җॊޑኧᏵᡉҢǴ༸ಒझҗ BP ೀࡕǴёаࢲϯ Jak2-Stat3 signaling pathwaysǶࣁΑ൨פ BP ࢂၸবঁ cytokineǴԶࢲϯ Jak2-Stat3 signaling pathwaysǴךॺճҔ real time PCR ٰϩፓ Jak2-Stat3 signaling
pathways ޑෞ cytokine ϐ୷Ӣ߄ໆǶკΜѤᡉҢஒ ES cells җ 5g/mlǵ10g/mlǵ20g/mlǵ40g/ml ޑ BP ೀբҔ 72 λਔࡕǴୀෳ LIF, EGF, IL5, IL11, EPO ک OSM ୷Ӣޑ߄ໆǶว ES cells җ 10g/ml ޑ BPೀբҔ 72 λਔࡕǴLIF, EGF, IL5, IL11, EPO ک OSM ୷Ӣޑ߄ໆᆶ
܄ჹྣಔ LIF OUT ࣬КዴჴԖᡉޑቚуǶ 3.9 BPჹ iPS cells ғԋϐቹៜ
җаޑኧᏵᡉҢǴBP ёаቚу Oct4 ک Sox2 ୷Ӣ߄ໆǴԶЪёа ᆢ༸ಒझޑԾך׳ཥૈΚکӭૈ܄ǶӢԜǴךॺճҔ MEF cells ᙯࢉ Oct4, Sox2, c-Myc, Klf4Ѥঁ୷ӢǴӕਔуΕ BP ၂კቚуౢғ iPS cells ޑਏǶ ךॺ٬ҔޑԴႵ MEF cells ࢂٰԾ Oct4-GFP ୷ӢᙯႵǴ܌аךॺΨᙖҗ ᢀჸ Oct4-GFP ޑ߄ໆٰჴǶკΜϖᡉҢ MEF cells 20g/ml BP ೀ
ࡕǴOct4-GFP ޑ߄ໆዴჴК܄ჹྣಔቚуӭǴԶ BP(T+P) (seeding ࡕុу BP)ѝԖӧಃϤϺ Oct4-GFP ޑ߄ໆК BP(T) (seeding ࡕόុ
у BP)ᡉǴ೭ΨཀښǴӧᙯࢉޑၸำύуΕ BP ೀቚу iPS cells ғ ԋޑਏǶ
ಃѤക ፕ
༸ಒझёаՉԾך׳ཥکϩϯԋӭᅿಒझᜪࠠǴ٠ЪᔈҔܭӭᅿᖏ
ݯᕍ(55)ǶךॺૈՉᡏѦᎦޑ܌Ԗ༸ಒझύǴES cells ڀԖӭૈ܄کϩ ϯԋΟঁЬाखቫಒझޑ܄Ǵ೭٤ख़ाޑ܄ёаౢғ଼நޑಒझҔܭ ᖏݯᕍǶӧ 2006 ԃǴВҁޑ Takahashi ი໗ᙖҗᙯࢉ Oct4, Sox2, c-Myc ک Klf4 ೭Ѥঁ୷ӢΕᡏಒझౢғ iPS cells (8)ǴԜ iPS cells ёаլܺ ES cells ܌य़ᖏޑٿঁख़ाϐୢᚒǺխࣝ௨ѾϸᔈکᙴᕍၰቺǴӧ҂ٰѬԋ ࣁߚதख़ाޑಒझݯᕍϐמೌǶՠࢂǴES ک iPS cells ޑᎦѸᙖҗ LIFٰࢲϯ Jak-Stat pathwayǴԶᆢځӭૈ܄(39)ǶԜѦǴiPS cells ғԋਏ
ߚதեࢂҞ iPS cells מೌޑനεज़ڋǶӧҁࣴزύǴךॺวঁપ
Ψࢂ BP ٬Ҕܭ༸ಒझޑ२ԛࣴزǶ
ٰᎦ ES celltǴว BP Ψёаᡣ Oct4, Sox2, c-Myc ک Klf4 ୷Ӣ߄ܴ
ᡉቚу(კϤ)Ƕ٠Ъ٬Ҕ BP ٰᎦ ES ک iPS cellsǴךॺҔΑ൳ঁ༸ಒझ
Ǵх֖ AP ࢉՅǵNanog ک SSEA1Ǵٰዴۓ BP ёаڗж LIF ٰᎦ ES ک iPS cellsǴЀځࢂ 10 ک 20 g/ml (კΎǵΖǵΐǵΜ)Ǵ೭ঁኧᏵᆶ Oct4 ک Sox2 ୷Ӣ߄ޑኧᏵठǶԜѦǴךॺճҔ 10 g/ml ޑ BP ڗж LIF ٰ
Ꭶ ES ک iPS cellsǴၸΟжᎦࡕǴճҔ embryoid body (EB)ԋ೭ঁ
מೌ(3, 8)Ǵٰෳ၂༸ಒझޑӭૈ܄Ǵ೭ঁኧᏵࡰрǴBP ёаڗж LIF ٰ
Ꭶ ES ک iPS cellsǶ
