中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/41365

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(1)ύ ୯ ᙴ ᛰ ε Ꮲ ୷ᘵᙴᏢࣴ‫܌ز‬ ᅺγᏢՏፕЎ ճҔલ਼‫ރ‬ᄊғԋคੰࢥགࢉϷठᕎ୷Ӣ‫ޑ‬ԴႵ ᇨᏤ‫܄‬ӭфૈ༸ಒझ Mouse-induced pluripotent stem cells generated under hypoxic conditions in the absence of viral infection and oncogenic factors. ࡰᏤ௲௤Ǻቅ၃ѳ ௲௤. ࣴ ‫ ز‬ғǺ೚ίએ. ύ๮҇୯΋ԃΎД.

(2) . ύЎᄔाǺ 2006 ԃǴВҁ‫ޑ‬΋ঁი໗ճҔϸᙯᒵੰࢥஒ Oct4, Sox2, c-Myc, Klf4 ೭ ѤঁᙯᒵӢηଌΕԋᡏಒझǴᇙբрᇨᏤ‫܄‬ӭфૈ༸ಒझǶᇨᏤ‫܄‬ӭф ૈ༸ಒझคፕӧӚБ‫ݤ‬೿‫ک‬खज༸ಒझΜϩ࣬՟ǴӢԜёаҔٰլܺख ज༸ಒझӧ଺ᖏ‫ݯ׉‬ᕍ΢‫܌‬ၶ‫֚ډ‬ᜤǶջ٬ӵԜǴᇨᏤ‫܄‬ӭфૈ༸ಒझ ϝԖԿϿ‫ঁٿ‬ሡाլܺ‫֚ޑ‬ᜤǺ1.٬Ҕੰࢥၩᡏஒ୷ӢකΕԋᡏಒझ‫ޑ‬ ࢉՅᡏύ 2.٬ҔΑठᕎ୷Ӣ c-Myc, Klf4Ƕӧ 2008 ԃǴOkita ฻ΓൔᏤ дॺёаӧό٬Ҕੰࢥၩᡏ‫ޑ‬௃‫ݩ‬Πԋф‫ޑ‬ғԋԴႵ‫ޑ‬ᇨᏤ‫܄‬ӭфૈ ༸ಒझǴՠࢂдॺӧ೭ঁჴᡍϝฅх֖‫ঁٿ‬ठᕎ୷ӢǺc-Myc, Klf4Ƕҁ ჴᡍύ‫ޑॺך‬ଷᇥࣁǺѝ٬Ҕ‫ঁٿ‬ᙯᒵӢηǺOct4, Sox2 ่ӝલ਼ᕉნǴ ᔈёаԋф‫ޑ‬ғԋᇨᏤ‫܄‬ӭфૈ༸ಒझǶ‫ॺך‬ճҔ፦ᡏᙯࢉख़ፄஒ‫ঁٿ‬ ߄౜፦ᡏȐϩձх֖Α Oct4, Sox2 ୷Ӣȑ໺ΕԴႵखजᠼᆢ҆ಒझ྽ύ ғԋᇨᏤ‫܄‬ӭфૈ༸ಒझǴԶؒԖ٬ҔੰࢥၩᡏǶᙯࢉϤλਔϐࡕǴᡣ ಒझೀ‫ ܭ‬3%લ਼ᕉნΠΒΜѤλਔǶ೭ঁ୏բᕴӅख़ፄΑѤԛǶӧಃ ΐϺஒԴႵखजᠼᆢ҆ಒझ୻Ꭶ‫ډ‬෷Ꭶቫಒझ΢ǴӧಃΒΜ΋Ϻਔ‫ॺך‬ ว౜ΑᇨᏤ‫܄‬ӭфૈ༸ಒझǶਥᏵಒझ‫׎ޑ‬ᄊǵᡵ‫܄‬ᕗለ䁙ࢉՅǵखज ౚჴᡍ‫ک‬྾जዦ‫׎‬ԋ฻ჴᡍǴ‫ॺך‬᛾ჴΑԜᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ڀ‬Ԗӭ ૈ‫܄‬Ǵ‫ک‬खज༸ಒझ࣬ᜪ՟ǶԜ΋Б‫ݤ‬ό໻ёаѐନੰࢥགࢉ‫ޑ‬ᅪቾǴ Ψ҂٬Ҕठᕎ୷ӢǴӢԜჹ҂ٰᔈҔ‫ܭ‬ᖏ‫׉‬΢Ԗ࣬྽ε‫ޑ‬ᔅշǶ . I.

(3) . Abstract Induced pluripotent stem (iPS) cells from somatic cells by introducting four transcriptional factors: Oct4, Sox2, c-Myc and Klf4 were reported in 2006. The morphology, proliferation, surface antigens, gene expression, telomerase activity, and epigenetic status of pluripotent cell-specific genes from the iPS cells were similar to those from embryonic stem (ES) cells. This novel technology has potential to overcome hurdles associated with ES cells due to their generation from mature somatic cells. However, at least two hurdles remain: integrating viral transgenes into the somatic genome, and introducing oncogenes: c-Myc and Klf4. In 2008, Okita et al. successfully generated mouse iPS cells without viral vectors, but they used four factors containing the oncogenes, c-Myc and Klf4. We tested our hypothesis that iPS cells can be generated by introducing only Oct4 and Sox2 under hypoxic conditions. We observed that the repeated transfection of two expression plasmids (one containing Oct4 and the other Sox2 cDNA) into mouse embryonic fibroblasts and combined hypoxic condition resulted in iPS cells generation. Six hours post-transfection, MEF cells were subjected to hypoxic conditions for 24 h (3% O2); this procedure was repeated four times. The MEF cells were passaged on feeder cells on day 9; iPS cell clones were observed 12 days post-passaging and designated as iPS-OSH cells. The morphology, positive alkaline phosphatase staining, embryonic body and teratoma formation indicated that the iPS-OSH cells had pluripotent capability similar to ES cells. This strategy avoids oncogenic factors and virus integration, thus decreasing the risk of cancer formation. ! . II.

(4) . ठᖴ ስ୉޸໒Ǵᙜሳख़ख़ǴΞ‫ډ‬Α೭ձᚆ‫ۑޑ‬࿯Ƕӧύ୯ᙴ‫ۺ‬ᅺγ੤‫ޑ‬ ೭‫ٿ‬ԃ္Ǵමၶ‫ډ‬Αᕴᕴ֚ᜤǴ‫۩܌‬Ԗգॺᡣ΋Ϫૈ໩ճǶ २Ӄाགᖴ‫ޑך‬Р҆ᒃǴЍ࡭‫ۺך‬ᅺγ੤Ǵᡣ‫ך‬ӧຳຳናና‫ޑ‬ғ‫ڮ‬ ਓ೼ύόԿ‫ܭ‬଎ைǶ‫׳‬གᖴாॺό៝΋Ϫ‫ޑ‬брᆶႴᓰǴᡣ‫ך‬ӧեပਔ Ψૈ߿ඪय़ჹࡷᏯǶ೭‫ٿ‬ԃύǴനाགᖴ‫ࡰޑך‬Ꮴ௲௤ɡቅ၃ѳԴৣǶ ᖴᖴாֶֶό॰‫ޑ‬௲ᏤǵΨགᖴாૈҭৣҭ϶‫ޑ‬ᡣ‫ך‬ӧ೭‫ٿ‬ԃ္ёаӧ ᇸ᚞ඍ‫ݗ਻ޑז‬ύᏢಞ‫ډ‬೚ӭ‫ޕ‬᛽аϷჴᡍ‫מ‬ѯǶ྽ฅǴΨाགᖴӧ೭ ‫ٿ‬ԃύഉՔ‫ࡋך‬ၸ‫ޑ‬ӕᏢॺǴᖴᖴգॺ‫ךک‬΋ଆոΚǵ΋ଆ៿ઢǶᗋԖ Ꮲ‫ॺۆ‬Ǵᖴᖴ‫ॺی‬ค‫ޑد‬௲ᏤǴᡣ‫ך‬Ꮲ཮೚ӭჴᡍ΢‫מޑ‬ѯǶനࡕǴा ᖴᖴᏢ‫ۂ׌‬ӧჴᡍ΢‫ޑ‬ᔅԆǴᖴᖴգॺᔅԆΑ‫ך‬೚ӭ٣ǶᗋԖ‫ي‬ᜐ‫ޑ‬೚ ӭΓǴᖴᖴգॺ‫ޑ‬ᔅշϷഉՔǶ ࣬ᆫ‫ޑ‬ਔӀᕴࢂццǴӧ೭‫ٿ‬ԃ္Ǵࡐ໒ЈǵΨࡐ۩ၮૈၶ‫ـ‬գॺǴ ࣁ‫ޑך‬Γғ‫׳‬బᝨ઱Ƕ . III.

