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[(f)-5.6(unc)-6.8(ti)-5.8(on)-512.5(an)-5.7(d)-512.5(m)-1.1(e)-6.8(c)-1.1(ha)-6.8(ni)-5.8(sm)-513.8(fo)-5.7(r)]TJ
/F7 1 Tf
12.919 0 TD
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[(MFN)-5.6(2)]TJ
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3.1851 0 TD
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[(in)-512.4(r)-5.5(e)-1(gu)-5.6(la)-6.7(ti)-5.7(ng)-512.4(MS)-5.4(C)]TJ
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[(d)-5.5(i).2(ff)-5.4(er)-5.4(en)-5.5(tia)-6.6(ti)-5.6(on)-333.8(f)-5.4(r).3(o)-5.5(m)-335(iP)-5.3(SC)-6.5(s.)]TJ
/F9 1 Tf
9.9626 0 0 9.9626 62.022 201.0897 Tm
0 Tc
[(Mater)-8.1(ials)-337.1(and)-336.9(Methods)]TJ
/F3 1 Tf
0 -1.673 TD
[(Cell)-336.9(cu)-6.4(lture)]TJ
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9.843 0 0 9.843 72 166.5638 Tm
[(iPSCs)-466.9(were)-468.3(reprogrammed)-474.1(from)-466.3(human)-468.7(gingival)-465.4(bro-)]TJ
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[(blasts)-405.1(in)-399.7(our)-400.7(previous)-407.3(studies)-400.4([7],)-404.1(which)-405.1(are)-403(derived)-401.8(from)]TJ
T*
[(wasted)-482(human)-480.2(gingival)-476.9(tissues)-480.8(from)-477.9(third)-478(molar)-480.3(removal)]TJ
0 -1.1174 TD
[(surgery)-360(or)-353.5(orthodontic)-359.2(therapy)-355.7(with)-356.9(informed)-356.9(consent.)-359(The)]TJ
0 -1.1116 TD
[(protocols)-466.1(were)-462.6(approved)-460.4(by)-463(the)-465.4(Biomedical)-466.4(Ethics)-458.3(Com-)]TJ
T*
[(mittee,)-561.2(Peking)-560.7(University)-560.4(Hospit)-7.5(al)-556.4(of)-560.9(Stomatology)-561.9(\(No.)]TJ
T*
[(PKUSSIRB-201414048\).)-580.6(The)-575.9(iPSCs)-576.4(were)-577.8(expanded)-576.8(and)]TJ
0 -1.1174 TD
[(passaged)-311.4(in)-301.8(TeSR-E8)-311.6(medium)-304.1(\(Stemcell)-314.3(Technologies\))-307(and)]TJ
0 -1.1116 TD
[(Matrigel)-363.7(\(Corning\))-362.3(precoated)-366(culture)-361.6(plates.)-363.8(All)-361.5(cells)-359.2(were)]TJ
T*
[(cultured)-397.3(in)-394(a)-393.9(cell)-398.6(culture)-396.2(incubator)-398.4(\(Thermo)-397.1(Fisher)-399.2(Scien-)]TJ
0 -1.1174 TD
[(tic\))-335(containing)-336.3(5%)-331.6(CO)]TJ
7.3823 0 0 6.5614 159.0236 55.3322 Tm
(2)Tj
9.843 0 0 9.843 165.9968 56.9763 Tm
[(at)-337.6(37)]TJ
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2.062 0 TD
()Tj
/F6 1 Tf
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(C.)Tj
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9.9626 0 0 9.9626 313.0582 725.0455 Tm
[(Const)-8.9(ruction)-340.9(of)-333.6(MFN)-8.8(2-KD)-337.3(iPSCs)-344.7(by)-330.8(lent)-7.4(ivirus)]TJ
/F6 1 Tf
9.843 0 0 9.843 323.0361 707.2439 Tm
[(The)-276.4(siRNA)-273.2(target)-279.9(gene)-271.7(sequence)-278.2(for)]TJ
/F7 1 Tf
15.004 0 TD
(MFN2)Tj
/F6 1 Tf
2.8914 0 TD
[(was)-270.1(designed)]TJ
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[(accor)-9(ding)-338.6(to)-336.4(the)-338.7(RNAi)-341.4(sequence)-341.6(design)-341.8(principles.)-339.5(The)-339.7(se-)]TJ
0 -1.1116 TD
[(quenc)-7.8(e)-376.6(was)-379.5(GGAAGAGCACCGTGATCAATG.)-392.1(The)-380(virus)]TJ
T*
[(vecto)-8(r)-513.7(framework)-521.1(was)-517.8(constructed)-9.9(,)-516(the)-517.2(plasmid)-517(transfor-)]TJ
0 -1.1174 TD
[(mation)-521.9(was)-517.8(conducted,)-518.1(and)-517.1(the)-517.2(transformed)]TJ
/F7 1 Tf
