegi u. 64, 1113 Budapes ,
Hunga y. Fax: +36 1 372 4353.
E-mail add ess: [email p o ec ed] (Á. Apá i).
Biochemical and Biophysical Resea ch Communica ions xxx (2011) xxx–xxx
Con en s lis s a ailable a SciVe se ScienceDi ec
Biochemical and Biophysical Resea ch Communica ions
jou nal homepage: www.else ie .com/loca e/ybb c
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Please ci e his a icle in p ess as: N. Va ga e al., Mesenchymal s em cell like (MSCl) cells gene a ed om human emb yonic s em cells suppo plu ipo en
cell g ow h, Biochem. Biophys. Res. Commun. (2011), doi:10.1016/j.bb c.2011.09.089
81
2. Ma e ials and me hods
82
2.1. HuES, MSCl cells, HFF-1 and bmMSC cul u es
83
The human emb yonic s em cell lines (HUES9 and HUES1 o ig-
84
inally p o ided by D . Douglas Mel on, Ha a d Uni e si y) we e
85
cul u ed on mi o ically inac i a ed mouse emb yonic fib oblas s
86
(MEF) [21]. Spon aneous di e en ia ion o he hESCs we e pe -
87
o med ia emb yoid body (EB) o ma ion as desc ibed p e iously
88
[21]. The cells we e ypsinized a day 80 and we e u he cul-
89
u ed in DMEM supplemen ed wi h 10% e al bo ine se um (FBS;
90
In i ogen) on a gela inized 10-cm pla e. The cells we e ypsinized
91
when confluen and spli o 1:3 a io. A e wo passages mos o
92
he cul u ed cells p esen ed fib oblas -like mo phology. The
93
hESC-de i ed fib oblas -like cells we e designa ed as hESC-de i ed
94
mesenchymal s em cell like (MSCl) cells. All MSCl cells used in his
95
s udy we e o polyclonal o igin.
96
HFF-1 cells we e ob ained om ATCC and we e cul u ed acco ding
97
o he manu ac u e ’s ins uc ions (h p://www.s emcells.a cc.o g).
98
Collec ion o bone ma ow samples om pa ien s wi h hema o-
99
logical diseases was app o ed by he Regional and Ins i u ional
100
e hical e iew boa d o he Medical and Heal h Science Cen e o
101
he Uni e si y o Deb ecen (p o ocol No.: UD-MHSC REC/IEC
102
2754-2008). Bone ma ow samples we e ha es ed om he iliac
103
c es unde medical examina ion. bmMSC isola ed and cul u ed
104
as desc ibed ea lie [22]. The cells we e used o pheno ypic and
105
unc ional analysis a e P5.
106
2.2. Cha ac e iza ion o MSCl, HFF-1 and bmMSC cells
107
Cell su ace exp ession o MSC ma ke p o eins on he su ace o
108
MSCl, HFF-1 and bmMSC cells was analyzed by h ee-colo flow
109
cy ome y using FITC-, APC- o PE-conjuga ed monoclonal an ibod-
110
ies (mAb) wi h iso ype-ma ched con ol mAbs; specific agains
111
hema opoie ic-, MSC ela ed-, endo helial ma ke s, cell adhesion
112
molecules and in eg ins as well (see Supplemen a y Table I o ull
113
lis ). Fluo escence in ensi ies we e measu ed by FACS Calibu flow
114
cy ome e and he da a we e analyzed by using he WinMDI ee-
115
wa e (Joseph T o e , La Jolla, CA). Resul s we e exp essed as
116
means o posi i e cells (%) ± SD. Plu ipo ency ma ke s o MSCs
117
we e es ed by immunos aining (Oc 4, SSEA4, PODXL) and by eal
118
ime PCR (Nanog, ABCG2) pe o med as desc ibed p e iously [21].
