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6.7246 0 0 5.9768 176.1448 176.3149 Tm
(22)Tj
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[(Cells)-526.9(from)-520(different)-530.3(donors)]TJ
-14.0053 -1.2203 TD
[(\(average)-352.9(age)-345.3(of)-351(30)]TJ
/F5 1 Tf
8.3526 0 TD
()Tj
/F4 1 Tf
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[(7\),)-344.3(at)-347.7(passages)-350.1(4)-347.3(and)-347.3(5,)-350.2(were)-349.9(used.)-343.6(The)]TJ
-9.0734 -1.2266 TD
[(ASC)-594.4(were)-596.5(obtained)-598.7(from)-595.9(healthy)-596.3(donors)-602(after)-595.4(informed)]TJ
0 -1.2203 TD
[(consent)-516.3(under)-514.2(a)-511.7(protocol)-519.5(reviewed)-515.1(and)-511.7(approved)-515.2(by)-514.7(the)]TJ
T*
[(Pennington)-259.9(Biomedical)-262.2(Research)-254.8(Center)-257.4(Institutional)-266.1(Review)]TJ
0 -1.2267 TD
(Board.)Tj
/F11 1 Tf
0 -2.4406 TD
[(Serum-free)-336.9(cultures)-331.1(under)-335.9(static)-336.3(conditions)]TJ
/F4 1 Tf
1.1128 -1.8336 TD
[(Before)-266(cell)-265.5(inoculation)-270.1(in)-266.2(the)-269.3(spinner)-266.5(asks,)-265.4(cryopreserved)]TJ
-1.1128 -1.2203 TD
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0 -1.2267 TD
(cm)Tj
6.7246 0 0 5.9768 73.8141 61.285 Tm
(2)Tj
8.9663 0 0 8.9663 77.159 57.2598 Tm
[(,)-401.3(o)0(n)-404.5(CELLstart)]TJ
/F5 1 Tf
6.6327 0 TD
()Tj
/F4 1 Tf
1.2899 0 TD
(CTS)Tj
/F5 1 Tf
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()Tj
/F4 1 Tf
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[(\(Invitrogen\))-408.1(pre-coated)-405.7(T-75)-400.8(or)]TJ
15.2698 74.5598 TD
[(T-175)-444.6(asks)-439.5(using)-444(StemPro)]TJ
/F5 1 Tf
6.7246 0 0 5.9768 423.9495 729.751 Tm
()Tj
/F4 1 Tf
8.9663 0 0 8.9663 433.2471 725.7825 Tm
[(MSC)-440(SFM)-438(XenoFree)-451.5(\(Invitro-)]TJ
-13.4046 -1.2203 TD
[(gen\).)-377.8(At)-374.9(70%)-380(cell)-373(conuence,)-381.9(MSC)-370.5(were)-381.5(detached)-375.2(from)-374.6(the)]TJ
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[(asks)-313.1(by)-312.3(adding)-307.7(TrypLE)]TJ
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()Tj
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1.195 0 TD
[(solution)-310(\(Invitrogen\))-319.5(for)-307.4(5)-170.2(min)-308(at)]TJ
-12.1779 -1.2203 TD
(37)Tj
/F5 1 Tf
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()Tj
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[(C.)-357.2(Cell)-354.9(number)-361.4(and)-353.7(viability)-358.8(were)-362.5(determined)-359.1(using)-355.4(the)]TJ
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/F5 1 Tf
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()Tj
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/F5 1 Tf
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()Tj
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[(pa)18.1(dd)19.3(le)21.9(s)]TJ
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()Tj
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(10)Tj
6.7246 0 0 5.9768 548.844 614.7211 Tm
0 Tc
(4)Tj
8.9663 0 0 8.9663 313.0582 599.7542 Tm
[(ce)13.5(ll)24.5(s/)14.7(m)15.1(L)0(.)]TJ
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[(Cu)23(lt)16.6(is)16.4(p)13.1(h)0(e)18.8(r)0(-)17.8(S)]TJ