19.3353 0 TD
[(E.)-518.3(coli)]TJ
/F6 1 Tf
3.404 0 TD
(was)Tj
-22.7393 -1.1116 TD
[(screened)-341.5(by)-330.5(ampicillin-containing)-341.1(agar)-335(plate.)-334.1(We)-339.7(identied)]TJ
T*
[(and)-390.4(sequenced)-394.5(the)-390.5(PCR)-391.1(product,)-392.5(and)-384.6(extracted)-394.9(the)]TJ
/F7 1 Tf
21.5298 0 TD
[(E.)-391.6(coli)]TJ
/F6 1 Tf
-21.5298 -1.1116 TD
[(solution)-302.7(with)-299.3(the)-304.1(correct)-299.2(sequencing)-308.2(result.)-299.2(Then,)-298.2(we)-303.7(added)]TJ
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[(pGag/Pol,)-573.1(pRev,)-568.7(and)-568.9(pVSV-G)-567.7(to)-566.8(package)-571.1(the)-569(lentiviral)]TJ
0 -1.1116 TD
[(particles)-376.4(and)-367.3(infected)-375.4(the)-367.5(293T)-368.5(cells,)-374.2(and)-367.3(concentrated)-376.4(the)]TJ
T*
.013 Tc
[(v)-5.4(i).3(r)-5.3(u).4(s)-356.5(i).3(n)-356.7(a)-.8(n)-356.7(u).4(lt)-5.5(ra)-6.5(cen)-5.4(tr)-5.3(if)-5.3(ug)-5.4(e)-352.1(\().4(B)-6.4(e)-.8(c)-6.5(k).4(ma)-6.5(n)-351(C)-.6(o)-5.4(u).4(l)-5.5(t).3(e)-6.5(r).4(\))-5.3(.)-353.3(For)-356.7(i).3(n)-5.4(f).4(e)-6.5(c)-.8(-)]TJ
0 -1.1174 TD
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0 -1.1116 TD
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T*
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0 -1.1174 TD
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[(a)-6.6(f).3(t)-5.6(e)-.9(r)-333.7(i)-5.6(nf)-5.4(ec)-6.6(tio)-5.5(n.)]TJ
/F3 1 Tf
9.9626 0 0 9.9626 313.0582 529.9086 Tm
0 Tc
[(Const)-8.9(ruction)-340.9(of)-333.6(MFN)-8.8(2-OE)-336.8(iPSCs)-344.7(by)-330.8(lent)-7.4(ivirus)]TJ
/F6 1 Tf
9.843 0 0 9.843 323.0361 512.0503 Tm
[(The)-391.6(linearized)-390.4(GV492)-393.2(vector)-389.2(\(Genechem\))-397.9(was)-385.3(obtained)]TJ
-1.0137 -1.1116 TD
[(by)-278.7(digestion)-276.2(with)-282(restriction)-279.6(enzymes)]TJ
/F7 1 Tf
15.4245 0 TD
(Bam)Tj
/F6 1 Tf
1.8316 0 TD
[(HI)-279.5(and)]TJ
/F7 1 Tf
3.0526 0 TD
(Age)Tj
/F6 1 Tf
1.5666 0 TD
[(I.)-275.2(The)]TJ
-21.8754 -1.1116 TD
[(target)-331.7(gene)-323.5(fragment)-329.2(\(NM_014874,)-327.2(2274)-165.7(bp\))-325.8(was)-327.7(amplied)]TJ
0 -1.1174 TD
[(by)-359.3(PCR)-350.8(using)-356.8(primers)-355.6(with)-356.9(homologous)-352(recombinat)-10.2(ion)-349(se-)]TJ
0 -1.1116 TD
[(quenc)-7.8(e)-307.5(\(underlined\))-315.2(added)-313.1(to)-313.4(the)-309.9(5end)-311.9(\(Primers:)]TJ
/F7 1 Tf
20.4009 0 TD
(MFN2)Tj
/F6 1 Tf
3.1102 0 TD
(1:)Tj
ET
313.058 454.734 139.805 .51025 re
f
BT
9.843 0 0 9.843 313.0582 457.2849 Tm
.0251 Tc
[(A)-5.7(GGTCGACT)-6(C)-.1(TAGAGGA)-5.7(TCC)]TJ
14.2034 0 TD
0 Tc
(CGCCACCATGTCCCT)Tj
-14.2034 -1.1174 TD
[(GCTC)-8.5(TTCTCTCG;)-531.4(and)]TJ
/F7 1 Tf
10.6382 0 TD
(MFN2)Tj
/F6 1 Tf
3.116 0 TD
(2:)Tj
ET
1 i
q 1 0 0 1 461.31 444.302 cm 0 0 m
90.879 0 l
90.879 -.51 l
0 -.51 l
f*
Q
BT
9.843 0 0 9.843 461.3101 446.2865 Tm
(TCCTTGTAGTCCAT)Tj
ET
313.058 432.85 20.239 .45352 re
f
BT
9.843 0 0 9.843 313.0582 435.3448 Tm
[(ACCTC)23.9(T)15(G)15.3(CT)]TJ
5.8634 0 TD
-.0152 Tc
[(GGGCT)-15.2(G)9.3(CAGG)-15.2(T)9.3(A).1(CTGG)-15.2(T)9.3(G).1(\).)-214.5(Th)-15.2(e)-202.9(l).8(i)-15.2(n)11.3(ear)-15.2(i)11.3(zed)]TJ
-5.8634 -1.1116 TD
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[(v)-15.7(e)9.7(cto)-15.7(r)-254(and)-270.2(t).4(arge)-15.7(t)-255.3(g).5(en)-15.7(e)-255.2(frag)-15.7(m)9.7(ent)-264.6(w)-15.7(e)8.9(r).5(e)-265.6(c)-15.7(o)9.7(nnec)-15.7(t)9.6(ed)-264.5(by)-270.2(a)-265.6(homo)-15.7(l)10.8(o-)]TJ