119
The di e en ia ion po en ial o MSCs was pe o med by using he
120
Gibco’s S emP o
Ò
Adipogenesis, Os eogenesis and Chond ogenesis
121
Di e en ia ion Ki s acco ding o he manu ac u e ’s guide.
122
2.3. Len i i al ansduc ion o MSCl cells
123
Fo i al-based gene deli e y, a hi d gene a ion len i i al ec-
124
o sys em was used, as desc ibed in [23]. De e mina ion o i us
125
i e s and he ansduc ion p ocedu es we e pe o med as de-
126
sc ibed p e iously [24]. The MSCl-2 cells we e ansduced by a
127
MOI o 2–5, wi h an eGFP encoding len i i al ec o and u he
128
handling was he same as o he pa en al cell line.
129
2.4. Mi ogen-induced cell p oli e a ion and cy okine sec e ion
130
Pe iphe al blood mononuclea cells (PBMCs) we e isola ed by
131
Ficoll g adien cen i uga ion (Ame sham Biosciences, Uppsala,
132
Sweden). Mi ogen-ac i a ed T lymphocy e p oli e a ion was in-
133
duced by concana alin A (ConA) o phy ohaemagglu inin (PHA)
134
used a a final concen a ion o 10
l
g/ml (Ame sham Pha macia
135
Bio ech) and 1
l
g/ml (Sigma–Ald ich), espec i ely added o
136
110
6
PBMCs. MSCs we e added o 1 10
6
PBMCs a 10
3
and
137
10
4
cell numbe s and co-cul u ed o 3 days. On day h ee p oli e -
138
a ion was de ec ed by he B DU colo ime ic assay di ec ly in he
139
cul u e pla e acco ding o he manu ac u e ’s ins uc ions (Roche).
140
IL-6, IL-10 and IFN-
c
cy okine sec e ion was quan i a ed by en-
141
zyme linked immunoso ben (ELISA) assay (Op EIA, BD Pha min-
142
gen) ollowing he supplie ’s ins uc ion. All expe imen s we e
143
pe o med in iplica es.
144
2.5. S a is ical analysis
145
Each expe imen was pe o med a leas h ee imes and each
146
sample was es ed in iplica es. Da a a e exp essed as mean + SD.
147
S a is ically significan di e ences we e de e mined by wo-way
148
ANOVA o pai ed s uden - es s.
⁄
p< 0.05,
⁄⁄
p< 0.01,
⁄⁄⁄
p< 0.001.
149
3. Resul s and discussion
150
3.1. Gene a ion o HUES9-de i ed MSCl cells – applica ion as eede
151
cells
152
Plu ipo en HUES9 cells we e spon aneously di e en ia ed ia
153
EB o ma ion (6 days), and hen he di e en ia ion p ocess was
Fig. 1. Plu ipo ency ma ke s o HUES9 cells cul u ed on di e en eede laye s, HUES9 cells we e g own on MEF (A) o MSCl-2-GFP (B) eede cells o wo days in eigh -well
chambe s o con ocal mic oscopy. Co-cul u e o HUES and eede cells we e fixed and s ained wi h he an ibodies ecognizing he ollowing p o eins: Oc 4, SSEA4, PODXL
( ed). An ibodies specific o undi e en ia ed cell ma ke s s ained only he HUES9 clumps, while he MEF o MSCl-2-GFP cells did no show any s aining wi h hese ma ke s.
Nuclei we e coun e s ained wi h DAPI (blue). (Fo in e p e a ion o he e e ences o colo in his figu e legend, he eade is e e ed o he web e sion o his a icle.)