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-10.319 -1.2203 TD
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6.7246 0 0 5.9768 408.8692 592.7809 Tm
(23)Tj
8.9663 0 0 8.9663 420.0377 588.8124 Tm
[(a)13.1(n)0(d)-493.3(p)0(r)16.9(e)12.7(co)16.9(a)13.1(t)0(e)18.6(d)-502.5(wi)20.1(th)-490.7(FB)17.9(S.)]TJ
6.7246 0 0 5.9768 521.0645 592.7809 Tm
(20)Tj
8.9663 0 0 8.9663 532.1762 588.8124 Tm
[(No)16.2(n-)]TJ
-24.4381 -1.2266 TD
[(po)14.8(ro)21.8(us)-318.8(pl)20.4(as)18.1(ti)15(c)-328.1(m)15.1(ic)16.2(ro)21.8(ca)20.2(rr)17.6(ie)21.9(rs)-318.2(\(S)13.7(ol)19(oH)22.5(il)18.2(l)-326(E)0(n)18.9(g)0(i)17.3(n)0(e)22(e)0(r)18.4(i)0(n)18.4(g)0(,)-317.2(I)0(n)19.8(c)0(.)16.8(\))-329.3(we)17.4(re)]TJ
0 -1.2203 TD
[(pr)16.9(ep)17.7(a)13.1(r)0(e)18.4(d)-692.2(a)13.1(c)0(c)20.4(o)0(r)15.5(d)0(i)25.1(n)0(g)-678(t)0(o)-679.8(m)0(a)21.9(n)0(u)14.2(f)12.2(ac)20.2(tu)17.1(re)18.4(r)16.4(s)-684.2(i)0(n)18.4(s)0(t)17.2(r)0(u)23.2(c)0(t)12.9(i)15.4(on)19.1(s.)-680.8(Th)18.9(e)12.8(n)0(,)]TJ
T*
[(pl)14.1(a)13.1(s)0(t)17.2(i)0(c)-407.5(m)0(i)17.9(c)13.4(ro)15.5(c)13.4(a)0(r)18.8(r)0(i)14.7(e)12.8(rs)-406.7(we)17.4(re)-411.6(c)13.4(o)0(a)16.7(t)12.2(ed)-407.6(wi)20.1(th)-414.8(a)-416.8(C)12.5(EL)19.3(Ls)21(ta)19(rt)-412.2(C)12.5(T)0(S)-412.5(s)0(o)21.2(-)]TJ
0 -1.2266 TD
[(lu)14.1(t)12.2(i)0(o)19(n)-370.1(\()12.2(d)0(i)18.8(l)0(u)20.4(t)0(e)18.6(d)-363.4(1:)16.5(1)13.1(0)0(0)-359.4(i)0(n)-354.7(P)0(B)20.9(S)-365.2(wi)13.8(t)12.2(h)-363.8(Ca)]TJ
6.7246 0 0 5.9768 457.1148 548.9573 Tm
(2)Tj
/F5 1 Tf
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(+)Tj
/F4 1 Tf
8.9663 0 0 8.9663 469.644 544.9321 Tm
[(an)16.1(d)-363.4(M)0(g)]TJ
6.7246 0 0 5.9768 500.9951 548.9573 Tm
(2)Tj
/F5 1 Tf
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(+)Tj
/F4 1 Tf
8.9663 0 0 8.9663 510.1227 544.9321 Tm
[(\))-367.2(f)12.2(or)-357.6(2)-157.6(h)-363.8(at)]TJ
-21.9784 -1.2203 TD
(37)Tj
/F5 1 Tf
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()Tj
/F4 1 Tf
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[(C,)-287.6(wi)20.1(th)-288.4(an)-287.4(in)18.4(te)18.6(rm)20.7(it)15(t)12.2(e)0(n)15.7(t)-297.6(a)13.1(g)0(i)17.3(t)12.2(at)19(io)19(n)-300.6(\()12.2(1)-157.6(m)0(i)17.9(n)-294.2(at)-290.8(3)13.1(0)0(0)-150.8(r)0(p)23.2(m)0(,)-285(1)0(0)-150.8(m)0(i)24.2(n)]TJ
-1.3658 -1.2203 TD
[(no)12.8(na)22.4(gi)17.3(t)12.2(a)0(t)19(e)0(d)16.1(\))-304(us)22.6(in)18.4(g)-307.9(a)-303(Th)18.9(er)18.4(mo)18.6(mi)17.9(xe)23.2(r)]TJ
/F5 1 Tf
6.7246 0 0 5.9768 448.4975 527.0172 Tm
()Tj
/F4 1 Tf
8.9663 0 0 8.9663 456.5479 523.0487 Tm
[(co)16.9(nf)21.4(or)21.8(t)-310.3(\()12.2(Ep)14.6(pe)24(nd)18.9(or)15.5(f)-304(A)0(G)18.8(\))12.2(,)]TJ
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[(an)16.1(d)-515.2(a)0(f)19(t)0(e)18.6(r)0(w)16.6(a)13.1(rd)-509.5(eq)21(ui)20.4(li)18.2(br)20.2(at)19(ed)-508.7(in)-506.4(pr)16.9(ew)17.4(a)13.1(r)0(m)20.7(e)0(d)-508.7(M)0(e)14(s)0(e)24.1(n)0(P)14.2(R)0(O)-506.8(R)0(S)22.6(/)]TJ
0 -1.2203 TD
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T*