(5) . Ҟᒵ! ύЎᄔाǺ ..................................................................................................... I Abstract ......................................................................................................... II ठᖴ ...............................................................................................................III Ҟᒵ ...............................................................................................................IV ಃ΋കǵᙁϟ .................................................................................................1 ಃ΋࿯ǵࣴ‫ز‬ङඳ ..................................................................................1 1.1.1 खज༸ಒझ (Embryonic Stem Cells, ES cells) ...................1 1.1.2 ᇨᏤ‫܄‬ӭфૈ༸ಒझ (Induced Pluripotent Stem Cells, iPS) ....................................................................................................2 1.1.3 લ਼ᕉნ (Hypoxia) ..............................................................3 1.1.4 Oct4 ..........................................................................................4 1.1.5 Sox2 ..........................................................................................5 ಃΒ࿯ǵჴᡍҞ‫ ޑ‬..................................................................................6 ಃΒകǵ‫׷‬਑ᆶБ‫ ݤ‬.....................................................................................7 2.1 ፦ᡏᄬᑐ Construction of the plasmid ...........................................7 2.2 ԴႵखजᠼᆢ҆ಒझϐ୻Ꭶ Mouse embryonic fibroblast cells (MEF) culture..........................................................................................7 2.2.1 ᇨᏤ‫܄‬ӭфૈ༸ಒझ‫୻ޑ‬Ꭶ Induced pluripotent stem cells (iPS) culture ......................................................................................8 2.3 ՋБᏀᗺ‫ ݤ‬Western Blot...............................................................9 2.4 લ਼ᕉნ Hypoxia.........................................................................10 . IV.

(6) . 2.5 ջਔᆫӝ䁙᜘ᙹϸᔈ Real-time PCR .........................................10 2.6 ፦ᡏᙯࢉ Transfection..................................................................11 2.7 ᡵ‫܄‬ᕗለ䁙ࢉՅ Alkaline phosphatase staining........................11 2.8 խࣝᑻӀࢉՅ-༸ಒझ኱૶ Immunofluorescent antibody assay-stem cell marker .........................................................................12 2.9 ᆫӝ䁙᜘ᙹϸᔈ PCR ...................................................................12 2.10 खजౚ‫׎ޑ‬ԋ‫ک‬ϩϯ Embryonic bodies formation and differentiation........................................................................................13 2.10.1 խࣝᑻӀࢉՅ-Οखቫ Immunofluorescent antibody assay-three germ layers .................................................................14 2.11 ྾जዦ‫׎ޑ‬ԋ Teratoma formation............................................15 2.12 ༾ତӈϩ‫ ݋‬Microarray analysis...............................................15 2.13 ಍ीϩ‫ ݋‬........................................................................................15 2.14 ճҔᇨᏤ‫܄‬ӭфૈ༸ಒझғԋઓ࿶༸ಒझ Generation of neural stem cells (NSC) from iPS-OSH ..............................................16 ಃΟകǵ่݀ ...............................................................................................18 ಃ΋࿯ǵ࿶җલ਼ᕉნೀ౛ၸࡕखजᠼᆢ҆ಒझ‫ ޑ‬Oct4, Sox2, c-Myc, Klf4 ೈқ‫୷ک‬Ӣ߄౜ ...............................................................18 ಃΒ࿯ǵճҔ Oct4, Sox2 ፦ᡏӧલ਼ᕉნΠғԋᇨᏤ‫܄‬ӭфૈ༸ಒ झ.............................................................................................................19 ಃΟ࿯ǵiPS-OSH ಒझύ༸ಒझ੝౦୷Ӣ‫߄ޑ‬౜ ...........................20 . V.

(7) . ಃѤ࿯ǵiPS-OSH ಒझӧᡏѦǵᡏϣ‫ޑ‬ӭૈ‫ ܄‬...............................21 ಃϖ࿯ǵճҔ Microarray ϩ‫ ݋‬iPS-OSH ‫ޑ‬ӄ୷Ӣ߄౜ ................22 ಃϤ࿯ǵ ճҔᇨᏤ‫܄‬ӭфૈ༸ಒझғԋઓ࿶༸ಒझ Generation of neural stem cells (NSC) from iPS-OSH ..............................................23 ಃѤകǵ૸ፕ ...............................................................................................24 ୖԵЎ᝘Ǻ ...................................................................................................26 კ߄ ...............................................................................................................30. . VI.

(8) . ಃ΋കǵᙁϟ ಃ΋࿯ǵࣴ‫ز‬ङඳ 1.1.1 खज༸ಒझ (Embryonic Stem Cells, ES cells) खज༸ಒझྍԾ៶ख‫ޑ‬ϣ೽ಒझ(inner cell mass, ICM) (1, 2)Ǵӧ୏‫ނ‬ ᡏύǴखज༸ಒझёа҅தϩϯԋΟखቫಒझ຾ԶғԋಔᙃǵᏔ‫(۔‬3-5)Ƕ ‫ॺך‬ஒԜૈΚᆀϐࣁӭૈ‫(܄‬Pluripotency)Ƕखज༸ಒझќ΋໨ૈΚࢂԾ ‫׳ך‬ཥ(self-renew)Ƕ΋૓‫ޑ‬ԋᡏಒझѝૈϩϯԋ‫ک‬Ծ‫࣬ي‬ӕ‫ޑ‬ಒझǴന ಖ‫و‬ӛ঒ΫǶՠࢂჹ‫ܭ‬खज༸ಒझԶ‫ق‬ǴѬ‫ڀ‬Ԗ࡭ុߥ࡭Ծᡏҁ‫ي‬ಒझ ό‫و‬ӛϩϯ‫ૈޑ‬ΚǴΨ‫ڀ‬ԖϩϯԋΟঁखቫӚᅿಒझ‫ૈޑ‬ΚǶ୷‫ܭ‬खज ༸ಒझ‫ޑ‬೭‫ૈঁٿ‬ΚǴᡣࣴ‫ز‬Ꮲ‫ॺޣ‬ᑈཱུ‫زࣴޑ‬ѬǴགྷᙖҗखज༸ಒझ ϩϯԋӚᅿಒझ‫ૈޑ‬Κٰှ،Ҟ߻ᙴᏢค‫ݯݤ‬ᘰ‫ੱੰޑ‬Ƕ‫ٯ‬ӵǺᑗֿ ੰǵ‫ߎۦ‬හМੱǵύ॥฻Ƕՠࢂखज༸ಒझࠅϝԖค‫ँݤ‬ઇ‫֚ޑ‬ᜤᗺǶ Γᜪ‫ޑ‬खज༸ಒझεӭٰԾ‫ܭ‬Γॺ଺ᡏѦΓπ‫ڙ‬ᆒਔǴӭᎩ‫ڙޑ‬ᆒռǶ ӵ݀ाҔ೭٤‫ڙ‬ᆒռٰ୻Ꭶрखज༸ಒझǴಃ΋ǵԖၰቺউ౛΢‫ޑ‬ୢ ᚒǴಃΒǵ೭٤खज༸ಒझჹ‫ੰܭ‬ΓԶ‫ࢂق‬Ѧٰ‫ނ‬ǴஒѬ෌Εᡏϣ‫ޑ‬၉ ཮Ԗխࣝ௨Ѿ‫ޑ‬౜ຝǶӢԜǴᗨฅखज༸ಒझჹ‫ܭ‬ᙴᏢᗨฅёૈ཮ԖΜ ϩख़ε‫ޑ‬ᔅշǴՠࢂࠅӢࣁа΢‫֚ޑ‬ᜤᗺԶค‫ݤ‬Ԗ‫׳‬຾΋‫ޑ؁‬ჴ፦ᔈ ҔǶ. . . 1.