0 -1.1174 TD
[(g)-15.7(o)10.9(us)-500.4(recomb)-15.7(i)10.8(n).5(at)-15.7(i)10.6(o).5(n)-494.8(r)-15.7(e)9.8(acti)-15.7(o)10.8(n.)-497.2(The)-501.8(foll)-15.7(o)10.8(wing)-500.6(step)-15.7(s)11.1(,)-497.2(suc)-15.7(h)-485.6(as)]TJ
0 -1.1116 TD
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[(t)-15.9(r)10.6(ans)-15.9(f)10.9(or)-15.9(m)9.5(a)-.9(tion)-15.9(,)-475.4(v).3(irus)-489.1(pack)-15.9(a)9.5(g).3(in)-15.9(g)10.7(,)-485.9(puri)-15.9()10.7(cati)-15.9(o)10.6(n,)-485.9(and)-489.3(i).1(nfec)-15.9(t)9.4(ion)-15.9(,)]TJ
T*
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[(w)-15.8(e)8.8(re)-254.2(the)-254.2(s).6(ame)-254.2(as)-252.9(the)-254.2(c)-.8(on)-15.8(s)11(t).3(ru)-15.8(c)9.6(t).3(ion)-253(o).4(f)]TJ
/F7 1 Tf
14.3993 0 TD
-.0151 Tc
(MFN2)Tj
/F6 1 Tf
2.5573 0 TD
-.0161 Tc
[(-KD)-254.2(i)-.1(PSC)-16.1(s)9.7(.)]TJ
/F3 1 Tf
9.9626 0 0 9.9626 313.0582 369.581 Tm
0 Tc
[(We)-9(stern)-337(blot)-337.5(analysi)-9.3(s)]TJ
/F6 1 Tf
9.843 0 0 9.843 323.0361 351.7794 Tm
[(The)-316.7(cells)-318.9(were)-318.6(lysed)-317.6(in)-319.1(RIPA)-317.1(lysis)-316.4(and)-315.5(Extraction)-320.2(buffer)]TJ
-1.0137 -1.1174 TD
[(\(Thermo)-616(Fisher)-612.3(Scientic\).)-611.2(All)-609.2(proteins)-614.8(were)-612.3(separated)]TJ
0 -1.1116 TD
[(by)-630(SDS-PAGE)-634.2(electrophoresis)-631.6(and)-632.3(transferred)-631.7(to)-630.1(poly-)]TJ
T*
[(vinylidene)-595.5(diuoride)-592.9(membrane)-590.7(\()-81.7(Merck)-596.2(Millipore\).)-592.9(The)]TJ
0 -1.1174 TD
[(blot)-500(was)-494.7(incubated)-497.5(by)-491.8(primary)-494(and)-499.8(secondary)-497(antibodies)]TJ
0 -1.1116 TD
[(after)-388(blocking)-384.6(in)-388.2(5%)-383.4(defatted)-386.9(milk.)-383.7(Immobilon)-387(ECL)-386.9(Ultra)]TJ
T*
[(Western)-432.7(HRP)-431.3(substrate)-432.5(\()-81.7(Merck)-434.9(Millipore\))-428.2(was)-431.4(applied)-430.8(to)]TJ
0 -1.1174 TD
[(visual)-7.8(ize)-334.1(the)-338.7(protein)-343.2(band.)-337.3(The)-339.7(antibodies)-336.1(were)-341.6(as)-337.2(follows:)]TJ
0 -1.1116 TD
[(MFN2)-288.3(\(1:1,000;)-283.2(Abcam\),)-285.1(GAPDH)-285.3(\(1:1,000;)-283.2(CST\),)-286.3(RUNX2)]TJ
T*
[(\(1:1,000;)-870.7(Proteintech\),)-867(OSX)-863.1(\(1:1,000;)-870.7(Abcam\),)-866.9(AKT)]TJ
T*
[(\(1:1,000;)-738.2(Proteintech\),)-740.3(phosphor-AKT)-735.9(\(pAKT,)-737.6(1:1,000;)]TJ
0 -1.1174 TD
[(Protei)-7.8(ntech\),)-432.9(pGSK-3)]TJ
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8.916 0 TD
(b)Tj
/F6 1 Tf
.9907 0 TD
[(\(1:1,000;)-432.9(CST\),)-436.1(GSK-3)]TJ
/F4 1 Tf
9.7569 0 TD
(b)Tj
/F6 1 Tf
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(\(1:1,000;)Tj
-20.6485 -1.1116 TD
[(CST\),)-1006.3(VDAC)-1004.5(\(1:1,000;)-1003.1(Proteintech\),)-1005.2(and)]TJ
/F4 1 Tf
20.5046 0 TD
(b)Tj
/F6 1 Tf
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(-catenin)Tj
-21.0517 -1.1116 TD
[(\(1:1,000;)-335(Proteintech\).)]TJ
/F3 1 Tf
9.9626 0 0 9.9626 313.0582 185.1023 Tm
[(Quan)-9.9(titative)-337.8(real-)-9.4(time)-335.3(polym)-8.2(erase)-339.2(chain)]TJ
0 -1.2007 TD
[(rea)-8.4(ction)-338.2(analysis)]TJ
/F6 1 Tf
9.843 0 0 9.843 323.0361 155.2818 Tm
[(TRIzol)-378.7(reagent)-374.2(\(Thermo)-374.1(Fisher)-376.1(Scientic\))-377.4(was)-379.5(added)-370.7(to)]TJ