[(ou)14.8(sl)20.4(y)-554.5(e)0(x)16.9(p)0(a)18.1(n)0(d)18.9(e)12.7(d)-553.1(u)0(n)14.2(d)0(e)22.4(r)-557.1(xe)23.2(no)12.8(-)12.2(f)0(r)17.8(e)12.8(e)-556.3(s)0(t)17.2(a)0(t)19(i)0(c)-546.6(c)13.4(on)12.8(d)16(i)0(t)15(i)0(o)19(n)0(s)-542.1(f)0(o)15.7(r)-557.1(t)12.2(w)0(o)]TJ
T*
[(pa)18.1(ss)16.3(a)13.1(g)0(e)14.6(s)0(,)-472.1(w)0(e)17.4(r)0(e)-481.1(s)0(e)24.1(e)0(d)16.1(e)0(d)-477.1(o)0(n)-480.4(2)13.1(0)-157.6(g)0(/)17.9(L)-483.5(of)-477.5(pr)16.9(e-)18.6(co)16.9(a)13.1(t)0(e)18.6(d)-489.9(pl)20.4(a)13.1(s)0(t)17.2(i)0(c)-477(m)0(i)17.9(-)]TJ
0 -1.2266 TD
[(cr)19(oc)16.9(ar)18.8(ri)21.1(er)18.4(s)-374.4(i)0(n)-367.3(1)0(5)-150.8(m)0(L)-367.3(o)0(f)-363.6(t)0(h)15.1(e)-372.9(re)18.4(sp)16.3(e)12.8(c)0(t)19.2(i)0(v)14.7(e)-372.9(me)15.2(d)16(i)0(u)14.1(m)-370.6(fo)15.7(r)-373.8(3)0(0)-150.8(m)0(i)24.2(n)0(,)-366.7(a)0(t)]TJ
0 -1.2203 TD
(37)Tj
/F5 1 Tf
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()Tj
/F4 1 Tf
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[(C)-417.5(a)13.1(nd)-404.7(5)13.1(%)-412.6(CO)]TJ
6.7246 0 0 5.9768 382.6771 455.8109 Tm
(2)Tj
8.9663 0 0 8.9663 385.9085 457.2849 Tm
[(,)-413.9(w)0(i)20.1(t)0(h)-402.2(g)0(e)14.6(n)0(t)21.4(l)0(e)-401.8(a)0(g)21.4(i)0(t)15(a)13.1(ti)15(on)-404.5(e)12.7(v)0(e)18.3(r)0(y)-403.5(5)-157.6(m)15.1(i)0(n)18.4(.)-413.9(Th)18.9(e)12.8(n)0(,)]TJ
-8.1249 -1.2203 TD
[(pr)16.9(ew)17.4(a)13.1(r)0(m)14.4(e)12.7(d)-224.3(m)0(e)15.2(d)0(i)25.1(u)0(m)-220.2(w)0(a)24.2(s)-228.9(a)13.1(d)0(d)19.3(e)0(d)-217.9(u)0(n)20.5(t)0(i)15(l)-224.8(re)18.4(a)13.1(c)0(h)16.3(i)0(n)18.4(g)-225.7(ha)22.4(lf)-219(of)-218.2(t)12.2(h)0(e)-218.3()0(n)18.9(a)0(l)]TJ
0 -1.2266 TD
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0 -1.2203 TD
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T*
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0 -1.2266 TD
[(th)15.1(e)-240.2(i)0(n)18.4(i)0(t)21.3(i)0(a)15.9(l)-237.5(24)-150.8(h,)-227.6(ag)21.4(it)15(a)13.1(t)0(i)15(o)0(n)-227.5(w)0(a)17.8(s)-241.6(se)17.8(t)-240.7(a)13.1(t)-240.7(4)0(0)-150.8(r)0(p)23.2(m)0(.)-234.4(A)0(f)23.4(t)0(e)18.6(r)-241(da)16.5(y)-238.4(3)0(,)-230(2)0(5)20(%)-241.9(of)]TJ
0 -1.2203 TD
[(th)15.1(e)-322.4(m)0(e)21.5(d)0(i)18.8(u)0(m)-315.1(v)0(o)15.5(l)15.4(um)13.7(e)-322.4(w)0(a)24.2(s)-323.8(re)18.4(ne)15.7(we)17.4(d)-319.1(d)0(a)16.5(i)0(l)24.5(y)0(.)]TJ
1.1128 -1.2203 TD
[(Cell)-291.7(counting)-290.5(on)]TJ
/F10 1 Tf
7.5875 0 TD
(Cultispher-S)Tj
/F4 1 Tf
5.3998 0 TD
[(microcarriers)-287.7(and)-290.4(nutrients/)]TJ
-14.1001 -1.2203 TD
[(metabolites)-323.2(analyses)-316.4(were)-318.3(performed)-316.2(according)-319.4(to)-319.4(protocols)]TJ
0 -1.2266 TD
[(described)-565.1(in)-557(the)-553.8(literature.)]TJ
6.7246 0 0 5.9768 423.9495 362.6078 Tm
(23)Tj
8.9663 0 0 8.9663 435.6849 358.6393 Tm
[(For)-558(cell)-556.3(counting)-562.4(on)-556.3(plastic)]TJ
-13.6765 -1.2203 TD
[(microcarriers,)-828.1(a)-821.5(microcarrier-cell)-830.5(suspension)-829(sample)-818.8(of)]TJ
T*
[(0.5)-166.3(mL)-588.6(was)-584.2(taken)-583.1(from)-589.5(the)-585.5(homogeneous)-588.6(culture)-588.3(in)-582.3(the)]TJ
0 -1.2266 TD
[(spinner)-367.7(ask.)-358.9(Microcarriers)-364.8(were)-356.2(washed)-362.8(with)-363.1(2)-163.9(m)0(L)-361(o)0(f)-357.3(pre-)]TJ