(9) . 1.1.2 ᇨᏤ‫܄‬ӭфૈ༸ಒझ (Induced Pluripotent Stem Cells, iPS) εऊӧΜԃа߻ǴWilmut ฻Γว౜Αԋᡏಒझёаᙖҗԋᡏಒझਡ ᙯ෗(somatic cell nuclear transfer, SCNT)೭໨‫ೌמ‬ᡂӣ҂ϩϯ‫ޑ‬खजਔ යಒझ(6)ǶՠࢂǴ΋‫ޔ‬аٰགྷճҔԋᡏಒझਡᙯ෗‫ٰೌמ‬ғԋੰΓ஑ឦ ಒझ‫ؒࠅزࣴޑ‬Ԗԋф‫ٯޑ‬η(7, 8)Ƕӧ 2006 ԃǴ΋ঁၨϿ‫ݾ‬᝼‫ޑ܄‬ཥ Б‫ݤ‬ౢғΑǶВҁ‫ ޑ‬Yamanaka ฻ΓճҔੰࢥၩᡏஒѤঁᙯ᝿ӢηǴх ֖ Oct4, Sox2, c-Myc, Klf4 ໺ΕԴႵखजᠼᆢ҆ಒझύ٬‫ځ‬ᡂӣ‫ڀ‬Ԗӭ ૈ‫ޑ܄‬༸ಒझǴ٠ஒ‫ځ‬ᆀϐࣁᇨᏤ‫܄‬ӭфૈ༸ಒझ(Induced Pluripotent Stem Cells, iPS) (9)ǶԴႵ‫ޑ‬ᇨᏤ‫܄‬ӭфૈ༸ಒझԖૈΚϩϯԋΟखቫ‫ޑ‬ ‫܌‬ԖಒझǴԶЪૈ୼ᙖҗᇙ೷൪ӝႵٰ᛾ჴ‫ځ‬ӧ୏‫ނ‬ᡏϣ‫ޑ‬ϩϯૈΚǶ ԶΓᜪ‫ޑ‬ᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ک‬Γᜪखज༸ಒझόᆅࢂӧᡏѦჴᡍ‫܈‬ ࢂᡏϣჴᡍࣣΜϩ࣬՟Ƕ೭٬ள‫ॺך‬ёа٬ҔᇨᏤ‫܄‬ӭфૈ༸ಒझٰբ ᛰ‫ނ‬໒วࣗԿࢂғԋੰΓ஑΋‫ޑ‬ᇨᏤ‫܄‬ӭфૈ༸ಒझаߡ‫ݯ‬ᕍҞ߻ᙴ Ꮲ΢ค‫ݯݤ‬ᘰ‫ੱੰޑ‬ǶғԋᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ޑೌמ‬วܴჹ‫ܭ‬Γᜪᙴ ᕍў΢ёఈԖख़ε‫ँޑ‬ઇǶҞ߻ς࿶Ԗ‫ߎۦ‬හМੱаϷ៑Θࠠ೦Ո‫୏ޑ‬ ‫ނ‬ኳԄ᛾ჴᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ڀ‬Ԗ‫ݯ‬ᕍ‫ޑ‬ወૈ(10, 11)Ƕՠࢂ೭٤ᇨᏤ ‫܄‬ӭфૈ༸ಒझ‫ޑ‬ғԋࢂ࿶җੰࢥၩᡏஒᙯ᝿Ӣη᏾ӝΕखजᠼᆢ҆ ಒझ‫ ޑ‬DNA ύǴӢԜό፾ӝҔٰբΓᜪ‫ޑ‬ᖏ‫׉‬၂ᡍǶ೭ࢂӢࣁӵ݀٬ Ҕ୷Ӣ᏾ӝ‫ٰࢥੰޑ‬ғԋᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ޑ‬၉Ǵёૈ཮೷ԋँᡂ‫ک‬ . . 2.

(10) . ค‫ݤ‬Ⴃය‫୷ޑ‬Ӣфૈ౦த(3, 12)ǶӢԜǴΨԖ‫ځ‬дჴᡍ࠻ճҔ፦ᡏᙯࢉ ‫ޑ‬БԄٰғԋᇨᏤ‫܄‬ӭфૈ༸ಒझ(13, 14)Ƕќ΋ঁ֚ᜤᗺࢂӢࣁѤঁᙯ ᝿Ӣη྽ύǴc-Myc ‫ ک‬Klf4 ឦ‫ܭ‬ᕎ࣬ᜢ୷ӢǶӧ٬Ҕ c-Myc ೭ঁठᕎ୷ Ӣ‫܌‬ғԋ‫ޑ‬ᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ុࡕޑ‬ჴᡍ྽ύǴว౜཮Ԗဍዦ‫ޑ‬ౢғ (3)Ƕ. 1.1.3 લ਼ᕉნ (Hypoxia) ༸ಒझǵғ෗ಒझ‫ځک‬д‫ޑ‬ӭૈ‫߻܄‬០ಒझ೿ғߏӧΜϩፄᚇ‫ޑ‬༾ ᕉნ྽ύǴ೭ঁ༾ᕉნ೏ᆀϐࣁғᄊ୔Տ(niches) (15)ǶҺՖಒ༾‫ޑ‬ᡂϯ ೿Ԗёૈቹៜ‫ډ‬೭΋٤ಒझ‫ޑ‬ғߏ‫ݩރ‬ǴࣗԿ೷ԋಒझ‫ޑ‬ϩϯ‫ࢂޣ܈‬೷ ԋಒझ঒ΫǶႽࢂ༸ಒझ‫୷ک‬፦ಒझ‫ޑ‬ௗ᝻ǵғߏᕉნ‫਻਼کࡋྕޑ‬ᐚ ࡋ฻฻Ǵ೿཮ჹ༸ಒझ೷ԋҥջ‫ޑ܄‬ቹៜǴёૈቹៜ༸ಒझ‫ޑ‬фૈ‫ޣ܈‬ ࢂ٬ள༸ಒझ໒‫ۈ‬຾ՉϩϯǶӧၸѐΨว౜Αલ਼ᕉნёаߦ຾ઓ࿶ૉ ༸ಒझ(neural crest stem cells)ǵ೷Ո༸ಒझ(hematopoietic stem cells)аϷ Γᜪखज༸ಒझ‫ޑ‬ғӸ(16-19)ǶќѦǴલ਼ᕉნΨ཮ፓှФ៶ख‫ޑ‬ϣಒ झი(inner cell mass, ICM)ϐಒझኧҞ(20)ǶԶલ਼ᕉნΨ཮ቚуՈᆅಒ झаϷ೷Ո߻០ಒझ‫ޑ‬ว৖(21, 22)Ƕғ‫ނ‬ᡏϣ‫ޑ‬લ਼‫ރ‬ᄊวғ‫ܭ‬ಒझಔ ᙃ‫ז‬ೲғߏǴຬၸՈనёа‫ٮ‬ᔈ‫ޑ‬௃‫ݩ‬Ƕ‫ٯ‬ӵǴวғӧखजว‫ػ‬ϐਔ‫ޑ‬ લ਼‫ރ‬ᄊǴ೭ঁਔং‫ޑ‬લ਼‫ރ‬ᄊࢂࡰ਼਻ӧᕉნΠ‫ޑޑ‬ᐚࡋࢂ 3~5%Ƕ. . . 3.

(11) . җၸѐ‫ޑ‬Ӛ໨ࣴ‫ز‬аϷखजว‫ޑػ‬ᕉნٰ࣮Ǵ‫ॺך‬ว౜༸ಒझӧғԋ‫ޑ‬ ਔংǴલ਼ᕉნࢂКၨԖճ‫(ޑ‬19)ǶќѦǴӃ߻‫زࣴޑ‬ΨࡰрǴલ਼ᇨ ᏤӢη(Hypoxic induced factor-2)Ǵ཮ቚу Oct4 ‫ޑ‬ೈқ߄౜ໆ(23)ǶӢ ԜǴ‫ޑॺך‬ଷᇥࢂǴ྽‫ॺך‬ճҔ፦ᡏᙯࢉ‫ೌמޑ‬ஒ፦ᡏᙯࢉΕԴႵखज ᠼᆢ҆ಒझϐࡕǴᏤठԴႵखजᠼᆢ҆ಒझᡂӣԋ‫ڀ‬Ԗӭфૈ‫ޑ‬ᇨᏤ‫܄‬ ӭфૈ༸ಒझ೭ঁၸำϐύǴલ਼ᕉნࢂჹ‫ځ‬Ԗ‫܌‬շ੻‫ޑ‬΋ঁ‫ނ‬౛ᕉ ნǶ. 1.1.4 Oct4 Oct4 Ξᆀࣁ Pou5f1, POU domain, class 5, transcription factor 1ǶΖᆫ ᡏ่ӝೈқৎ௼‫ ک‬Sox ೭‫ঁٿ‬ᙯᒵӢηৎ௼ӧ༸ಒझࢂӵՖ௓‫ڋ‬Ծ‫׳ך‬ ཥаϷӵՖ௓‫ڋ‬ϩϯ‫ޑ‬ᆸѠ΢Ǵ‫ת‬ᄽΑЬा‫ف‬ՅǶΖᆫᡏ่ӝೈқৎ௼ ‫ޑ‬ԋ঩ന߃೏ว౜ࢂᙖҗѬॺ่ӝ‫ ډ‬DNA ‫ޑ‬Ζঁᡵ୷ჹ΢೏ว౜‫ډ‬Ԗ ೚ӭፓ௓୔ୱ‫୷ޑ‬ӢǶ‫ঁ؂‬Ζᆫᡏ่ӝೈқৎ௼‫ޑ‬ԋ঩ࣣ֖Ԗ΋ঁᚈ POU ่ᄬୱǴεऊࢂ 150~160 ঁữ୷ለ‫܌‬ಔԋǴࢂॄೢ‫ ک‬DNA ଺่ӝ ‫୔ޑ‬ୱǶӧ POU ᙯᒵӢη‫ޑ‬ৎ௼ύǴPOU ่ᄬୱ೏ଯࡋߥӸǴᙖҗѬ ॺ‫ޑ‬চࠠԋ঩ Oct1, Pit1, Unc86 ٰ߄౜ POU ่ᄬୱǶՠࢂǴOct1 ൳Я Ӹӧ‫܌‬Ԗ‫ޑ‬ಒझ྽ύǴӢԜ Oct4 ൩ԋΑεৎቶ‫ޑزࣴݱ‬ᚒ‫׷‬ǴӢࣁ Oct4 ൳Яѝӧ༸ಒझ྽ύ೏ว౜(2, 24, 25)ǶԜѦǴӧ 2006 ԃ Oct4 ς೏᛾ჴ ࢂѤঁҔٰғԋᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ޑ‬ᙯᒵӢηϐ΋(9)Ƕന߈ǴΨԖ‫ځ‬ . . 4.