ࣁΑࣴز BP ೀࡕځբҔޑᐒڋǴךॺճҔ microarray ϩפр BP Ьाፓޑ৩ǶΟӜޑ৩ǺPPAR signaling pathwayǵ ECM-receptor interaction ک Jak-Stat signaling pathway (߄Β)Ƕӧ 2010 ԃǴMo et al.ࣴزᡉ Ң PPAR ፓ LIF ၸ Tyk2-Stat3 ৩ǴᇨᏤԴႵޑखज༸ಒझғߏᆶԾ ך׳ཥ(59)ǶךॺୀෳΑ ES cells BP ೀࡕ PPAR ޑ୷Ӣ߄Ǵว
PPAR ୷Ӣ߄ᆶڋಔ࣬К٠ؒԖৡ౦ǴਥᏵ೭ঁচӢǴךॺஒख़ܭ Jak-Stat signaling pathway Ƕ ך ॺ ዴ ᇡ ༸ ಒ झ BP ೀ ࡕ Ǵ phosphorylation-Jak2 ک phosphorylation-Stat3 ޑೈқ፦߄ቚу(კΜΟ)Ƕ LIFޑૻ৲ၸ Jak ࢲϯΑ࣬ᜢޑૻ৲ሀǴ٠Ъࢲϯ Stat3 (39)Ǵჹܭ ES cells ޑԾך׳ཥૈΚ LIF ࢂתᄽঁόё܈લޑفՅ(31)ǶךॺޑኧᏵࡰ
рǴ BP ೀёаࢲϯ Jak2 ک Stat3 ٬ ES cells ᆢԾך׳ཥૈΚǶԜѦǴ
Jak-Stat signaling pathwayёаၸ LIF, EGF, EPO, IL5, IL11 ک OSM (17-21) ύǴԖঁλϩη[valproic acid (VPA)]ёаҔٰගଯ iPS cells ޑғԋਏ
(10)ǶVPA ٠όቚу Oct4 ک Sox2 ޑ୷Ӣ߄ǴՠࢂѬቚуੰࢥ൪Ε
ךॺዴۓΑ BP ၸፓୖᆶܭ Jak2-Stat3 signaling pathways ύޑಒझ ᐟનǴቚу Jak2 ک Stat3 ޑೈқ፦߄Ǵᡣ༸ಒझᆢӭૈ܄کԾך׳ཥ
ૈΚǹԜѦǴBP ޑೀࡕёаቚу iPS cells ғԋޑਏǶஒٰ BP ёаว
ԋࣁ଼ந१ࠔǴҔٰࢲϯ༸ಒझǴԶЪӧ҂ٰ BP ёаҔܭׯ๓ iPS cell ғԋϐਏǶ
ୖԵЎ
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კ߄
߄ǵջਔۓໆᆫӝ䁙ೱᙹϸᔈׇӈំ߄
Primer Forward SequenceǺ(5’-3’) Reverse SequenceǺ(5’-3’) Taqman Probe
Sox2 AGGGCTGGACTGCGAACTG TTTGCACCCCTCCCAATTC c-Myc CATTCAAGCAGACGAGCA CGAGTTAGGTCAGTTTATGCAC
Klf4 CCTTTCAGTGCCAGAAGT ACTACGTGGGATTTAAAAGTGC Jak2 CAATGATAAACAAGGGCAAATGAT CTTGGCAATCTTCCGTTGCT Stat3 CCCCGTACCTGAAGACCAAGT CCGTTATTTCCAAACTGCATCA
EGF GAGTCTGCCTGCGGATGGT GCTGCAGGGAGGGAGACA EPO CCCCCACGCCTCATCTG TGCCTCCTTGGCCTCTAAGA
IL5 TCCCTGCTACTCTCCCCAAA CAACCTTCTCTCTCCCCAAGAA IL11 CATGCCACACCCCAAACAA CCCCTCACCCAGGTCTACTG LIF CCTACCTGCGTCTTACTCCATCA TGTTTTCCCCAAAGGCTCAA OSM CGGTCCACTACAACACCAGATG GCGATGGTATCCCCAGAGAA