-1.0137 -1.1116 TD
[(the)-499.9(cell)-496.6(samples)-500.6(to)-497.7(extract)-501(total)-496.6(RNA.)-499.1(According)-502.8(to)-497.7(the)]TJ
T*
[(manufactur)-10(ers)-431.5(instructions,)-432.6(reverse)-437.2(transcription)-431.6(was)-437.1(per-)]TJ
0 -1.1174 TD
[(forme)-7.8(d)-461.9(with)-460.6(the)-465.4(PrimeScript)-470.7(RT)-465.2(Kit)-465.2(\(TaKaRa,)-467(Japan\))-463.7(to)]TJ
0 -1.1116 TD
[(obtain)-405.5(cDNA.)-403.3(Quantitative)-404.1(real-time)-406.6(polymerase)-407.3(chain)-399.6(re-)]TJ
T*
[(action)-585.2(\(qRT-PCR\))-584.2(was)-581.1(performed)-583.6(by)-583.9(Power)-577.6(Up)-584.8(SYBR)]TJ
0 -1.1174 TD
[(Green)-469.5(master)-465.1(mix)-465.4(\(Thermo)-466.3(Fisher)-468.3(Scientic\))-469.6(in)-463.1(CFX384)]TJ
0 -1.1116 TD
[(Touch)-474.4(Real-Time)-472.1(PCR)-471.7(Detection)-469.7(System)-475.3(\(Bio-Rad\).)-471.6(The)]TJ
T*
[(primer)-481.4(sequences)-486.5(are)-477.9(shown)-478.5(in)-480.4(Table)-481.5(1;)]TJ
/F7 1 Tf
17.7745 0 TD
(ACTB)Tj
/F6 1 Tf
2.9259 0 TD
[(was)-483.2(em-)]TJ
-20.7004 -1.1174 TD
[(ployed)-336.2(as)-337.2(an)-331.7(internal)-339.8(control.)]TJ
/F9 1 Tf
8.9663 0 0 8.9663 62.022 749.6502 Tm
(182)Tj
48.2185 0 TD
[(DENG)-335.4(ET)-338.7(AL.)]TJ
ET
endstream
endobj
59 0 obj
<>stream
/GS1 gs
BT
/F3 1 Tf
9.9626 0 0 9.9626 59.7543 498.6707 Tm
0 0 0 rg
0 Tc
0 Tw
[(Im)-7(munouor)-6.9(esce)-7.2(nce)]TJ
/F6 1 Tf
9.843 0 0 9.843 69.7323 480.8692 Tm
[(The)-264.8(cells)-267.1(were)-266.8(xed)-264.6(in)-267.3(4%)-262.4(paraformaldehyde)-272.4(solution)-262.4(for)]TJ
-1.0137 -1.1116 TD
[(30)-169.2(min,)-434.3(gently)-434.2(rinsed)-433.9(three)-434.1(times)-434.2(with)-431.8(phosphate-buffered)]TJ
0 -1.1174 TD
[(saline)-348.8(\(PBS\),)-343.7(permeabilized)-350(in)-347.9(0.3%)-341.9(Triton)-342.1(X-100)-346.2(\(Sigma-)]TJ
0 -1.1116 TD
[(Aldrich\))-420(in)-411.3(PBS)-418.7(for)-412.2(20)-169.2(min,)-417(and)-413.4(blocked)-413.3(with)-420.2(5%)-412.2(bovine)]TJ
T*
[(serum)-468.4(albumin)-465.4(\(BSA\))-462.2(\(Sigma-Aldr)-9.6(ich\))-460.7(in)-463.1(PBS)-464.8(for)-464(1)-168.1(h)-461.9(at)]TJ
0 -1.1174 TD
[(room)-368.5(temperature.)-367.2(The)-362.8(cells)-365(were)-370.4(stained)-361.4(by)-365.1(primary)-367.3(anti-)]TJ
0 -1.1116 TD
[(body)-413.3(MFN2)-409.2(\(1:100;)-411.1(Abcam\))-414.2(at)-412.4(room)-408.8(temperature)-415.6(for)-412.2(1)-168.1(h)0(,)]TJ
T*
[(then)-282.2(incubated)-284.4(by)-284.4(secondary)-283.9(antibody)-280.9(DyLight)-286.5(488)-279.8(\(ZSGB-)]TJ
0 -1.1174 TD
[(Bio\))-454.8(for)-452.5(1)-168.1(h)0(.)-453.8(The)-454.9(nucleus)-453.5(was)-454.4(stained)-453.6(by)-451.5(DAPI)-455.2(\(Thermo)]TJ
0 -1.1116 TD
[(Fisher)-324.3(Scientic\).)-323.2(Images)-321(were)-324.4(observed)-321.9(and)-315.5(recorded)-323.3(by)-319(a)]TJ
T*
[(confocal)-340.7(microscope)-338.2(\(Leica\).)]TJ
/F3 1 Tf
9.9626 0 0 9.9626 59.7543 349.3416 Tm
[(Flow)-339.2(cy)-7.2(tometry)]TJ
/F6 1 Tf
9.843 0 0 9.843 69.7323 331.5401 Tm
[(The)-282.1(cells)-284.4(were)-284(digested)-281.9(with)-282(Accutase)-290.7(\()-81.7(Merck)-285.2(Millipore\))]TJ
-1.0137 -1.1174 TD
[(and)-269.4(stained)-269.3(with)-264.7(antibody)-263.7(for)-268.2(30)-169.2(min)-263.8(in)-267.3(the)-263.8(dark)-264.7(at)-268.4(4)]TJ
/F11 1 Tf
21.1554 0 TD
()Tj
/F6 1 Tf