0 -1.2203 TD
[(warmed)-312.1(PBS,)-315.6(and)-309.4(1)-170.2(m)0(L)-310.4(o)0(f)-313.1(TrypLE)-306.3(Express)-319.6(\(1)]TJ
/F5 1 Tf
20.227 0 TD
()Tj
/F4 1 Tf
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[(\))-310.3(was)-312.3(added.)]TJ
-20.9478 -1.2203 TD
[(Microcarrier)-401.4(suspension)-405.3(was)-394.5(then)-399.2(incubated)-400.6(at)-398.3(37)]TJ
/F5 1 Tf
22.5222 0 TD
()Tj
/F4 1 Tf
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[(C)-398.5(for)-396(5)]TJ
-22.9206 -1.2266 TD
[(7)-170.2(min)-339.6(at)-335.1(650)-169.3(rpm)-334.7(using)-342.8(Thermomixer)-340(confort.)-341.6(Subsequently,)]TJ
0 -1.2203 TD
[(4)-170.2(m)0(L)-398.9(o)0(f)-401.6(the)-408.4(corresponding)-405.9(medium)-401.3(was)-400.8(added)-400.4(to)-401.6(stop)-404.3(en-)]TJ
T*
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T*
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T*
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[(tu)17.1(re)-247.2(ov)15.5(e)12.7(r)0(n)14.9(i)0(g)23.7(h)0(t)15.1(.)-255.8(Th)18.9(e)12.7(n)0(,)-252.9(s)0(a)24.5(m)0(p)13.7(l)15.4(es)-247.8(we)17.4(re)-247.2(wa)17.8(sh)20.6(ed)-249.5(t)12.2(w)0(i)13.8(c)13.4(e)-259.1(a)13.1(nd)-253(a)-252.4(D)0(N)17.6(A)]TJ
T*
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T*
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[(\(0.1)-166.8(mg)]TJ
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($)Tj
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(mL)Tj
/F5 1 Tf
6.7246 0 0 5.9768 211.3511 318.7842 Tm
(-)Tj
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(1)Tj
8.9663 0 0 8.9663 219.5716 314.8157 Tm
[(\))-1031.1(\(Sigma\))-1028.8(in)-1031.2(di-)]TJ
-17.8243 -1.2203 TD
[(methylformamide)-545.4(\(Fischer)-544.1(Scientic\))-548.8(and)-543.3(0.6)-166.3(mg)]TJ
/F6 1 Tf
22.2946 0 TD
($)Tj
/F4 1 Tf
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(mL)Tj
/F5 1 Tf
6.7246 0 0 5.9768 278.0787 307.8424 Tm
(-)Tj
/F4 1 Tf
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(1)Tj
8.9663 0 0 8.9663 291.118 303.8739 Tm
(of)Tj
-25.8038 -1.2266 TD
[(Red)-285.3(Violet)-278.6(LB)-277.9(salt)-283(\(Sigma\))-276.3(for)-282.2(45)-169.7(min)-276.4(and)-277.8(washed)-280.6(four)-283.5(times)]TJ
0 -1.2203 TD
[(with)-521.2(distilled)-515(water.)-517.3(Cells)-514.3(were)-520.6(then)-519.3(observed)-518.6(under)-514.2(the)]TJ
T*
[(microscope)-400.5(\(Leica)-391.4(Microsystems\))-397.5(for)-396(ALP)-397.8(staining,)-395.5(as)-392.8(a)-391.5(re-)]TJ
0 -1.2267 TD
[(sult)-297.5(of)-294.1(osteogenic)-296(commitment.)-296.3(Cells)-293(were)-293(then)-298(stained)-295.6(with)]TJ
0 -1.2203 TD
[(silver)-452.3(nitrate)-450.6(\(2.5%)-446.6(w/v\))-448.4(\(Sigma\))-447.1(for)-446.5(30)-163.4(min)-447.1(at)-448.9(room)-446.4(tem-)]TJ
T*
[(perature)-395.9(for)-389.6(von)-386.3(Kossa)-393.4(staining)-392.6(to)-388.9(evaluate)-390.6(the)-389.5(deposits)-393(of)]TJ
T*
[(calcium)-339.2(in)-329.4(the)-338.9(cultures.)]TJ
/F11 1 Tf
0 -2.447 TD
[(Adipogenic)-340.4(differentiation)]TJ
/F4 1 Tf
1.1128 -1.8336 TD
[(Cells)-545.9(retrieved)-546.3(from)-545.3(microcarriers)-546.9(were)-545.9(plated)-545.2(at)-543.8(3000)]TJ