͊-Actin GTGCGTGACATCAAAGAGAAGC TGGATGCCACAGGATTCCATAC
Taqman-Oct4 GAGGCTACAGGGACACCTTTC GTGCCAAAGTGGGGACCT Roche Universal Probes 6 Taqman-͊-Actin CTAAGGCCAACCGTGAAAAG ACCAGAGGCATACAGGGACA Roche Universal Probes 64
߄ΒǵճҔ KEGG ک Babelomics ၗϩᡉ߄ޑ୷ӢኧᏵᆶғނф
ૈϩᜪǶ
I: number of upregulated genes; D: number of downregulated genes.
კǵLIF ၸࢲϯ Jak/Stat3 signaling pathwaysǴፓ༸ಒझޑғނࢲ܄Ǵ კᘏڗԾЎക(62)Ƕ
კΒǵn-Butylidenephthalide ޑϯᏢ่ᄬ
კΟǵճҔ real-time PCR ୀෳ MEF cells ϩձೀ 18 ᅿόӕᐚࡋޑύᛰ բҔ 24 λਔࡕǴOct4 ک Sox2 ୷Ӣ߄ໆǶ
კѤǵճҔ MTT assay ϩ BP ჹ MEF cells ғߏૈΚޑቹៜǶMEF cells
ೀόӕᐚࡋޑ BP ϩձբҔ 24 ک 72 λਔǶӚಔ࣬ჹ֎Ӏॶޑѳ֡ॶᆶ
ྗৡǶ*P 0.05 versus control (DMSO*Ƕ
კϖǵճҔ real-time PCR ୀෳ MEF cells ೀόӕᐚࡋ BP բҔ 24 λਔࡕǴ Oct4, Sox2, c-Myc, Klf4୷Ӣ߄ໆǶ*P 0.05 versus control (DMSO*Ƕ!
კϤǵճҔ real-time PCR ୀෳ ES cells ೀόӕᐚࡋ BP բҔ 72 λਔࡕǴ Oct4, Sox2, c-Myc, Klf4୷Ӣ߄ໆǶ*P 0.05 versus control (LIF OUT).
(A) (B)
(C)
კΎǵ(A) ES cells ೀόӕᐚࡋ BP բҔ 72 λਔࡕǴճҔ alkaline phosphatase stainingϩ९ᕒკǶ(B) Οԛᐱҥჴᡍޑѳ֡ॶr SD Ƕ*P 0.05 versus LIF OUT. (C) ES cellsೀόӕᐚࡋޑBP բҔ72 λਔࡕǴalkaline phosphatase stainingܭ 40 ७ᡉ༾᜔კǶ
LIF OUT LIF BP 5
BP 40 BP 20
BP 10
(A) (B)
(C)
კΖǵ(A) iPS cells ೀόӕᐚࡋ BP բҔ 72 λਔࡕǴճҔ alkaline phosphatase stainingϩ९ᕒკǶ(B) Οԛᐱҥჴᡍޑѳ֡ॶr SDǶ*P 0.05 versus LIF OUT. (C) iPS cellsೀόӕᐚࡋޑ BP բҔ 72 λਔࡕǴalkaline phosphatase stainingܭ 40 ७ᡉ༾᜔კǶ
(A)
LIF OUT LIF BP 5 BP 10 BP 20 BP 40
Merge Merge Merge Merge Merge Merge
Nanog
DAPI DAPI DAPI DAPI DAPI DAPI
Nanog Nanog Nanog Nanog Nanog
100ȫm 100ȫm 100ȫm 100ȫm 100ȫm 100ȫm
100ȫm 100ȫm 100ȫm 100ȫm 100ȫm 100ȫm
100ȫm 100ȫm 100ȫm 100ȫm 100ȫm 100ȫm