.3917 0 TD
[(C.)-270.5(The)]TJ
-21.5471 -1.1116 TD
[(cells)-514.7(were)-508.7(then)-506.8(washed)-510.7(twice)-515.8(with)-506.6(PBS,)-508.5(resuspended)-510.5(in)]TJ
T*
[(PBS,)-341.5(and)-344.3(analyzed)-347.7(by)-336.3(CytoFLEX)-347.1(S)-340.8(ow)-343.8(cytometer)-343.2(\(Beck-)]TJ
0 -1.1174 TD
[(man)-455(Coulter\))-460.5(and)-453.7(Cytexpert)-457(software)-455.4(\(ver.)-458.2(2.3;)-450.4(Beckman)]TJ
0 -1.1116 TD
[(Coulter\).)-481.2(The)-478(antibodies)-485.8(were)-479.9(as)-475.5(follows:)-482.1(CD34-PE)-477.2(\(BD)]TJ
T*
[(Biosciences\),)-356.2(CD45-APC-A750)-358.6(\(eBioscience\),)-358.7(CD73-FITC)]TJ
T*
[(\(eBioscience\),)-617.9(CD90-FITC)-607.1(\(BioLegend\),)-613.4(and)-603.5(CD146-PE)]TJ
0 -1.1174 TD
(\(BioLegend\).)Tj
/F3 1 Tf
9.9626 0 0 9.9626 59.7543 221.9527 Tm
[(Thre)-9(e)-330(ger)-8.4(m)-333.6(layer)-337.4(differ)-8(entiatio)-9.1(n)-330(o)0(f)-339.2(iPSCs)]TJ
/F6 1 Tf
9.843 0 0 9.843 69.7323 204.0944 Tm
[(Neural)-493.7(ectoderm,)-489.4(hematopoietic)-498.8(progenitor)-490.4(cell)-490.8(\(meso-)]TJ
-1.0137 -1.1116 TD
[(derm\),)-518.1(and)-511.3(endoderm)-519.2(differentiation)-521.7(were)-514.4(performed)-514.5(ac-)]TJ
0 -1.1174 TD
[(cording)-492.8(to)-491.9(the)-488.4(protocols)-494.9(described)-491.4(in)-491.9(our)-487.1(previous)-493.7(study)]TJ
0 -1.1116 TD
[([18].)-422.5(qRT-PCR)-426.5(and)-419.2(ow)-424.5(cytometry)-422.6(analyses)-426.7(were)-422.3(carried)]TJ
T*
[(out)-337.5(3)-329.4(days)-333.7(after)-336.2(differentiation.)]TJ
/F3 1 Tf
9.9626 0 0 9.9626 59.7543 140.2582 Tm
[(Extrac)-7.6(ellu)-7.8(lar)-336.7(oxygen)-337.2(con)-6.4(sumption)-341.7(assay)]TJ
/F6 1 Tf
9.843 0 0 9.843 69.7323 122.3999 Tm
.013 Tc
[(Ce)-6.5(ll)-5.5(s)-581.2(w)-.5(er)-5.3(e)-576.7(s)-5.2(ee)-6.5(de)-6.5(d)-575.6(o)-5.4(n)-575.6(a)-582.5(9)-5.4(6-w)-6.2(el)-5.5(l)-575.7(p)-5.4(la)-6.5(te.)-583.7(T)-.8(he)-582.5(r)-5.3(e)-.8(la)-6.5(ti)-5.5(ve)]TJ
-1.0137 -1.1116 TD
[(o)-5.5(x).3(i)-5.6(d).3(at)-5.6(iv)-5.5(e)-582.6(p)-5.5(hos)-5.3(ph)-5.5(or)-5.4(yl)-5.6(ati)-5.6(o).3(n)-587.2(\(ba)-6.6(sa)-6.6(l)-581.6(a)-6.6(nd)-581.4(m)-6.7(a)-.9(xi)-5.6(mal)-587.3(re)-6.6(sp)-5.5(ir)-5.4(a-)]TJ
0 -1.1174 TD
.0154 Tc
[(tion\))-515.6(r)-2.9(ate)-516.8(w)1.9(as)-515.4(m)-4.2(easured)-515.6(by)-515.6(uoresc)-4.1(e)1.6(nce)-516.8(i)2.7(nte)-4.1(n)2.8(sity)-515.6(in)-515.6(a)]TJ
0 -1.1116 TD
.0144 Tc
[(uore)-5.1(s)2(ce)-5.1(nc)-5.1(e)-373.8(m).6(ic)-5.1(roplate)-373.8(r)-3.9(ea)-5.1(der)-372.6(\()-84.6(M)-4.9(o)1.8(lecula)-5.1(r)-372.6(D).9(e)-5.1(v)1.8(ic)-5.1(es\))-372.6(w).9(ith)]TJ
T*
.013 Tc
[(t)-5.5(h).4(e)-438.5(M)-.5(it)-5.5(oc)-6.5(hon)-5.4(dr)-5.3(ia)-6.5(l)-431.7(S).5(t)-5.5(r).4(e)-6.5(s).6(s)-437.2(Tes)-5.2(t)-431.7(C)-6.4(omp)-5.4(l).3(e)-6.5(t).3(e)-432.8(A)-6.2(ss)-5.2(ay)-431.6(K)-6.2(i).3(t)-437.5(\().4(A)-6.2(b).4(-)]TJ
T*
.0146 Tc
[(c)-4.9(am\))-562.4(acc)-4.9(ording)-556.7(to)-556.7(the)-557.9(m).8(a)-4.9(n)2(ufa)-4.9(c)]TJ
14.0422 0 TD
.0151 Tc
[(ture)-4.4(rs)-556(p)2.5(rotocols.)-552.8(B)-4.3(asal)]TJ
-14.0422 -1.1174 TD
[(respiration)-584.9(r)-3.1(ate)-586.1(r)-3.1(epre)-4.3(sents)-590.5(t)]TJ
12.3085 0 TD
.0132 Tc
[(he)-593.8(cel)-5.3(lu)-5.2(lar)-592.6(e)-6.3(ner)-5.1(g).6(y)-592.7(r).6(e)-6.3(q).6(u)-5.2(i).5(r)-5.1(e)-.6(-)]TJ
13.2012 44.9199 TD
.0172 Tc