2N. Va ga e al. / Biochemical and Biophysical Resea ch Communica ions xxx (2011) xxx–xxx
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30 Sep embe 2011
Please ci e his a icle in p ess as: N. Va ga e al., Mesenchymal s em cell like (MSCl) cells gene a ed om human emb yonic s em cells suppo plu ipo en
cell g ow h, Biochem. Biophys. Res. Commun. (2011), doi:10.1016/j.bb c.2011.09.089
154
comple ed by u he cell cul u ing on adhe en su ace in he
155
p esence o 10% FBS o 80 days. The di e en ia ed fib oblas -like
156
cells we e sepa a ed om he o he cell ypes by sho ypsiniza-
157
ion. A e 2–3 passages on adhe en su ace he cells achie ed uni-
158
o m, fib oblas like mo phology, and hese cells ( e med MSCl
159
cells) could be p opaga ed a leas o 15 passages wi hou mo -
160
phological o ka io ypical changes (Supplemen a y Fig. 1). By using
161
he same p o ocol, mo e han fi e polyclonal MSCl cul u es we e
162
independen ly gene a ed om he HUES9 cell line.
163
In he ollowing expe imen s we es ed he capaci y o he MSCl
164
cellsas au ogenic eede cells o main ainingplu ipo ency o hESCs.
165
When undi e en ia ed HUES1 o HUES9 cells we e passaged o
166
mi omycin ea ed MSCl cul u es, each MSCl cell line suppo ed
167
he g ow h o hese plu ipo en hESCs wi hou any change in he
168
exp ession o plu ipo ency ma ke s o he a e o cell g ow h, as
169
compa ed o hose seen wi h cells g owing on MEF (Supplemen a y
170
Fig. 2). Recen publica ions ha e al eady desc ibed a ious me hods
171
o he es ablishmen o au ogenic eede cells om hESC [25–27].
172
Ou p esen me hod p o ides means o c ea e applicable eede s
173
o hESC wi hou cell so ing o mechanical sepa a ion, and he de-
174
i ed eede cells demons a e he capaci y o p omo e hESC
175
expansion.
176
In o de o allow a as sepa a ion o hESCs om he human
177
MSCl cells, we ha e gene a ed eGFP exp essing MSCl cells by using
178
a len i i al gene deli e y me hod. The MSCl-GFP cells we e so ed
179
o eGFP posi i i y, ea ed wi h mi omycin and used as eede o
180
hESCs. As documen ed in Fig. 1, he MSCl-GFP cells p omo ed he
181
g ow h o HUES9 cells in an undi e en ia ed s a e. This Figu e also
182
documen s ha he plu ipo ency ma ke s Oc 4, SSEA4 and PODXL
183
we e exp essed in he plu ipo en HUES9 cells, while hese ma k-
184
e s we e absen in he MSCl-GFP eede cells.
185
We also ex ended hese s udies wi h RT-PCR measu emen s o
186
plu ipo ency ma ke s exp ession in he undi e en ia ed hESC
187
and in he MSCl cells. The ansc ip ion ac o s Oc 4 and Nanog
188
and he ABCG2 cell su ace p o ein showed high le els o gene
189
exp ession in he HUES9 cells, while hey we e close o de ec ion
190
le els in he MSCl cells (da a no shown).