-1.1128 -1.2203 TD
(cells/cm)Tj
6.7246 0 0 5.9768 93.8834 181.8141 Tm
(2)Tj
8.9663 0 0 8.9663 100.5732 177.8456 Tm
[(on)-379.2(CELLstart)-372(CTS-precoated)-382.1(12-well)-374.8(plates)-379.1(using)]TJ
-4.5525 -1.2266 TD
[(StemPro)-285.8(MSC)-282(SFM)-279.9(XenoFree.)-283.7(The)-284.5(adipogenic)-286.3(differentiation)]TJ
0 -1.2203 TD
[(was)-293.3(induced)-291.7(at)-284.5(80%)-291.5(cell)-284.5(conuence)-296.8(after)-291.9(culture)-291.1(for)-288.5(14)-289.9(days,)]TJ
T*
[(using)-526.2(StemPro)]TJ
/F5 1 Tf
6.7246 0 0 5.9768 119.3385 148.9323 Tm
()Tj
/F4 1 Tf
8.9663 0 0 8.9663 129.3165 144.9638 Tm
[(Adipogenesis)-525.3(Differentiation)-529.3(Kit)-519(\(Invitro-)]TJ
-7.7582 -1.2266 TD
[(gen\).)-403.1(The)-398.3(medium)-394.9(was)-394.5(changed)-398.6(twice)-403.2(a)-391.5(week)-397.9(for)-402.3(14)-397.4(days.)]TJ
0 -1.2203 TD
[(The)-708.1(assessment)-707.8(of)-705.1(differentiation)-714.2(toward)-703.6(an)-704.7(adipocytic)]TJ
T*
[(phenotype)-548.1(was)-533.6(performed)-543.8(based)-539.3(on)-537.3(the)-541.2(accumulation)-542.7(of)]TJ
T*
[(lipids,)-385.9(using)-380.7(Oil)-383.2(Red-O)-383.7(stain.)-384.2(Cells)-381.5(were)-387.8(washed)-381.8(with)-382.1(cold)]TJ
0 -1.2266 TD
[(PBS)-281(and)-271.5(xed)-279.9(in)-272.5(2%)-273(formaldehyde)-283.7(for)-275.8(30)-163.4(min.)-279.3(After)-274.8(xation,)]TJ
0 -1.2203 TD
[(cells)-405.9(were)-406.8(then)-405.5(washed)-407.1(with)-401(distilled)-407.5(water)-406.9(and)-404.2(incubated)]TJ
T*
[(with)-489.6(Oil)-490.7(Red-O)-484.9(solution)-493.4(\(Sigma\))-485(\(0.3%)-490.9(in)-487.5(isopropanol\))-491.4(at)]TJ
0 -1.2267 TD
[(room)-338.9(temperature)-336.9(for)-332.7(1)-170.2(h)0(.)]TJ
/F11 1 Tf
28.0042 74.5598 TD
[(Chondrogenic)-334(differentiation)]TJ
/F4 1 Tf
1.1128 -1.8336 TD
[(Expanded)-337.8(BM)-343.2(MSC)-345.2(and)-341(ASC)-341.5(were)-343.6(plated)-342.9(as)-342.3(small)-340.6(drop-)]TJ
-1.1128 -1.2203 TD
[(lets)-378.3(\(510)]TJ
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(m)Tj
/F4 1 Tf
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[(L\))-382.8(with)-382.1(high)-381.5(cell)-385.6(densities)-383.1(\()]TJ
/F12 1 Tf
12.4435 0 TD
(*)Tj
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(2)Tj
/F5 1 Tf
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()Tj
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(10)Tj
6.7246 0 0 5.9768 496.7432 702.3683 Tm
(7)Tj
8.9663 0 0 8.9663 503.5463 698.3998 Tm
(cells)Tj
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1.8842 0 TD
($)Tj
/F4 1 Tf
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(mL)Tj
/F5 1 Tf
6.7246 0 0 5.9768 538.8093 702.3683 Tm
(-)Tj
/F4 1 Tf
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(1)Tj
8.9663 0 0 8.9663 547.0298 698.3998 Tm
(\))Tj
-26.3412 -1.2266 TD
[(on)-638.4(ultra)-644.2(low)-640.2(attachment)-642.6(culture)-638.9(plates)-644.7(\(Corning\).)-641.9(After)]TJ
0 -1.2203 TD
[(30)-163.4(min,)-740.9(StemPro)-747.3(Chondrogenesis)-744.4(Differentation)-753.4(Kit)-740.3(\(In-)]TJ
T*
[(vitrogen\))-534.9(was)-527.3(added.)-529.8(The)-531.1(medium)-527.7(was)-533.6(changed)-531.4(twice)-529.6(a)]TJ
0 -1.2266 TD