(12) . д‫ޑ‬ᙯᒵӢηಔӝ೏ҔٰғԋᇨᏤ‫܄‬ӭфૈ༸ಒझǴՠࢂ‫ঁ؂‬ಔӝ൳Я ೿ค‫ݤ‬όѐ٬Ҕ‫ ډ‬Oct4 ‫ ک‬Sox2 ೭‫ঁٿ‬ᙯᒵӢηǶନߚࢂႽઓ࿶༸ಒझ ೭ᜪёаҗಒझϣྍ‫߄܄‬ၲ೭٤ᙯᒵӢη‫ޑ‬௃‫ݩ‬Ǵωёаόѐ٬Ҕ Oct4 ‫ ک‬Sox2 (26, 27)Ƕ༸ಒझ‫ޑ‬੝‫ڀࢂ܄‬ԖԾ‫׳ך‬ཥ‫ૈޑ‬ΚаϷᆢ࡭ӄૈ ‫܄‬ǶԶ Oct4 ჹ‫ܭ‬ᆢ࡭खज༸ಒझаϷғ෗ಒझ‫ޑ‬όϩϯ‫ת‬ᄽ΋ঁΜϩ ख़ा‫فޑ‬Յ(1, 2)ǶOct4 ӧፓှखज༸ಒझ‫ޑ‬ϩϯаϷᆢ࡭៶खϣಒझ (ICM)‫ޑ‬ӭૈ‫܄‬Ψ೿‫ת‬ᄽΑࡐᜢᗖ‫فޑ‬ՅǶ‫ׯ‬ᡂ Oct4 ‫߄ޑ‬౜ໆ཮೷ԋख ज༸ಒझѨѐӭૈ‫܄‬ԶᏤठค‫ݤ‬ϩϯԋΟঁखቫ‫ޑ‬ಒझ(28, 29)Ƕӧ 2005 ԃǴว౜ Oct4 ࢂ‫ ک‬NanogǵSox2 ่ӝϐࡕω‫ڀ‬ԖૈΚѐ‫ࢲࢂ܈ڋ׭‬ϯ ༸ಒझǴ‫ࢂޣ܈‬ѐ௓‫ڋ‬༸ಒझ‫ޑ‬ϩϯ(30)ǶOct4 ‫߄ޑ‬౜‫ک‬खजว‫ػ‬аϷ ঁᡏрғࡕ‫ޑ‬᏾ঁғ‫ڮ‬৲৲࣬ᜢǶᖐ‫ٯ‬Զ‫ق‬Ǵफ़ե Oct4 ‫୷ޑ‬Ӣ߄౜཮ ቹៜϣखቫ‫ޑ‬ϩϯբҔǶ྽ Oct4 ‫߄ޑ‬౜ѨதϐਔǴखजӧ๱‫׉‬ϐ߻൩ ό཮Ԗ҅த‫ޑ‬ว‫(ػ‬31)Ƕჹ‫ܭ‬ԋᡏԶ‫ق‬Ǵ΋૓ᇡࣁ Oct4 ѝ཮߄౜ӧғ෗ ಒझ྽ύǶՠࢂǴࡕٰว౜ Oct4 Ψр౜ӧԋᡏ༸ಒझ྽ύǴႽࢂ೷Ո ༸ಒझ‫ک‬ӭૈ‫܄‬ԋᡏ߻០ಒझ྽ύ(multipotent adult progenitor cells, MAPCs) (32, 33)ǶҞ߻Ԗᜢ‫ ܭ‬Oct4 ߄౜‫ޑ‬ϩηᐒᙯᗋ҂ΜϩమཱǶ. 1.1.5 Sox2 Sox ৎ௼‫ک‬Ζᆫᡏ่ӝೈқৎ௼΋ኬǴς࿶೏ቶ‫زࣴݱ‬೚ΦΑǶӧ থ٢୏‫ނ‬྽ύǴSox ᙯᒵӢη‫ޑ‬ৎ௼ԋ঩εऊԖΒΜᅿ(34)ǶSox ೈқߥ . . 5.

(13) . ࡭ଯख़ፄ‫ ޑ‬DNA ่ӝୱǴऊΖΜঁữ୷ለǶ೭٤่ᄬୱ೏ᆀࣁ high mobility group (HMG) box ่ᄬୱǴ೭ࢂӢࣁѬॺ‫ׇޑ‬ӈ‫ ک‬HMG DNA ่ӝೈқ‫ ޑ‬DNA ่ӝ่ᄬୱࡐ࣬՟ǶԾவ໒‫ۈ‬լໜ Sox ৎ௼ SRY ϐࡕǴ Sox ৎ௼൩ς࿶೏᛾ჴࢂ൳Я‫܌‬Ԗথ٢ᜪ‫ޑ‬ว‫ػ‬΢Μϩᜢᗖ‫ޑ‬΋ঁ୷Ӣ (34)Ƕӧၸѐ‫ޑ‬൳ԃ္ǴSox2 ԋΑ΋ঁ೏ᑈཱུࣴ‫ޑز‬ᙯᒵӢηǶ೭ࢂӢ ࣁѬ‫ ک‬Oct4 ΋ኬǴࢂғԋᇨᏤ‫܄‬ӭфૈ༸ಒझਔόё‫܈‬લ‫ޑ‬ᙯᒵӢη ϐ΋ǶԜѦǴOct4 ‫ ک‬Sox2 ς࿶ӧ୷ӢѨࢲ‫ޑ‬ჴᡍ΢೏᛾ჴǴࢂথ٢୏ ‫ނ‬ӧว‫ػ‬ၸำύόёલϿ‫୷ޑ‬Ӣ(1, 35)ǶSox2 ‫ ࢂ܈‬Oct4 ୷ӢѨࢲ཮Ꮴ ठ๱‫ޑ߻׉‬खजԝΫǶӢԜǴOct4 ‫ ک‬Sox2 ‫ޣٿ‬ჹ‫ܭ‬खज༸ಒझ‫ޑ‬Ծ‫ך‬ ‫׳‬ཥаϷᆢ࡭ӭૈ‫ࢂࣣ܄‬Ѹा‫ޑ‬Ƕ. ಃΒ࿯ǵჴᡍҞ‫ޑ‬ Ҟ߻ᇨᏤ‫܄‬ӭૈ༸ಒझ‫ޑ‬౟ᓍࣁ٬ҔੰࢥၩᡏаϷठᕎ୷ӢǴҁჴ ᡍ‫ޑ‬Ҟ‫פࣁޑ‬р΋ঁӼӄ‫ޑ‬ᇨᏤ‫܄‬ӭૈ༸ಒझғԋБ‫ݤ‬ǶࣁΑᗉխ٬Ҕ ੰ ࢥ ၩ ᡏ а Ϸ ठ ᕎ ୷ Ӣ Ǵ ‫ ॺ ך‬ჴ ᡍ ࠻ ᒧ ᏷ ٬ Ҕ ፦ ᡏ ᙯ ࢉ (plasmid transfection) ‫ޑ‬Б‫ݤ‬բࣁࣴ‫ޑز‬БԄǴ٠Ъᗉ໒Α c-Myc ‫ ک‬Klf4 ೭‫ঁٿ‬ ठᕎ୷ӢǴ໻٬Ҕ Oct4 ‫ ک‬Sox2 ೭‫୷ঁٿ‬Ӣଛӝલ਼ᕉნٰғԋᇨᏤ‫܄‬ ӭфૈ༸ಒझǶ. . . 6.