191
3.2. Mul i-lineage di e en ia ion po en ial
192
MSCs should fi he unc ional and pheno ypic c i e ia defined
193
by he Mesenchymal and Tissue S em Cell Commi ee o he In e -
194
na ional Socie y o Cellula The apy [28]. We fi s es ed whe he
195
he MSCl cells could be di e en ia ed owa d he canonical meso-
196
de mal (os eoblas ic, chond ogenic, adipogenic) di ec ions. We
197
ound ha all ou es ablished MSCl cell lines exhibi ed he
198
po en ial o di e en ia e o a leas one di ec ion, bu one o hem
199
(MSCl-2) showed obus di e en ia ion po en ial owa d all h ee
200
di ec ions (Supplemen a y Fig. 3). To comple e a mo e de ailed
201
cha ac e iza ion we nex compa ed he di e en ia ion capaci y o
202
MSCl cells wi h he comme cially a ailable human o eskin fib o-
203
blas cells (HFF-1) widely used as a eede cell line o hESC cul-
204
u es, and ha o MSCs isola ed om human bone ma ow. All
205
hese MSCs o di e en o igin could be di e en ia ed in i o o
206
adipogenic, oes eogenic and chond ogenic di ec ions. Followed
207
by a h ee-week adipogenic induc ion pe iod, a la ge numbe o
Fig. 2. Di e en ia ion o MSCl-2, HFF-1 and bmMSC cells. Adipogenic di e en ia ion is demons a ed by in acellula lipid acuoles s ained in ed by Oil Red O ( ed) and wi h
nuclei coun e s ained wi h DAPI (blue) (A). Os eogenic di e en ia ion o MSCs is demons a ed by he o ma ion o calcium-hyd oxyapa i e-posi i e a eas s ained in ed by
Aliza in Red (B). High densi y cul u es showed he de elopmen o chond ogenic pheno ype when cul u ed in mic omass; pink ex acellula ma ix s aining ma ks
p o eoglycans s ained wi h oluidine blue (C). Magnifica ion: 200. (Fo in e p e a ion o he e e ences o colo in his figu e legend, he eade is e e ed o he web e sion
o his a icle.)
N. Va ga e al. / Biochemical and Biophysical Resea ch Communica ions xxx (2011) xxx–xxx 3
YBBRC 27406 No. o Pages 7, Model 5G
30 Sep embe 2011
Please ci e his a icle in p ess as: N. Va ga e al., Mesenchymal s em cell like (MSCl) cells gene a ed om human emb yonic s em cells suppo plu ipo en
cell g ow h, Biochem. Biophys. Res. Commun. (2011), doi:10.1016/j.bb c.2011.09.089
208
MSCl-2 and bmMSC cells showed oil ed posi i e s aining, cha ac-
209
e is ic o adipocy es (Fig. 2A), while only a small numbe o HFF-1
210
cells became oil ed posi i e. Simila ly, when di e en ia ion was
211
induced by os eogenic induc ion medium o 2–3 weeks, MSCl-2
212
and bmMSC cells showed dense calcium deposi s s ained wi h aliz-
213
a in ed, while he HFF-1 cells exhibi ed weak os eogenic po en ial
214
(Fig. 2B). A e 3 weeks o di e en ia ion sec ions made om chon-
215
d ogenic mass cul u es showed me ach omazy upon oluidin-blue
216
s aining indica ing chond ocy e o ma ion om all h ee cell ypes
217
(Fig. 2C). Based on hese da a we sugges ha MSCl-2 cells
218
gene a ed om HUES9 cells e ained hei mul ilineage di e en i-
219
a ion po en ial, simila o ha ound in bmMSCs [4,29], whe eas
220
HFF-1 cells possessed educed di e en ia ion capaci y.
221
3.2.1. Cell su ace ma ke s o MSCl cells
222
In he fi s se o expe imen s summa ized in Fig. 3 we com-
223
pa ed he pheno ypic cha ac e is ics o h ee di e en HUES9-de-
224
i ed polyclonal MSCl cell lines: MSCl-2, MSCl-3 and MSCl-4,
225
which could be cul u ed o a long pe iod o ime, o he exp es-
226
sion o he key cell su ace molecules o MSCs. As shown by flow
Fig. 3. Exp ession o common MSC ma ke s in di e en MSCl cell lines. Flow cy ome y measu emen s o cell su ace ma ke exp ession o HUES9-de i ed MSCl cells. Single
cell suspensions om MSCl-2, MSCl-3 and MSCl-4 cells we e ob ained by gen le ypsiniza ion. Non- iable cells we e excluded by Top o3 o 7AAD s aining. Monoclonal
an ibodies specific o CD44 (conjuga ed o FITC), CD73 and CD90 (conjuga ed o PE) we e used o de ec MSC ma ke s. An i CD34-PE was used o demons a e he absence o
hema opoie ic cells in he MSCl cul u es. Dashed lines show s aining wi h he ele an iso ype-ma ched con ol mAbs.