[(ment)4.5(s)-496.3(un)4.6(der)-496.5(b)4.6(as)4.8(al)-496.6(con)4.6(d)4.6(i)-1.3(t)4.5(i)-1.3(o)4.6(n)-1.2(s)4.8(.)-498.8(Maxi)4.5(mal)-496.6(r)4.6(es)4.8(pi)4.4(rat)4.5(i)-1.3(o)4.6(n)-496.5(rate)]TJ
0 -1.1116 TD
.0178 Tc
[(r)5.2(e)-1.7(f)5.2(e)4(rs)-731.9(to)-732.1(t)5.1(h)-.6(e)-733.2(c)4(el)5.1(lu)5.2(lar)-732(m)4(ax)5.2(imu)5.2(m)-733.3(resp)5.2(ir)5.2(at)5.1(i)5.1(o)-.6(n)-732.1(p)-.6(o)5.2(t)5.1(en)5.2(ti)5.1(al)]TJ
0 -1.1174 TD
.0147 Tc
[(treated)-401.1(w)-4.5(ith)-401.1(FCCP.)]TJ
/F3 1 Tf
9.9626 0 0 9.9626 310.8472 454.9605 Tm
0 Tc
[(Derivation)-343.8(and)-335.7(mul)-7.4(tilineag)-9.5(e)-330(dif)-7.4(ferentia)-7.9(tion)]TJ
0 -1.2007 TD
[(of)-333.6(iPSC-)-10.9(MSCs)]TJ
/F6 1 Tf
9.843 0 0 9.843 320.8251 425.14 Tm
[(According)-260.9(to)-267.3(the)-263.8(manufacturers)-268.8(instructions,)-265.6(on)-261.4(day)-263.7(13,)]TJ
-1.0137 -1.1116 TD
[(the)-384.7(TeSR-E8)-386.5(medium)-384.7(was)-385.3(replaced)-387.9(by)-382.3(3)-168.1(m)0(L)-384.8(STEMdiff)]TJ
/F11 1 Tf
23.0734 0 TD
()Tj
/F6 1 Tf
.8812 0 TD
(-)Tj
-23.9546 -1.1116 TD
[(ACF)-356.4(mesenchymal)-359(induction)-351.3(medium)-355.9(\(Stemcell)-360.4(Technolo-)]TJ
0 -1.1174 TD
[(gies\).)-568.7(On)-573.3(day)-574.7(45,)-565.4(the)-574.8(medium)-569.1(was)-575.4(replaced)-572.3(by)-572.4(2)-168.1(m)0(L)]TJ
0 -1.1116 TD
(MesenCult)Tj
/F11 1 Tf
4.3947 0 TD
()Tj
/F6 1 Tf
.887 0 TD
[(-ACF)-472.7(Plus)-470.7(medium)-471.1(\(Stemcell)-475.6(Technologies\).)]TJ
-5.2816 -1.1116 TD
[(On)-636.6(day)-632.3(6,)-638.1(the)-638.2(cells)-635.7(were)-635.4(passag)-8.5(ed)-631.2(and)-638(cultured)-639.2(with)]TJ
0 -1.1174 TD
[(MesenCult-ACF)-504.2(Plus)-499.5(medium.)-503.4(Flow)-500.6(cytometry)-503.3(and)-499.8(total)]TJ
0 -1.1116 TD
[(RNA)-317.1(extraction)-314.4(were)-318.6(performed)-312.9(on)-313.2(day)-315.5(8.)-315.6(On)-314.1(day)-315.5(6)-312.1(t)0(o)-313.3(day)]TJ
T*
[(21,)-374.3(cells)-376.5(were)-376.2(passaged)-374.8(at)]TJ
/F4 1 Tf
11.2545 0 TD
(*)Tj
/F6 1 Tf
.9964 0 TD
[(80%)-373(conuence)-377.3(and)-378.8(cultured)]TJ
-12.2509 -1.1116 TD
[(with)-333.8(MesenCult-ACF)-337.2(Plus)-338.2(medium.)]TJ
1.0137 -1.1174 TD
[(Multilineage)-496.3(differentiation)-498.6(was)-500.5(performed)-497.2(on)-491.8(day)-494(21.)]TJ
-1.0137 -1.1116 TD
[(For)-412(the)-413.5(osteogenic)-412(differentiation)-418(of)-416.9(iPSC-MSCs,)-413.2(the)-413.5(me-)]TJ
T*
[(dium)-276.5(was)-275.9(replaced)-278.5(by)-278.7(DMEM)-279(supplemented)-277.2(with)-282(10%)-275.1(FBS)]TJ
0 -1.1174 TD
[(\(Gibco\),)-255.2(10)-163.5(mM)-252(sodium)]TJ
/F4 1 Tf
9.9009 0 TD
(b)Tj
/F6 1 Tf
.5529 0 TD
[(-glycerophosphate,)-257.2(50)]TJ
/F4 1 Tf
9.0715 0 TD
(m)Tj
/F6 1 Tf
.5529 0 TD
[(M)-255.4(vitamin)]TJ
-20.0783 -1.1116 TD
[(C,)-541.2(and)-545.9(0.1)]TJ
/F4 1 Tf
4.867 0 TD
(m)Tj
/F6 1 Tf
.5529 0 TD
[(M)-543.4(dexamethasone)-551.2(\(Sigma-Aldrich\).)-548.6(Alizarin)]TJ
-5.4199 -1.1116 TD
[(Red)-333.8(staining)-332.7(was)-339.2(performed)-335.9(on)-330.5(day)-338.5(42.)]TJ
1.0137 -1.1174 TD
[(Fo)21(r)-202.7(a)0(d)19.7(i)0(p)15(o)0(g)20.8(e)0(n)19.7(i)0(c)-193.6(d)0(i)14.9(f)0(f)20.9(e)0(r)19.7(e)0(n)19.7(t)0(i)20.6(a)0(t)19.5(i)0(o)15(n)0(,)-194.6(t)0(h)20.7(e)-203.8(me)18.4(di)20.7(um)-199.3(wa)18.8(s)-202.5(r)0(e)13.9(p)0(l)20.7(a)0(c)18.5(e)0(d)-193.4(b)0(y)]TJ
-1.0137 -1.1116 TD