(14) . ಃΒകǵ‫׷‬਑ᆶБ‫ݤ‬. 2.1 ፦ᡏᄬᑐ Construction of the plasmid mOct4 ‫ ک‬mSox2 cDNA ٰԾѱय़΢‫ ޑ‬clones (Thermo scientific)Ƕ೭ ‫ ঁٿ‬clones ΢ϩձх֖Ԗ Oct4 ‫ ک‬Sox2ǴёаճҔ EcoR1 Ϫрள‫ॺךډ‬ ሡा‫ޑ‬Тࢤ cDNAǴ٠ஔԿ pcDNA 3.1 ၩᡏ΢Ƕ೭‫ঁٿ‬፦ᡏǴ pcDNA-Oct4 ‫ ک‬pcDNA-Sox2 ٬Ҕӧ፦ᡏᙯࢉ‫ޑ‬ჴᡍ΢Ƕ. 2.2 ԴႵखजᠼᆢ҆ಒझϐ୻Ꭶ Mouse embryonic fibroblast cells (MEF) culture ҁჴᡍύ‫܌‬٬Ҕ‫ޑ‬ಒझࣁԴႵखजᠼᆢ҆ಒझ Mouse Embryonic Fibroblast Cells (MEF)Ǵ‫ڗ‬Ծᚶѫ 13.5 Ϻ‫ ޑ‬C57BL/6 ‫ޑ‬जႵǶ२Ӄஒᚶ ѫ 13.5 Ϻ‫ޑ‬ᚶѫ҆Ⴕ឴࣊Ǵӆ٬ҔѦࣽЋೌஒजႵவᚶѫ҆Ⴕη৐ϣ‫ڗ‬ рǴ٠Ъ੆‫ݰ‬ӧးԖ phosphate-buffered saline (PBS)‫୻ޑ‬ᎦҝύǶҔ PBS wash ϐࡕѐନѤަǵᓐ‫ک׀‬ϣ᠌ǴഭΠ‫ޑ‬ಔᙃҔཥᗲ‫ ޑ‬PBS wash Οԛ ϐࡕҔ୊Θஒ‫ځ‬୊࿗Ǵஒ 0.1mM trypsin/1mM EDTA solution (GIBCO BRL)у‫୻ډ‬Ꭶҝ྽ύǴ٠Ъ֎ଆಔᙃ‫ܫ‬ΕᚆЈᆅ྽ύǶᇸᇸའਗᚆЈᆅ 20 ϩដϐࡕуΕಒझ୻Ꭶనύ‫ک‬բҔǴ٠Ъᓉ࿼٬εი༧ϐಔᙃ༧؇ᐘ Կᆅ‫ۭي‬೽Ƕ֎ଆ΢మనԿќ΋ᚆЈᆅ྽ύ‫ܫ‬ΕᚆЈᐒ྽ύ ᚆЈǶѐ ନᚆЈϐࡕ‫ޑ‬΢మ‫ࡕڹ‬ǴӆуΕ medium ѺණಔᙃǴҔ pipette ౽‫ډ‬ಒझ . . 7.

(15) . ୻Ꭶҝ྽ύǴനࡕ‫ܫ‬ΕΒ਼ϯᅹಒझ୻Ꭶጃ(5% CO2, 37ʚ)྽ύ୻Ꭶ‫܌‬ ளǶќѦӧҁჴᡍ‫܌‬٬Ҕ‫ޑ‬෷Ꭶಒझ feeder cells ࢂஒԴႵखज҆ಒझ (MEF Cells)Ҕ֖Ԗ Mitomycin C (Roche)‫ޑ‬ಒझ୻Ꭶన୻Ꭶ 2.5 λਔ(37 ʚ)ϐࡕ‫܌‬ள‫ޑډ‬ಒझǶӢࣁ Mitomycin C ೭ঁᛰ‫ޑނ‬բҔ٬ளԴႵखज ᠼᆢ҆ಒझ‫ޑ‬ғߏຼයύЗǴӢԜёаҔٰ྽բ༸ಒझ‫ޑ‬෷ᎦቫಒझǴ ᗉխ༸ಒझӢࣁௗ᝻‫ډ‬ಒझ୻Ꭶҝ‫ۭޑ‬೽ԶϩϯǴӕਔΨග‫ٮ‬༸ಒझ΋ ٤ғߏӢηǶԴႵखजᠼᆢ҆ಒझ(MEF Cells)‫ک‬෷Ꭶቫಒझ(Feeder Cells) ‫ޑ‬ಒझ୻Ꭶనࣣࣁ DMEM (GIBCO BRL)బу 10% fetal bovine serum (FBS) (GIBCO BRL) ǵ 1% penicillin and streptomycin (p/s) (GIBCO BRL)ǵ1% non-essential amino acids (NEAA) (GIBCO BRL)Ƕ. 2.2.1 ᇨᏤ‫܄‬ӭфૈ༸ಒझ‫୻ޑ‬Ꭶ Induced pluripotent stem cells (iPS) culture ᇨᏤ‫܄‬ӭфૈ༸ಒझ Induced pluripotent stem cells (iPS)‫୻ޑ‬ᎦБԄ ‫ک‬ԴႵखज༸ಒझ mouse embryonic stem cells (mES)࣬ӕǶӧႣ଺ᇨᏤ ‫܄‬ӭфૈ༸ಒझ Induced pluripotent stem cells (iPS)୻Ꭶ‫߻ޑ‬΋ϺǴѸ໪ Ӄྗഢ෷Ꭶቫಒझ(Feeder Cells)Ƕ२Ӄӧಒझ୻Ꭶҝ‫ۭޑ‬೽Ҕ 0.1%‫ޑ‬ Gelatin (Sigma)଺༡ቫǴ΋λਔϐࡕѐନ Gelatin ٠ЪуΕ෷Ꭶቫಒझ‫ޑ‬ ୻ᎦనǴ٠Ъஒ෷Ꭶቫಒझᎎ‫ ډ‬Gelatin ϐ΢Ƕ႖Ϻߡёаᝩុ଺ᇨᏤ ‫܄‬ӭфૈ༸ಒझ‫୻ޑ‬Ꭶ୏բǶᇨᏤ‫܄‬ӭфૈ༸ಒझӧ଺ಒझ୻Ꭶ‫ޑ‬ਔ . . 8.

(16) . ংǴಒझόёаѺණԋൂ΋ಒझǴ೭ёૈ཮ᏤठᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ޑ‬ ғߏ‫ݩރ‬όӳǶᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ک‬ԴႵखज༸ಒझ࣬ӕǴӧ଺ಒझ ୻Ꭶ‫ޑ‬ၸำύࣣሡा‫؂‬Ϻࣁ‫׳ځ‬ඤ୻ᎦనǶᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ޑ‬ಒझ ୻Ꭶనࣁ DMEM (GIBCO BRL)బу HyClone FBS (Thermo)ǵ1mM -mercaptoethanol (GIBCO BRL)ǵ1% non-essential amino acids (NEAA) (GIBCO BRL)ǵLIF (millipore)Ƕ. 2.3 ՋБᏀᗺ‫ ݤ‬Western Blot ஒςࢉՅϐϩηໆ኱‫ۓ‬ೈқ፦аϷೈқ፦ኬࠔǴҗѰԿѓ٩‫ݙׇ‬Ε ኬࠔኲϐύǴ຾Չႝ‫ݚ‬Ƕ٠ӧႝ‫ݚ‬ኲϣॹΕ‫୼ى‬ໆ‫ ޑ‬Running bufferǶ΢ ጤ‫ޑ‬೽ҽ٬Ҕ 80mAǴΠጤ೽ҽ٬Ҕ 120VǶ่‫״‬ϐࡕǴλЈ‫ڗޑ‬рጤᡏǴ ੆Ε PBST ϐύǶӃஒ transfer membrane ‫ک‬ᘠર੆‫ݰ‬ӧ Transfer buffer ྽ύǴ‫ډޔ‬રֹӄዎᔸࣁЗǶஒጤᡏλЈ‫ܫޑ‬࿼‫ ډ‬transfer membrane ϐ ΢Ǵ٬ transfer membrane ᎞߈ཱུ҅ǵጤᡏٗ΋य़᎞߈ॄཱུǴ໒‫ۈ‬຾Չ transferǴ400mVǵ1.5 λਔǶᙯӑֹԋϐࡕǶஒ transfer membrane ࿼‫ܭ‬ blocking solution ϐύǴҔਁᑲᏔ᎜ᕏ 20 ϩដǶϐࡕа 1:1000 ‫ޑ‬К‫ٯ‬у Ε߃ભ‫ל‬ᡏǴӧհ‫܊‬྽ύ blocking overnightǶ႖ϺճҔ PBST ஒ transfer membrane ࢱΟԛǴ‫؂‬ԛΜϩដǶϐࡕӆуΕа 1:10000 ‫ޑ‬К‫ٯ‬Βભ‫ל‬ᡏǴ ճҔ᎜ᑲᏔ᎜ᕏ 60 ϩដǴനࡕӆҔ PBST ஒ transfer membrane ࢱΟԛǴ. . . 9.