4N. Va ga e al. / Biochemical and Biophysical Resea ch Communica ions xxx (2011) xxx–xxx
YBBRC 27406 No. o Pages 7, Model 5G
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Please ci e his a icle in p ess as: N. Va ga e al., Mesenchymal s em cell like (MSCl) cells gene a ed om human emb yonic s em cells suppo plu ipo en
cell g ow h, Biochem. Biophys. Res. Commun. (2011), doi:10.1016/j.bb c.2011.09.089
227
cy ome y, he selec ed cell lines exp essed simila high le els o
228
CD44, CD73, and CD90 wi h some a iabili y in he magni ude o
229
CD90 exp ession and none o hese cells exp essed CD34, an ea ly
230
hema opoie ic s em cell ma ke (Fig. 3).
231
Fo ade ailedcha ac e iza ion,weha ecompa ed heexp ession
232
o he cell su ace ma ke s in bmMSC, HFF-1 and MSCl-2 cells, by
233
using flow cy ome y analysis. As documen ed in Table 1, all he
234
h ee cell ypes showed a uni o mly high exp ession o all well
235
known MSC ma ke s, e.g. CD44, CD73, CD90, and CD105. Because
236
none o hese ma ke s a e conside ed as MSC specific, we u he
237
analyzed he exp ession o in eg ins, cell adhesion molecules and
238
endo hel- ela ed su ace p o eins on MSCl-2 cells as compa ed o
239
ha o he o he cell ypes. No di e ence in he exp ession pa e n
240
o hese cell su ace ma ke s could be shown (Supplemen a y Table
241
II). Impo an ly, none o he MSCs showed measu able exp ession o
242
he hema opoie ic ma ke s CD14, CD34, CD45, CD117, CD133 o
243
HLA-R. The unique p ope yo HFF-1 cells was hei high cellsu ace
244
exp essiono CD14,whichwasunde ec ableinbmMSCo MSClcells
245
(Table 1). Thus in line wi h p e ious publica ions [17,20,30], he de-
246
ailed pheno ypic analysis o HUES9-de i ed MSCl-2 and adul
247
bmMSCs cells e ealed close pheno ypic simila i y.
248
3.2.2. Immunosupp essi e e ec s o MSCl-2, HFF-1 and bmMSCs
249
Due o hei clinical u ili y, he immunosupp essi e p ope ies
250
o MSCs ha e ex ensi ely been s udied and MSCs we e shown o
251
supp ess immune esponses bo h in i o and in i o media ed by
252
mul iple mechanisms [31–33] alida ing MSC as a he apeu ically
253
ele an cell ype. Nume ous s udies ha e demons a ed ha hu-
254
man MSCs dec ease allo eac i e esponses, in e e e wi h dend i ic
255
cell (DC) and T-cell unc ions, and a e able o gene a e an immuno-
256
supp essi e mic oen i onmen . Unde in i o condi ions bmMSCs
257
a e capable o supp ess a ious T-cell e ec o unc ions [33,34]
258
and inhibi mi ogen-s imula ed lymphocy e p oli e a ion [35,36].
259
In he p esen s udy mi ogen-induced T-cell p oli e a ion was
260
used o compa e he immunosupp essi e p ope ies o MSCl-2,
261
HFF-1 and bmMSC cells. Human PBMC o heal hy dono s was used
262
as esponde cells, and ConA o PHA as mi ogenic ac i a o s. We
263
ound ha all cell ypes esponded o mi ogenic ac i a ion and
264
had an e ec on lymphocy e-p oli e a ion induced by ConA o
265
PHA (Fig. 4). As shown in Fig. 4A, he p esence o MSCls supp essed
266
he mi ogenic esponse o lymphocy es in a dose-dependen man-
267
ne and he addi ion o bo h 10
3
and 10
4
MSCl-2 cells o 1 10
6
268
PBL e ficien ly educed ConA-induced lymphocy e p oli e a ion,
269
whe eas he PHA-induced mi ogenic esponse was significan in
270
he p esence o 10
4
MSCl-2 cells, only (Fig. 4A). A simila e ec
271
could be de ec ed when using bmMSCs as supp esso s (Fig. 4C).