[(week)-271.4(for)-275.8(14)-270.9(days.)-276.7(The)-271.9(assessment)-277.9(of)-275.1(differentiation)-278(toward)-273.7(a)]TJ
0 -1.2203 TD
[(chondrocytic)-433.3(phenotype)-440.6(was)-426.1(performed)-436.3(based)-431.8(on)-436.1(the)-427.4(syn-)]TJ
T*
[(thesis)-395.6(of)-395.3(proteoglycans)-396.9(by)-388.2(chondrocytes,)-396.7(using)-399.7(Alcian)-390(Blue)]TJ
T*
[(stain.)-498(Cells)-501.6(were)-495.3(washed)-501.9(with)-495.9(cold)-501.8(PBS)-496(and)-499.1(xed)-501.2(in)-493.8(2%)]TJ
0 -1.2266 TD
[(formaldehyde)-612.5(for)-610.9(30)-163.4(min.)-614.4(After)-609.9(xation,)-612.1(cells)-614.6(were)-609.1(then)]TJ
0 -1.2203 TD
[(washed)-375.5(with)-382.1(distilled)-375.9(water)-381.6(and)-379(incubated)-381.6(with)-375.7(1%)-380.5(Alcian)]TJ
T*
[(Blue)-330.3(solution)-341.6(\(Sigma-Aldrich\))-335.9(at)-335.1(room)-332.6(temperature)-343.3(for)-332.7(1)-163.9(h)0(.)]TJ
/F11 1 Tf
0 -2.447 TD
[(RNA)-332.3(isolation)]TJ
/F4 1 Tf
1.1128 -1.8336 TD
-.0188 Tc
[(Cells)-260.4(w)-1.5(ere)-259(h)-3.2(arvested)-262.1(for)-259.8(R)2.3(NA)-266.8(isolation)-262.5(a).7(t)-265.9(t)-.3(he)-265.3(beginning)-263.5(a).7(nd)]TJ
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[(at)-228(the)-233.8(e).2(nd)-230.6(of)-234.3(the)-233.8(s)-1.2(pinne)-6.1(r)-228.3(as)5.1(k)-232(cultures)-228.9(as)-235.2(indicated)-230.6(b)-4.4(efore.)-236.8(Total)]TJ
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[(\(Roche\))-246.6(a)1(c)-5.1(c)1.2(ording)-250.5(to)-248.9(the)-246(m)-3.4(anufac)]TJ
14.1444 0 TD
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[(turers)-254(i)3.1(nstructions)5.3(.)-255.6(RNA)-254(w)5(as)]TJ
-14.1444 -1.2203 TD
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[(quan)-4.1(tied)-218.7(b)1.2(y)-220.1(U)-2.1(V)-218.5(s)-2(pe)-7(ctroph)-4.1(otom)]TJ
13.8661 0 TD
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[(etry)-212.8(\(N)-6(anoDrop)-222.4(T)3.6(echnologies\))]TJ
-13.8661 -1.2203 TD
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[(at)-443.1(260)-170.3(nm.)-445.6(C)-.2(omplementa)-5.9(r)5.6(y)-447.1(DNA)-450.4(w)]TJ
15.8326 0 TD
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[(as)-449.9(synthesized)-445.2(u)-1(sing)-446.6(the)]TJ
-15.8326 -1.2266 TD
[(Transcriptor)-354.3(First)-354.1(S)-4.3(trand)-356.6(c)1.2(DN)]TJ
12.8924 0 TD
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[(A)-360.4(S)2.9(ynthes)6.4(is)-354(Kit)-359.5(\().9(Roche)-352.6(Applied)]TJ
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[(Scienc)-5.7(e\))-247.2(with)-250.1(anc)-5.7(h)2.8(ored-)-6.9(o)3.4(ligo\(dT\))]TJ
6.7246 0 0 5.9768 435.5148 472.2518 Tm
-.0195 Tc
(18)Tj
8.9663 0 0 8.9663 444.1888 473.7259 Tm
-.0192 Tc
[(pr)5.4(imers)-254.5(a).3(nd)-256.1(1)]TJ
/F12 1 Tf
6.0384 0 TD
0 Tc
(m)Tj
/F4 1 Tf
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[(go)246.3(fR)244.8(N)249.1(A)247.7(.)]TJ
/F11 1 Tf
-21.441 -2.447 TD
0 Tc
[(Quantitative)-332.7(real-time-polymerase)]TJ
0 -1.2203 TD
[(chain)-332.2(reaction)-336(analysis)]TJ
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1.1128 -1.8336 TD