(17) . ‫؂‬ԛΜϩដǶௗ๱ஒ transfer membrane λЈ֨ଆǵጬଳǴуΕ ECL բ ҔϐࡕǴ຾ՉᓸТǵࢱТϐࡕᢀჸ่݀Ƕ. 2.4 લ਼ᕉნ Hypoxia ਼਻ϩᓸ(3%)ࢂճҔේ਻(N2)‫֖ޑ‬ໆٰ௓‫ڋ‬Ƕஒ hermetic chambers (Billups-Rothenberg Inc., Del Mar, CA)‫ܫ‬Ε௓‫ڋ‬ᆢ࡭ӧ 37ʚ‫୻ޑ‬Ꭶጃ྽ ύǶ΋ՠஒന٫‫਻਼ޑ‬ᐚࡋ(3%)ፓ௓ӳϐࡕǴϐࡕ‫ޑ‬ჴᡍߡӧ 3%਼਻ǵ 5%Β਼ϯᅹǵ92%ේ਻‫ޑ‬Ο਻୻Ꭶጃ (ThermoForma, Waltham, MA)ϣ຾ ՉǶ. 2.5 ջਔᆫӝ䁙᜘ᙹϸᔈ Real-time PCR Ҕ TRIzol (Invitrogen, Carlsbad, CA)ஒಒझ‫܌‬Ԗ‫ ޑ‬RNA բ๧‫ڗ‬ϐࡕ ӆճҔ SuperScript II Reverse Transcriptase Kit (Invitrogen)ᙯԋ dEOBǶӃ ஒ cDNA ଺ 40 ७ีញаߡ຾Չ Real-Time PCRǶஒ primer reverse(6M), primer forward(6M), SYBR Mix(2X)(Roche)٩К‫ٯ‬ଛᇙǶӃӧ 96 Ͼዬϐ ύǴуΕኬࠔ cDNAǴӆуΕ primer ‫ ک‬SYBR Mix షӝనǶᚆЈ΋ϩដǵ 1000rpm ϐࡕ΢ᐒǶReal-time PCR ࢂҔٰዴᇡ Oct4, Sox2, c-Myc ‫ ک‬Klf4 ‫୷ޑ‬Ӣ߄౜Ƕനࡕ٬Ҕ Excel ଺ϩ‫݋‬ǶReal-Time PCR ‫܌‬٬Ҕ‫ ޑ‬Primer sequences ӈ‫ ܭ‬Table 1Ƕ!. . . 10.

(18) . 2.6 ፦ᡏᙯࢉ Transfection ஒ MEF ႣӃ୻Ꭶӧ 6-well cell culture plate ΢Ǵ٠ЪႣӃྗഢ plasmids (Oct4ǵSox2)ǶӃஒ medium ‫׳‬ඤԋ Opti-MEM (GIBCO BRL)Ǵ ஒ plasmid уΕ fugene(Roche)྽ύӧ࠻ྕϐΠ incubate15 ϩដϐࡕѳ֡ ᅀΕ 6-well cell culture plate ϐύǶ‫ܫ‬Ε incubator ྽ύ culture 6 λਔ(37 ʚǴ5%CO2)Ƕஒ Opti-MEM ‫׳‬ඤԋ MEF medium ϐࡕ‫ܫ‬Ε Hypoxia incubator (ThermoForma, Waltham, MA)ύ୻Ꭶ 24 λਔ(37ʚǴ5%O2)ࡕǴ ஒ 6-wll cell culture plate ‫ܫ‬ӣ CO2 incubator ྽ύ(37ʚǴ5%CO2 )Ƕ႖Ϻ ӆ଺Π΋ԛ transfectionǶҁჴᡍӧԜӅ଺ΑѤԛ transfection (კ΋) Ƕ. 2.7 ᡵ‫܄‬ᕗለ䁙ࢉՅ Alkaline phosphatase staining Alkaline phosphatase (AP) ࢉՅࢂ٬Ҕ Alkaline phosphatase staining kit (Vector)ǶӃஒಒझҔ PBS wash ‫ٿ‬ԛࡕǴӧ࠻ྕΠஒಒझҔ 80%ଚ ᆒ‫ ۓڰ‬overnightǴ႖Ϻ٬Ҕ Alkaline phosphatase staining kit (Vector)଺ࢉ ՅǶ౽ନ 80%ଚᆒǴ٠аΒԛН੆ዎమࢱಒझࡕ౽ନǶӆԛуΕΒԛНǴ ‫ྕ࠻ܭ‬Π੆‫ݰ‬բҔ 2-3 ϩដࡕ౽ନǶ‫୻ܭ‬ᎦҝϣуΕ 100mM Tris-HClǴ ‫ ྕ ࠻ ܭ‬Π ੆ ‫ ݰ‬բ Ҕ 5 ϩ ដ ࡕ ౽ ନ Ƕ ௗ ๱ ‫ ୻ ܭ‬Ꭶ ҝ ϣ у Ε Alkaline Phosphatase Substrate working sol’n ‫ྕ࠻ܭ‬ΠբҔǴࡑಒझև౜፾྽आՅ மࡋȐऊ 20-30 ϩដȑ Ƕ౽ନ alkaline phosphatase substrate working sol’nǴ. . . 11.

(19) . а 100mM Tris-HCl buffer ੆ዎమࢱࡕǴ‫ܭ‬ᡉ༾᜔Πᢀჸ‫ྣܡ‬Ƕalkaline phosphatase substrate working sol’n ‫ޑ‬ଛᇙӵΠǺ‫ ڗ‬5ml 100mM Tris-HCl buffer ᅀΕ red alkaline phosphatase Substrate Kit ‫ ޑ‬Reagent 1 ‫ٿ‬ᅀǴషӝ ֡ϬࡕӆᅀΕ Reagent 2 ‫ٿ‬ᅀǴࡑషӝ֡ϬࡕӆᅀΕ Reagent 3 ‫ٿ‬ᅀ٠ష ӝǶ. 2.8 խࣝᑻӀࢉՅ-༸ಒझ኱૶ Immunofluorescent antibody assay-stem cell marker ࣁΑዴᇡ࿶ reprogrammed ϐࡕ‫׎܌‬ԋ‫ޑ‬ಒझ‫ڀ‬Ԗ༸ಒझ‫ޑ‬੝‫܄‬Ǵӧ ೭္٬ҔΑϤঁ stem cell marker ଺խࣝᑻӀࢉՅǶх֖ Nanog (Novus)ǵ Oct4 (Cell Signaling)ǵSox2 (millipore)ǵSSEA1 (millipore)ǵTra-1-60ǵ Tra-1-80ǶӃஒಒझҔ 0.1% Tween-20 (1x PBS) wash ΜϩដǵΟԛǴࡕ Ҕ 0.3% Triton-X 100 (1X PBS) wash ΟΜϩដǴӆҔ 0.1% Tween-20 (1x PBS) wash ΜϩដǵΟԛǶ5% FBS(1X PBS) blocking ‫ঁٿ‬λਔϐࡕа 1:100 ‫ޑ‬К‫ٯ‬уΕ΋‫ ל‬overnightǶಃΒϺҔ 0.1% Tween-20 (1x PBS) wash ϖϩដࡕа 1:500 ‫ޑ‬К‫ٯ‬Β‫ל‬΋ঁλਔǴӆҔ 0.1% Tween-20 (1x PBS) wash ΜϩដǴനࡕҔ DAPI ࠾Тጤ(UltraCruz™ Mounting Medium)࠾ТǴ ٠ҔᑻӀᡉ༾᜔ᢀჸǶ. 2.9 ᆫӝ䁙᜘ᙹϸᔈ PCR . . 12.