[(DM)19.1(EM)-223.2(su)15.3(pp)20.8(le)19.6(me)18.4(nt)20.7(ed)-228(wi)19.8(th)-227(10)20.8(%)-238.3(F)0(B)20(S)0(,)-223.2(1)0(0)]TJ
/F4 1 Tf
16.4152 0 TD
(m)Tj
/F6 1 Tf
.5472 0 TD
.2324 Tc
[(Mi)232.4(n)253.1(s)232.4(u)247.7(l)232.4(i)253(n)232.4(,)9(2)232.4(0)253.2(0)]TJ
/F4 1 Tf
5.8979 0 TD
0 Tc
(m)Tj
/F6 1 Tf
.5472 0 TD
(M)Tj
-23.4074 -1.1116 TD
[(in)20.7(do)20.8(me)18.4(th)20.7(ac)18.5(in)15(,)-608.2(0)0(.)18.5(5)]TJ
/F4 1 Tf
7.597 0 TD
(m)Tj
/F6 1 Tf
.5414 0 TD
-.0201 Tc
[(M)-632.6(I)-20.1(B)-6(M)-20.1(X)-623.1(\()-20.1(Si)-20.1(gm)-20.1(a-)-20.1(A)-5.9(l)-20.1(dr)-20.1(ic)-20.1(h\))-20.1(,)-623.6(a)-20.1(nd)-631.7(1)]TJ
/F4 1 Tf
14.7218 0 TD
0 Tc
(m)Tj
/F6 1 Tf
.5472 0 TD
(M)Tj
-23.4074 -1.1116 TD
[(de)19.7(xa)19.7(me)18.4(th)20.7(as)19.9(on)15.1(e.)-213(Oi)19.8(l)-225.9(R)0(e)18.6(d)-225.7(O)-226.6(s)0(t)20.9(a)0(i)19.5(n)0(i)20.7(n)0(g)-215.3(w)0(a)18.8(s)-225.5(pe)13.9(rf)20.9(or)20.9(me)18.4(d)-225.7(o)0(n)-215.3(d)0(a)19.7(y)-225.7(42)20.8(.)]TJ
0 -1.1174 TD
[(Fo)21(r)-202.7(c)0(h)19.7(o)0(n)20.8(d)0(r)15.1(o)0(g)20.8(e)0(n)19.7(i)0(c)-193.6(d)0(i)20.7(f)0(f)20.9(e)0(r)19.7(e)0(n)13.9(t)0(i)20.6(a)0(t)19.5(i)0(o)20.7(n)0(,)-194.6(t)0(h)20.7(e)-203.8(me)18.4(di)20.7(um)-193.5(wa)18.8(s)-202.5(r)0(e)19.7(p)0(l)20.7(a)0(c)18.5(e)0(d)-193.4(b)0(y)]TJ
0 -1.1116 TD
[(DM)19.1(EM)-488.1(su)21(pp)20.8(le)19.6(me)18.4(nt)20.7(ed)-487.2(wi)19.8(th)-486.1(10)20.8(%)-497.5(F)0(B)20(S)0(,)-488.2(1)0(0)20.8(0)]TJ
/F4 1 Tf
18.2064 0 TD
(m)Tj
/F6 1 Tf
.5414 0 TD
[(g/)20.7(mL)-488.4(so)21(di)14.9(um)]TJ
-18.7478 -1.1116 TD
[(py)20.8(ru)20.9(va)19.7(te)19.6(,)-274.1(4)0(0)]TJ
/F4 1 Tf
5.0973 0 TD
(m)Tj
/F6 1 Tf
.5414 0 TD
[(g/)20.7(mL)-258.1(pr)15.1(ol)20.7(in)20.7(e)-267.2(\()0(S)15.3(i)0(g)20.7(m)0(a)18.4(-)0(A)20(l)0(d)20.7(r)0(i)15(c)0(h)-256.8(\))0(,)-257.9(5)0(0)]TJ
/F4 1 Tf
13.8866 0 TD
(m)Tj
/F6 1 Tf
.5472 0 TD
[(g/)20.7(mL)-258(vi)20.7(ta)19.5(-)]TJ
-20.0726 -1.1174 TD
[(mi)19.5(n)-611.6(C)0(,)-604.6(1)0(0)-146.2(n)0(g)20.8(/)0(m)19.5(L)-612.8(TG)18.8(F-)]TJ
/F4 1 Tf
10.2465 0 TD
(b)Tj
/F6 1 Tf
.5414 0 TD
.6174 Tc
[(1\()617.4(R)637.2(&)617.4(D)14.1(S)617.4(y)638.4(s)617.4(t)638.3(e)617.4(m)635.9(s)617.4(\))632.7(,)9.2(a)617.4(n)637.1(d0)617.4(.)641.7(1)]TJ
/F4 1 Tf
12.0723 0 TD
0 Tc
(m)Tj
/F6 1 Tf
.5472 0 TD
(M)Tj
-23.4074 -1.1116 TD
[(de)19.7(xa)19.7(me)18.4(th)20.7(as)19.9(on)15.1(e.)-322.5(Al)19.8(ci)19.5(an)-325.9(Bl)19.7(ue)-325.9(st)20.9(ai)19.5(ni)14.9(ng)-319(wa)18.8(s)-335(p)0(e)19.7(r)0(f)15.2(o)0(r)20.9(m)0(e)18.4(d)-335.2(on)-319(da)19.7(y)]TJ
T*
[(42)20.8(.)-464.2(M)0(u)19.9(l)0(t)20.6(i)0(l)20.6(i)0(n)15(e)0(a)18.5(g)0(e)-452.6(d)0(i)20.7(f)0(f)15.1(e)0(r)19.7(e)0(n)19.7(t)0(i)20.6(a)0(t)19.5(i)0(o)15(n)-461.9(ma)18.4(rk)20.9(er)19.7(s,)]TJ
/F7 1 Tf
17.1351 0 TD
[(RU)18.8(NX)18.6(2)]TJ
/F6 1 Tf
3.0642 0 TD
(,)Tj
/F7 1 Tf
.7142 0 TD
[(PP)18.4(AR)18.4(G)]TJ
/F6 1 Tf
3.1275 0 TD
(,)Tj
-24.041 -1.1174 TD
[(an)19.7(d)]TJ
/F7 1 Tf
1.6358 0 TD
[(SO)20(X9)]TJ
/F6 1 Tf
2.2981 0 TD
[(,)-210.8(w)0(e)18.8(r)0(e)-199.2(d)0(e)19.7(t)0(e)19.6(c)0(t)13.8(e)0(d)-199.2(a)0(c)18.5(c)0(o)19.7(r)0(d)15.1(i)0(n)20.7(g)0(l)20.7(y)-208.4(by)-203.8(qR)19.8(T-)19.7(PC)20(R)-215.2(o)0(n)-198(d)0(a)19.7(y)-214.2(42)20.8(.)]TJ
-3.9339 -1.1116 TD