[(F)14.6(o)0(r)-370.2(r)0(e)18.4(a)13.1(l-)15(t)12.2(i)0(m)17.9(e)-379.3(po)21.2(ly)17.3(me)21.5(ra)18.8(se)-367.9(ch)16.3(a)13.1(i)0(n)-373.6(r)0(e)18.4(a)13.1(ct)19.2(io)19(n)-376.4(\()0(R)14.3(T)0(-)21.8(P)0(C)17.4(R)0(\))20.6(,)-382.3(a)-372.6(t)0(w)16.9(o)0(-)]TJ
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[(st)17.2(e)12.7(p)-425(PC)17.4(R)-421.5(r)0(u)16.9(n)-420.7(wa)17.8(s)-424.9(p)0(e)17.7(r)0(f)17.8(o)0(r)21.8(m)0(e)21.5(d)-426.6(in)-411.6(a)-423.1(L)0(i)18.8(g)0(h)17.5(t)12.2(Cy)20.7(cl)16.2(er)]TJ
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6.7246 0 0 5.9768 498.3306 417.4298 Tm
()Tj
/F4 1 Tf
8.9663 0 0 8.9663 507.4581 413.4613 Tm
[(\()12.2(R)0(o)18.3(c)0(h)16.3(e)-423.5(Di)20.4(-)]TJ
-21.9279 -1.2266 TD
[(a)13.1(g)0(n)17.5(o)0(s)21.2(t)0(i)15(c)13.4(s\))-476(us)16.3(in)18.4(g)-478.6(a)-486.4(S)14.2(Y)0(B)18.1(R)]TJ
/F5 1 Tf
6.7246 0 0 5.9768 409.6062 406.4881 Tm
()Tj
/F4 1 Tf
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[(G)13.9(r)0(e)18.4(e)0(n)-477.5(P)0(C)23.8(R)-484.8(ma)21.9(st)17.2(er)-474.8(mi)17.9(x)-482.7(\()12.2(Ro)18.3(ch)16.3(e)12.8(\))0(,)]TJ
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[(c)13.4(o)0(n)19.1(t)0(a)19(i)0(n)18.4(i)0(n)18.4(g)-371.1(1)-372.6(L)0(i)18.8(g)0(h)17.5(t)12.2(Cy)20.7(cl)16.2(e)12.8(r)-373.8(Fa)21.4(st)17.2(St)20(ar)18.8(t)]TJ
/F5 1 Tf
6.7246 0 0 5.9768 445.1526 395.4897 Tm
()Tj
/F4 1 Tf
8.9663 0 0 8.9663 453.77 391.5212 Tm
[(DN)17.6(A)-374.4(M)0(a)20.7(s)0(t)17.2(e)12.7(r)-373.7(P)0(l)20.4(u)0(s)-363.1(S)0(Y)16.3(B)0(R)]TJ
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[(G)13.9(r)0(e)18.4(e)0(n)-351(m)0(a)21.9(s)0(t)17.2(e)0(r)-348.4(m)0(i)17.9(x)0(,)-352.8(4)-151.3(mM)-350.4(Mg)15.8(C)12.5(l)]TJ
6.7246 0 0 5.9768 434.1542 379.0487 Tm
(2)Tj
8.9663 0 0 8.9663 437.4424 380.5794 Tm
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[(,0)370.1(.)357(5)]TJ
/F12 1 Tf
1.9917 0 TD
0 Tc
(m)Tj
/F4 1 Tf
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[(M)-359.2(o)0(f)-351(e)12.8(ac)20.2(h)-363.8(p)0(r)23.2(i)0(m)17.9(e)0(r)18.4(,)-357(an)16.1(d)]TJ
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(2)Tj
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(m)Tj
/F4 1 Tf
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[(L)-224.3(o)0(f)-218.2(t)12.2(em)15.2(pl)20.4(a)13.1(t)0(e)-215.3(r)0(e)18.4(s)0(u)16.3(l)0(t)21.3(i)0(n)18.4(g)-225.7(fr)17.8(om)-215.3(t)12.2(h)0(e)-218.3(c)13.4(DN)17.6(A)-229(s)0(y)19.6(n)0(t)21.4(h)0(e)15.7(s)0(i)20.4(s)-222.6(re)18.4(ac)20.2(ti)15(on)-214.8(in)]TJ
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-.0131 Tc
(20)Tj
/F12 1 Tf
1.1444 0 TD
0 Tc
(m)Tj
/F4 1 Tf
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[(L)-312.8(o)0(f)-306.7()0(n)18.9(a)13.1(l)-313.4(v)0(o)15.5(l)15.4(um)13.7(e)12.8(.)-312.7(Th)18.9(is)-308.3(t)12.2(w)0(o)20.9(-)0(s)17.2(t)0(e)18.6(p)-317.5(pr)23.2(og)18.1(ra)18.8(m)-313.7(c)13.4(on)12.8(si)20.4(st)23.5(ed)-306.4(in)-304.1(an)]TJ
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[(in)18.4(it)21.3(ia)22.3(l)-465.1(d)0(e)16.1(n)0(a)22.4(t)0(u)17.1(r)0(a)18.8(t)0(i)21.3(o)0(n)-461.4(s)0(t)23.5(e)0(p)-456.5(a)0(t)-461.6(9)13.1(5)]TJ
/F5 1 Tf
13.4425 0 TD
()Tj
/F4 1 Tf