(B)
LIF OUT LIF BP 5 BP 10 BP 20 BP 40
Merge Merge Merge Merge Merge Merge
SSEA1 SSEA1 SSEA1 SSEA1 SSEA1 SSEA1
DAPI DAPI DAPI DAPI DAPI DAPI
100ȫm 100ȫm 100ȫm 100ȫm 100ȫm 100ȫm
100ȫm 100ȫm 100ȫm 100ȫm 100ȫm 100ȫm
100ȫm 100ȫm 100ȫm 100ȫm 100ȫm 100ȫm
კΐǵ(A) ES cells ೀόӕᐚࡋ BP բҔ 72 λਔࡕǴӧᑻӀᡉ༾᜔Πᢀჸ Nanog-GFPޑ߄ໆǶ(B) ES cells ೀόӕᐚࡋ BP բҔ 72 λਔࡕǴӧᑻ Ӏᡉ༾᜔Πᢀჸ SSEA1 ޑ߄ໆǶ
(A)
LIF out LIF BP5
BP10 BP20 BP40
100ȫm 100ȫm 100ȫm
100ȫm 100ȫm 100ȫm
(B)
LIF OUT LIF BP 5 BP 10 BP 20 BP 40
Merge Merge Merge Merge Merge Merge
SSEA1 SSEA1 SSEA1 SSEA1 SSEA1 SSEA1
DAPI DAPI DAPI DAPI DAPI DAPI
100ȫm 100ȫm 100ȫm 100ȫm 100ȫm 100ȫm
100ȫm 100ȫm 100ȫm 100ȫm 100ȫm 100ȫm
100ȫm 100ȫm 100ȫm 100ȫm 100ȫm 100ȫm
კΜǵ(A) iPS cells ೀόӕᐚࡋ BP բҔ 72 λਔࡕǴӧᑻӀᡉ༾᜔Πᢀჸ Nanog-GFPޑ߄ໆǶ(B) iPS cells ೀόӕᐚࡋ BP բҔ 72 λਔࡕǴӧᑻ Ӏᡉ༾᜔Πᢀჸ SSEA1 ޑ߄ໆǶ
Ectoderm
DAPI
Merge Geta-4
Endoderm
Merge Ƞ-SMA
DAPI
Mesoderm
Merge Tuj-1 DAPI
50ȫm 50ȫm 50ȫm
50ȫm 50ȫm 50ȫm
50ȫm 50ȫm 50ȫm
კΜǵճҔ BP ڗж LIF Ꭶ ES cells Οжϐࡕ٬ϐԋ EB ϩϯғԋΟ खቫಒझǶ
კΜΒǵճҔ real-time PCR ୀෳ ES cells ೀόӕᐚࡋ BP բҔ 72 λਔࡕǴ Jak2ک Stat3 ୷Ӣ߄ໆǶ*P 0.05 versus LIF OUT.
(A)
(B)
კΜΟǵ(A) ES cells ೀόӕᐚࡋ BP բҔ 72 λਔࡕǴճҔ western blot ϩ Jak2 ک phosphorylation-Jak2 ೈқ፦߄ໆǶ(B) ES cells ೀόӕᐚࡋ BPբҔ 72 λਔࡕǴճҔ western blot ϩ Stat3 ک phosphorylation-Stat3 ೈ қ፦߄ໆǶ
კΜѤǵճҔ real-time PCR ୀෳ ES cells ೀόӕᐚࡋ BP բҔ 72 λਔࡕǴ LIF, EGF, IL5, IL11, EPO, OSM୷Ӣ߄ໆǶ*P 0.05 versus LIF OUT.
0 positive clonesीբკǶControlǺᙯ Oct4, Sox2, c-Myc, Klf4ǹBP (T)Ǻ ᙯࢉ Oct4, Sox2, c-Myc, Klf4 ӧᙯࢉၸำύу BPǴϐࡕᎦ߾όуǹBP (T+P)Ǻᙯࢉ Oct4, Sox2, c-Myc, Klf4 ӧᙯࢉၸำکϐࡕᎦࣣុу BPǶ