[(Th)19.6(e)-284.5(t)0(i)20.6(m)0(e)18.4(l)0(i)20.6(n)0(e)-274.1(o)0(f)-272.9(t)0(h)20.7(e)-284.5(di)20.7(ff)20.9(er)13.9(en)19.7(ti)20.6(at)19.6(io)20.7(n)-283.3(a)0(n)19.7(d)-283.3(in)20.7(du)20.8(ct)13.8(io)20.7(n)-283.3(p)0(r)20.9(o)0(c)19.7(e)0(s)19.9(s)-283.1(wa)18.8(s)]TJ
T*
[(su)21(mm)18.4(ar)19.7(iz)19.5(ed)-262.5(in)-261.5(Su)21(pp)20.8(le)19.6(me)18.4(nt)14.9(ar)19.7(y)-266(F)0(i)15.1(g)0(.)-258(S)0(1)21(.)]TJ
/F3 1 Tf
9.9626 0 0 9.9626 310.8472 96.3779 Tm
[(RNA-sequ)-12.6(encing)]TJ
/F6 1 Tf
9.843 0 0 9.843 320.8251 78.5763 Tm
[(One)-322.1(microgram)-325.8(total)-318.1(RNA)-322.9(of)-324.7(the)-321.4(control)-325.9(and)]TJ
/F7 1 Tf
18.8918 0 TD
(MFN2)Tj
/F6 1 Tf
2.6149 0 TD
(-KD)Tj
-22.5205 -1.1174 TD
[(iPSCs)-380.5(was)-379.5(collected)-383.6(and)-378.8(processed)-381.6(by)-382.3(the)-379(NEBNext)-381.6(Ultra)]TJ
0 -1.1116 TD
[(RNA)-466.9(Library)-465.1(Prep)-466.1(Kit)-471(for)-464(Illumina)-466.6(\(NEB\))-469.3(to)-463.1(generate)-468.6(a)]TJ
ET
59.754 719.49 490.167 .22668 re
f
59.754 532.176 490.167 .22681 re
f
59.754 700.384 490.167 .22681 re
f
BT
/F10 1 Tf
9.845 0 0 9.845 101.9338 725.1588 Tm
(Table)Tj
/F6 1 Tf
3.0002 0 TD
(1.)Tj
/F10 1 Tf
1.2496 0 TD
[(Primer)-335.2(Sequences)-333.2(Used)-332.7(for)-332.2(Quantitative)-337.3(Real-Time)-334.5(Polymerase)-338.2(Chain)-330.8(Reaction)]TJ
/F7 1 Tf
-8.5342 -1.8715 TD
[(Gene)-13182.7(Forward)-331.2(primer)-336(\(5)]TJ
/F11 1 Tf
23.063 0 TD
()Tj
/F7 1 Tf
.2476 0 TD
(3)Tj
/F11 1 Tf
1.002 0 TD
()Tj
/F7 1 Tf
.2476 0 TD
[(\))-12537.4(Reverse)-340.7(primer)-336(\(5)]TJ
/F11 1 Tf
20.2643 0 TD
()Tj
/F7 1 Tf
.2534 0 TD
(3)Tj
/F11 1 Tf
.9962 0 TD
()Tj
/F7 1 Tf
.2534 0 TD
(\))Tj
-46.3275 -1.9118 TD
(MFN2)Tj
/F6 1 Tf
12.3003 0 TD
[(CACAAGGTGAGTGAGCGTCT)-8267.2(CGTTGAGCACCTCCTTAGCA)]TJ
/F7 1 Tf
-12.3003 -1.0077 TD
(PAX6)Tj
/F6 1 Tf
12.3003 0 TD
[(CGAGACTGGCTCCATCAGAC)-8319.2(CTTTTCGCTAGCCAGGTTGC)]TJ
/F7 1 Tf
-12.3003 -1.0135 TD
(SOX1)Tj
/F6 1 Tf
12.3003 0 TD
[(CAACCAGGACCGGGTCAAAC)-8111.2(CCTCGGACATGACCTTCCACT)]TJ
/F7 1 Tf
-12.3003 -1.0135 TD
(T)Tj
/F6 1 Tf
12.3003 0 TD
[(TATGAGCCTCGAATCCACATAGT)-6425.2(CCTCGTTCTGATAAGCAGTCAC)]TJ
/F7 1 Tf
-12.3003 -1.0135 TD
(MESP1)Tj
/F6 1 Tf
12.3003 0 TD
[(TCGAAGTGGTTCCTTGG)-10577.3(TGCTTGCCTCAAAGTGTC)]TJ
/F7 1 Tf
-12.3003 -1.0077 TD
(SOX17)Tj
/F6 1 Tf
12.3003 0 TD
[(CCTTCACGTGTACTACGGCG)-8527.2(GTTCAAATTCCGTGCGGTCC)]TJ
/F7 1 Tf
-12.3003 -1.0135 TD
(GATA4)Tj
/F6 1 Tf
12.3003 0 TD
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T*
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T*
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T*
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[(\(Fig.)-382.2(2B\))-378.7(and)-378.8(CD34-)]TJ
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ET
q
1 i
191.622 732.019 m
552.189 732.019 l
552.189 428.088 l
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W n
q
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Q
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[(-OE)-265.4(i)-15.6(P)11.2(S).9(Cs)-15.6(.)]TJ
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[(KD)-15.5(,)]TJ
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[(kno)-16.3(c)9.3(kdow)-16.3(n)9.7(.)]TJ
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(-OE,)Tj
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