(20) . Ҕ TRIzol (Invitrogen, Carlsbad, CA) ஒಒझ‫܌‬Ԗ‫ ޑ‬RNA բ๧‫ڗ‬ϐࡕ ӆճҔ SuperScript II Reverse Transcriptase Kit (Invitrogen)ᙯԋ cDNAǶ ᆫӝ䁙᜘ϸᔈచҹࣁႣᡂ‫ ܄‬94ʚΠ 1.5 ϩដǴฅࡕа 35 ঁൻᕉᘉቚ 94 ʚ30 ࣾǴ60ʚ60 ࣾǴ72ʚ60 ࣾǴᒿࡕ 2 ϩដ‫ۯޑ‬՜΋‫؁‬ӧ 72ʚǴനࡕ ಖЗ‫ ܭ‬10ʚǶPCR ճҔΑ ExTaq (Takara)ٰᔠෳ Ecat1, Eras, Nanog, c-Myc, Esg1, Klf4, Oct3/4, Rex1, Sox2 ‫ ک‬beta-actin ‫୷ޑ‬Ӣ߄౜ǶPrimers ӈ߄‫ ܭ‬Table 1Ƕ. 2.10 ख ज ౚ ‫ ׎ ޑ‬ԋ ‫ ک‬ϩ ϯ. Embryonic bodies formation and. differentiation ஒ iPS Ҕ 0.1mM trypsin/1mM EDTA solution (GIBCO BRL)բҔ 5 ϩ ដϐࡕҔ medium ύ‫ک‬٠ᚆЈǴѐ΢మనࡕуΕ EB formation medium ஒ ಒझѺණԿΟ‫ډ‬ϖᗭಒझࣁ΋იǴ‫ܫ‬Ε Ultra Low Cluster Plate (Costar) ྽ύ culture ΒԿΟϺ‫׎‬ԋ Embryonic bodiesǶ‫׎‬ԋ Embryonic bodies ϐ ࡕǴӃҔ 0.1% gelatin coated ӧ 24-well ΢ 1 ঁλਔǴ΋ঁ well ྽ύ‫ܫ‬Ε ϖԿϤᗭ Embryonic bodiesǴ‫ܫ‬Ε incubator ྽ύ culture ΟϺ٬‫ځ‬ຠߕǵ ϩϯԋΟঁखቫ‫ޑ‬ಒझǶEB formation medium ࣁ DMEM(GIBCO BRL) బу 20% fetal bovine serum (FBS) (GIBCO BRL), 1mM -mercaptoethan (GIBCO. BRL),. 1%. L-glutamin. insulin-transferin-selenium-Gsupplement . . 13. (GIBCO. (ITSG). (GIBCO. BRL), BRL),. 1% 1%.

(21) . non-essential amino acids (NEAA) (GIBCO BRL)Ƕ EB differentiation medium ࣁ DMEM (GIBCO BRL)బу 20% fetal bovine serum (FBS) (GIBCO BRL), 1% L-glutamine (GIBCO BRL), 1% non-essential amino acids (NEAA) (GIBCO BRL), 1% penicillin and streptomycin (p/s) (GIBCO BRL), 1mM -mercaptoethanol (GIBCO BRL)Ƕ. 2.10.1 խࣝᑻӀࢉՅ-Οखቫ Immunofluorescent antibody assay-three germ layers Embryonic bodies ϩϯϐࡕǴࣁΑዴᇡ‫ࢂځ‬ց‫ڀ‬ԖϩϯԋΟঁखቫಒ झ‫ૈޑ‬ΚǴҁჴᡍ٬ҔխࣝᑻӀࢉՅٰ᛾ჴǶϣखቫಒझճҔ Anti-Actin, smooth muscle (ASM) (abcam)଺ࢉՅǵύखቫಒझճҔ(Gata4) (abcam) ଺ࢉՅǵѦखቫಒझճҔ Anti--Tubulin III (Tuj1) (sigma)଺ࢉՅǶӃஒ ಒझҔ 0.1% Tween-20 (1x PBS) wash ΜϩដǵΟԛǴࡕҔ 0.3% Triton-X 100 (1X PBS) wash ΟΜϩដǴӆҔ 0.1% Tween-20 (1x PBS) wash Μϩ ដǵΟԛǶ5% FBS(1X PBS) blocking ‫ঁٿ‬λਔϐࡕа 1:100 ‫ޑ‬К‫ٯ‬уΕ ΋‫ ל‬overnightǶಃΒϺҔ 0.1% Tween-20 (1x PBS) wash ϖϩដࡕа 1:500 ‫ޑ‬К‫ٯ‬уΕΒ‫ל‬΋ঁλਔǴӆҔ 0.1% Tween-20 (1x PBS) wash ΜϩដǴ നࡕҔ DAPI ࠾Тጤ(UltraCruz™ Mounting Medium)࠾ТǴ٠ҔᑻӀᡉ༾ ᜔ᢀჸǶ. . . 14.

(22) . 2.11 ྾जዦ‫׎ޑ‬ԋ Teratoma formation iPS-OSH Cells ѺΕᇘႵ (SCID mice) ङ೽‫ޑ‬ҜΠǴ‫ݙ‬৔ϐࡕ‫ޑ‬Ϥ‫ډ‬ Ζ‫ڬ‬ճҔѦࣽЋೌஒ tumors ‫ڗ‬ΠǶ٠ஒ tumors ੆‫ݰ‬ӧ 4%‫ޑ‬ᅽଭ݅྽ύǴ ௗ๱଺հএϪТ٠ЪҔ hematoxylin ‫ ک‬eosin ࢉՅǶӧᡉ༾᜔Πᢀჸ΋ᗭ teratoma ྽ύࢂցӕਔ‫ڀ‬ԖΟঁखቫ‫ޑ‬ಔᙃಒझӸӧǶ. 2.12 ༾ତӈϩ‫ ݋‬Microarray analysis ճҔ TRIzol reagent(Invitrogen, Carlsbad, CA)ஒ Mouse embryonic fibroblast cells (MEF)ǵEmbryonic stem cells (ES)ǵInduced pluripotent stem cells from Japan (iPS J) (9)ǵInduced pluripotent stem cells OSH (iPS-OSH) ‫ޑ‬ӄ೽ RNA ๧‫ڗ‬рٰϐࡕǴϩ‫ ݋‬MEF ‫ځک‬дΟᅿಒझ‫୷ޑ‬Ӣ߄౜ໆǶ ٩Ᏽᇙ೷ቷ୘‫ ޑ‬protocol ஒኬࠔᆶ Agilent Mouse G3 Whole Genome Oligo 8×60K Microarray (Agilent) ຾ՉᚇҬǶ Arrays ‫ޑ‬௟ඔࢂճҔ Microarray Scanner System (Agilent)ǶኧᏵ‫ޑ‬ϩ‫ࢂ݋‬ճҔ GeneSpring GX ೬ᡏ (Agilent)Ƕ. 2.13 ಍ीϩ‫݋‬ ࣬ᜢኧᏵϐ໔ৡ౦߾٬Ҕ one-way ANOVA ϩ‫݋‬ǶӆճҔ Tukey HSD ٣ࡕᔠ‫ݤۓ‬ϩ‫݋‬Ǵऩ p<0.05 ߾߄Ң಍ी΢Ԗᡉ๱ৡ౦Ƕ. . . 15.