272
P oli e a ion o lymphocy es could also be supp essed by HFF-1
273
cells when ConA bu no PHA was used o s imula ion (Fig. 4B).
274
In o de o elici hei immunosupp essi e unc ion, MSCs
275
should acqui e inc eased p oduc ion o ce ain cy okines such as
276
IL-6, IL-10 and IFN
c
[37,38]. IL-6 was shown o inc eases PGE
2
p o-
277
duc ion and hus plays a key ole in he inhibi ion o DC ma u a ion
278
and T-cell p oli e a ion [31,39–42]. Dependen on local IFN
c
le els
279
MSC-media ed immunosupp ession can be enhanced [43], and
280
bmMSCs can d i e DC di e en ia ion o IL-10 sec e ing cells [33],
281
which a e po en inhibi o s o T lymphocy e p oli e a ion [44].
282
In he nex se o expe imen s we sough o measu e he concen-
283
a ion o sec e ed IL-6, IL-10 and IFN
c
cy okines in he supe na an
284
o mi ogen-ac i a ed PBMCs co-cul u ed wi h MSCl-2, HFF-1 and
285
bmMSC cells (Supplemen a y Fig. 4). Ou esul s e ealed ha
286
bmMSCs induced significan inc eases in IL-6 (Supplemen a y
287
Fig. 4C), IFN
c
(Supplemen a y Fig. 4F) and IL-10 (Supplemen a y
288
Fig. 4I) cy okine le els, whe eas MSCl-2 cells we e unable o do so
289
e en hough hey e ficien ly inhibi ed T lymphocy e p oli e a ion
290
(Fig. 4A, D, and G). The p esence o HFF-1 cells induced a sligh
291
inc ease in IL-6 le els (Supplemen a y Fig. 4B), bu he concen a-
292
ion o sec e ed IFN
c
and IL-10 did no change (Suppleman a y
293
Fig. 4E and H). These esul s indica e ha he p oduc ion o he
294
unc ionally ele an cy okines is no an absolu e equi emen o
295
exe ing he immunosupp essi e e ec s o MSCs, and he MSCl-2
296
cells may use di e en means o inhibi T-lymphocy e associa ed
297
e ec o unc ions.
298
Al hough hede ailedimmunologicalcha ac e iza iono HUES9-
299
de i ed MSCl-2 needs u he in es iga ions, ou p esen esul s
300
indica e a s ong immunosupp essi e e ec by he HUES9-de i ed
301
MSCl-2 cells. Ou cu en expe imen s ein o ce p e ious obse a-
302
ions [20,30,45] demons a ing ha MSCs de i ed om human
303
emb yonic s emcells ha e he capaci y o supp esspe iphe al blood
304
T lymphocy e p oli e a ion and also ex end hem by demons a ing
305
he lack o MSCs-induced enhancemen o cy okine sec e ion upon
306
hei immunosupp essi e ac ion.