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[(Cf)461.4(o)477.1(l)461.4(l)485.9(o)461.4(w)476(e)474.1(db)461.4(y4)474.5(5)-6(r)461.4(o)483.2(u)461.4(n)475.6(d)461.4(s)8.2(o)461.4(f)]TJ
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0 Tc
[(c)13.4(y)0(c)15.3(l)0(i)24.6(n)0(g)-330.3(b)0(e)21(t)0(w)16.9(e)0(e)19.2(n)-338.5(10)-150.8(s)-336.4(a)13.1(t)-341.9(9)13.1(5)]TJ
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()Tj
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[(C)12.5(,)-338(10)-144.5(s)-342.7(a)13.1(t)-341.9(t)12.2(he)-332.1(re)18.4(sp)22.6(ec)19.8(ti)15(ve)-329.4(a)13.1(n)0(n)18.5(e)0(a)19.6(l)0(i)18.2(n)0(g)]TJ
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[(t)12.2(e)0(m)15.2(p)0(e)24(r)0(a)18.8(t)0(u)17.1(r)0(e)-278.8(a)13.1(nd)-278.3(1)13.1(0)-157.6(s)-285.8(a)13.1(t)-285(7)0(2)]TJ
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()Tj
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[(C.)-275(Th)18.9(e)-284.4(f)0(o)15.7(l)0(l)18.2(o)0(w)20.9(i)0(n)18.4(g)-282.6(pr)16.9(im)24.2(er)18.4(s)-285.8(f)0(o)15.7(r)-285.2(e)12.8(a)0(r)18.8(l)0(y)]TJ
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[(di)18.8(f)12.2(f)0(e)18.6(r)0(e)18.4(n)0(t)15.1(i)15.4(at)19(io)19(n)-237.3(c)13.4(el)15.5(l)-231.2(m)0(a)21.9(r)0(k)20.2(e)0(r)18.4(s)-235.3(we)17.4(re)-221.9(us)16.3(e)12.8(d)0(:)-227.2(R)0(G)22.3(C)0(3)19.3(2)-233.5(\()12.2(e)0(a)13.2(r)0(l)21.1(y)-232.1(os)21.2(te)18.6(oc)16.9(yt)20.4(e)]TJ
T*
[(c)13.4(e)0(l)15.5(l)-326(ma)15.6(rk)20.2(e)12.8(r)0(,)-319.8(1)0(6)19.9(6)-157.6(bp)19.5(\))-335.6(\()12.2(Fw)19.2(\))-329.3(5)]TJ
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()Tj
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[(-)12.2(G)0(C)20(C)-329(AC)17.4(T)-331.8(T)0(C)22.1(C)-329(AC)17.4(T)-331.8(A)0(C)17.4(G)-327.5(AG)18.8(G)]TJ
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[(AG)18.8(-)12.2(3)]TJ
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()Tj
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[(,\()320.6(R)308.4(e)329.6(\)5)]TJ
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3.0982 0 TD
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()Tj
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[(-G)19.7(CT)-300.3(G)13.9(G)0(G)-301(G)13.9(TA)-295.2(GA)18.8(G)-308.6(T)0(C)22.1(T)-306.5(GT)17.2(T)-306.5(G)13.9(G-)13.4(3)]TJ
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()Tj
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[(;F)306.4(A)325.9(B)306.4(P)327.3(4)]TJ
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[(\(e)-13.4(a)6.2(r)-13.4(ly)-365.6(ad)-13.4(i)5.4(p)-13.4(ocy)-13.4(t)7(e)-367.4(c)-13.4(ell)-371(m)-13.4(a)8.5(r)-13.4(k)6.8(e)-13.4(r)5(,)-370.4(21)-13.4(5)-157.9(b)-13.4(p\))-367.9(\(F)-13.4(w)5.8(\))-368(5)]TJ
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()Tj
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[(-T)15.5(C)12.5(A)-355.5(TA)20.9(C)-354.3(T)0(G)17.2(G)]TJ
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[(G)13.9(C)0(C)-322.8(A)0(G)18.8(G)-333.9(AA)22.5(T-)15.5(3)]TJ
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