307
As a summa y, we ha e p epa ed and cha ac e ized human MSCl
308
cells ob ained om he human emb yonic s em cell line, HUES9. In
309
p e iouss udies a iousp o ocols ha ebeenapplied o ob ainMSCs
310
om hESC, including co-cul u e wi h mouse OP9 cells [46],
311
emb yoid body di e en ia ion [47], spon aneous di e en ia ion in
312
monolaye [20], selec ion o cell popula ions by FACS [17], using
313
Rho-associa edkinaseinhibi o [30],o e exp essiono HOXB4gene
314
[18] and mechanical sepa a ion [19]. In he p esen s udy we ha e
315
de eloped a no el, simplified me hod o ob ain pheno ypically and
316
unc ionally homogeneous hESC-de i ed MSC popula ions. Ou de -
317
i a ion p o ocol equi ed nei he he use o xenogenic eede cell,
318
no selec ion (manual o FACS), no chemical induce s. These MSCl
319
cells showed long e m s abili y and could be main ained in cul u e
320
o e 30 passages wi hou de ec able pheno ypic o unc ional
321
changes. Cell su ace ma ke s, mul ipo en di e en ia ion po en ial
322
and immunosupp essi e e ec s o MSCl-2 p o ed o be simila o
323
bmMSCs bu was di e en om ha o HFF-1. Ou da a sugges ha
324
MSCl-2 cells may be success ully applied as a model sys em o
325
s udying human MSC ea u es and may be used in bio echnology
326
applica ions.
327
4. Disclosu es
328
The au ho s indica e no po en ial conflic s o in e es .
Table 1
Exp ession o common pheno ypic ma ke s by MSCs o a ious o igins.
MSCl-2 HFF-1 bmMSC
CD44/H-CAM 96.97 ± 2.81 93.23 ± 9.44 89.69 ± 10.04
CD105/Endoglin 89.61 ± 3.95 95.73 ± 6.19 86.08 ± 8.85
CD73/NT5E 99.51 ± 0.25 99.21 ± 0.22 93.57 ± 6.44
CD90/Thy-1 96.24 ± 2.41 87.38 ± 7.87 89.78 ± 7.67
CD34 0.00 1.24 ± 2.49 0.00
CD14 1.04 ± 1.88 85.27 ± 10.47 0.09 ± 0.19
CD45/P o ein y osine phospha ase ecep o C 0.00 0.00 0.00
CD117/c-ki 0.00 0.00 0.00
CD133/P ominin 1 0.00 0.00 0.00
HLA-DR 0.00 0.00 0.00
N. Va ga e al. / Biochemical and Biophysical Resea ch Communica ions xxx (2011) xxx–xxx 5
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Please ci e his a icle in p ess as: N. Va ga e al., Mesenchymal s em cell like (MSCl) cells gene a ed om human emb yonic s em cells suppo plu ipo en
cell g ow h, Biochem. Biophys. Res. Commun. (2011), doi:10.1016/j.bb c.2011.09.089

329
Acknowledgmen s
330
This wo k has been suppo ed by G an s om OTKA (NK72057),
331
ETT (213-09), STEMKILL (OM00108/2008), KMOP-1.1.2-07/1-2008-
332
0003 and TÁMOP-4.2.2-08/1-2008-0015. The au ho s wish o
333
hank Beá a Ha asz i o help in cell cul u ing, o Monika Bá kai
334
o helping wi h he len i i al ansduc ion o MSCl cells o Gyö gy
335
Vá ady o helping wi h FACS and Emma Ádám and And ás Kozma
336
o ka yo yping and FISH analysis.
337
Appendix A. Supplemen a y ma e ial
338
Supplemen a y da a associa ed wi h his a icle can be ound, in
339
he online e sion, a doi:10.1016/j.bb c.2011.09.089.
340
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Fig. 4. E ec o MSCl-2, HFF-1 and bmMSC cells on mi ogenic lymphocy e
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N. Va ga e al. / Biochemical and Biophysical Resea ch Communica ions xxx (2011) xxx–xxx 7
YBBRC 27406 No. o Pages 7, Model 5G
30 Sep embe 2011
Please ci e his a icle in p ess as: N. Va ga e al., Mesenchymal s em cell like (MSCl) cells gene a ed om human emb yonic s em cells suppo plu ipo en
cell g ow h, Biochem. Biophys. Res. Commun. (2011), doi:10.1016/j.bb c.2011.09.089