(23) . 2.14!ճҔᇨᏤ‫܄‬ӭфૈ༸ಒझғԋઓ࿶༸ಒझ!Generation of neural stem cells (NSC) from iPS-OSH २Ӄஒ୻ᎦΑᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ޑ‬ಒझ୻ᎦҝҔ PBS ؑࢱ‫ٿ‬ ԛǴௗ๱уΕ 1c.c ‫ ޑ‬0.1mM trypsin/1mM EDTA solution (GIBCO BRL) ӧ 37ʚΠ୻Ꭶ 1 ϩដǶуΕ 10c.c ‫ ޑ‬EB formation medium ࡕǴஒಒझѺ ණԋൂ΋ಒझǶᚆЈϐࡕǴஒಒझᎦӧ 15 cm ‫ޑ‬ಒ๵୻Ꭶҝ྽ύǴಒझ ஏࡋࣁ‫ ؂‬c.c ‫ ޑ‬medium ύ֖Ԗ 10^5 ᗭಒझǶ‫ܫ‬ΕΒ਼ϯᅹಒझ୻Ꭶጃ (5% CO2, 37ʚ)྽ύ୻ᎦϤϺǴ‫׎‬ԋखजౚ(EB)Ƕஒ୻ᎦϤϺ‫ޑ‬खजౚ ౽ΕᚆЈᆅύǴᓉ࿼ 10 ϩដǶѐନε೽ҽ‫ޑ‬΢మనϐࡕǴуΕ 30c.c ‫ޑ‬ PBSǴᓉ࿼ 5 ϩដǶѐନ΢మనϐࡕǴуΕ trypsin ӧ 37ʚΠ୻Ꭶ 5 ϩ ដǶௗ๱уΕ FBS ύЗϸᔈǴ٠ЪѺණखजౚǶஒ֖Ԗಒझ‫ޑ‬నᡏ౽Ε ཥ‫ޑ‬ᚆЈᆅ྽ύǴ٠ЪуΕ MHM mediumǴ2 ϩដϐࡕӆஒᚆЈᆅύ‫ޑ‬ నᡏ౽Կཥ‫ޑ‬ᚆЈᆅ྽ύǴ੮Πۭ೽ 1c.c ‫ޑ‬నᡏҔٰѐନၸε‫ޑ‬ಒझ იǶᚆЈϐࡕǴஒಒझᎦӧ‫ س‬10cm ‫ޑ‬ಒझ୻Ꭶҝ྽ύǴಒझஏࡋࣁ‫؂‬ c.c ‫ ޑ‬medium ύ֖Ԗ 5*10^4~10^5 ᗭಒझǶ‫ܫ‬ΕΒ਼ϯᅹಒझ୻Ꭶጃ(5% CO2, 37ʚ)྽ύ୻ᎦϤϺǴ‫׎‬ԋઓ࿶ౚ(neurosphere)Ǵӧ୻Ꭶ‫ޑ‬ಃΟϺਔ уΕ 1μg/ml bFGFǶஒ୻ᎦϤϺ‫ޑ‬ઓ࿶ౚ౽ΕᚆЈᆅύǴᚆЈϐࡕѐନ ΢మనǴуΕ 1c.c ‫ ޑ‬MHM mediumǴ٠Ҕ P1000 ‫ ޑ‬pipetman ஒઓ࿶ౚ ѺණǶуΕ MHM medium ࡕᚆЈǴௗ๱ஒಒझ୻Ꭶӧ 24-well ‫ޑ‬ಒझ୻. . . 16.

(24) . Ꭶҝ྽ύǶ ಒझஏࡋࣁ‫ ؂‬well ྽ύ֖Ԗ 3.2*10^6 ᗭಒझǶ ‫ܫ‬ΕΒ਼ ϯᅹಒझ୻Ꭶጃ(5% CO2, 37ʚ)྽ύ୻ᎦϖϺǴ٬ઓ࿶ౚϩϯԋઓ࿶༸ ಒझǶӧҁჴᡍ྽ύ٬Ҕ‫ ޑ‬EB formation medium ࣁ͉MEM (GIBCO BRL)బу 10% fetal bovine serum (FBS) (GIBCO BRL)ǵ1% penicillin and streptomycin (p/s) (GIBCO BRL) ǵ 100mM -mercaptoethan (GIBCO BRL)ǵ200ng/ml NogginǶMHM medium ࣁ DMEM/F12 (GIBCO BRL)బ у 3mM Sodium bicarbonate (Sigma)!ǵ 5mM HEPES (Sigma) ǵ25g/ml Insulin (Sigma) ǵ 100g/ml Transferrin (Sigma)! ǵ 20nM Progestenone (Sigma)!ǵ30ng Sodium selenate (Sigma*ǵ60nM Putrescine (Sigma*ǵ2% B27 (GIBCO BRL)!ǵ1 g/ml bFGF (GIBCO BRL)Ƕ. . . 17.

(25) . ಃΟകǵ่݀ ಃ΋࿯ǵ࿶җલ਼ᕉნೀ౛ၸࡕखजᠼᆢ҆ಒझ‫ ޑ‬Oct4, Sox2, c-Myc, Klf4 ೈқ‫୷ک‬Ӣ߄౜ ‫ॺך‬२Ӄाዴᇡೀӧલ਼ᕉნΠ‫ޑ‬ԴႵखजᠼᆢ҆ಒझϐ Oct4, Sox2, c-Myc ‫ ک‬Klf4 ‫ޑ‬ೈқ߄౜Ƕ‫ॺך‬٬ԴႵखजᠼᆢ҆ಒझೀӧલ਼ ᕉნΠǴႣෳ Oct4, Sox2, c-Myc, Klf4 ‫ޑ‬ೈқ߄౜ໆ཮ଯ‫҅ܭ‬த਼਻ᕉ ნΠ‫ޑ‬ಒझೈқ߄౜ໆǶ‫ॺך‬ஒಒझϩࣁѤಔǴӧલ਼ᕉნΠ႟λਔǵ ѤλਔǵΖλਔаϷΒΜѤλਔ‫ޑ‬ਔংϩձ‫ڗܜ‬ಒझ‫ޑ‬ೈқ፦Ǵ࿶җՋ БᏀᗺ‫( ݤ‬Western Blot)ٰዴᇡલ਼ᕉნჹ‫ ܭ‬Oct4, Sox2, c-Myc, Klf4 ೭ Ѥঁᙯ᝿Ӣη‫ޑ‬ೈқ߄౜ໆࢂցԖ‫܌‬ቹៜǶ‫ॺך‬ว౜Ǵखजᠼᆢ҆ಒझ ೀ‫ܭ‬લ਼ᕉნΠ‫ޑ‬ਔংǴOct4, Sox2, c-Myc, Klf4 ೭Ѥঁᙯ᝿Ӣη‫ޑ‬ೈқ ߄౜ໆܴᡉଯ‫ֹܭ‬ӄؒԖ࿶ၸલ਼ᕉნೀ౛‫ޑ‬ಒझȐკΒȑ Ƕ ௗ๱Ǵ‫ॺך‬ஒ Oct4, Sox2 ೭‫ঁٿ‬ᙯ᝿ӢηճҔ፦ᡏᙯࢉ(Plasmid transfection)‫ޑ‬БԄ໺ΕԴႵखजᠼᆢ҆ಒझύǴ࿶ၸϤঁλਔ‫ޑ‬ᙯࢉϐ ࡕǴ‫ע‬ಒझϩձᎦӧ҅த਼਻ᕉნ‫୻ޑ‬ᎦጃύаϷલ਼ᕉნ‫୻ޑ‬Ꭶጃ྽ ύǶΒΜѤλਔϐࡕ‫ڗܜ‬Ӛಔಒझ‫ ޑ‬RNAǴճҔ Real-time PCR ٰᢀჸ ಒझӧલ਼ᕉნϐΠ RNA ‫߄ޑ‬౜ໆ‫ک‬ӧ҅த਼਻ᕉნϐΠԖϙሶό ӕǶ‫ॺך‬ଷ೛ಒझ྽ύ‫ک‬खज༸ಒझၨԖᜢ߯‫୷ޑ‬Ӣӧલ਼ᕉნΠ߄౜ ໆ཮КၨଯǶ࿶җ Real-time PCR ჴᡍ‫ॺך‬ёа࣮‫ډ‬ǴӕਔճҔ፦ᡏᙯ . . 18.

(26) . ࢉ໺Ε Oct4, Sox2 ೭‫୷ঁٿ‬Ӣ‫ޑ‬ԴႵखजᠼᆢ҆ಒझϐ RNA ߄౜ໆӧ લ ਼ ᕉ ნ ྽ ύ ߄ ౜ ໆ ଯ ‫ ҅ ܭ‬த ਼ ᕉ ნ Π ‫ ޑ‬ಒ झ RNA ߄ ౜ ໆ Ƕ வ Real-time PCR ‫ޑ‬ኧᏵკ߄ȐკΟȑ྽ύёа࣮‫ډ‬Ǵ࿶ၸલ਼ᕉნೀ౛ၸ ࡕ‫ޑ‬खजᠼᆢ҆ಒझϐ Oct4, c-Myc ೭‫୷ঁٿ‬Ӣ߄౜ໆܴᡉଯ‫҅ܭ‬த਼ ਻ᕉნΠ‫ޑ‬ಒझ୷Ӣ߄౜ໆǶՠࢂǴSox2 ‫୷ޑ‬Ӣ߄౜ӧ࿶ၸલ਼ᕉნೀ ౛ϐࡕࠅؒԖܴᡉ΢ϲǶԶ Klf4 ୷Ӣ߄౜ӧ҅த਼ᕉნΠ‫ک‬લ਼ᕉნΠ ߾ؒԖϼεৡ౦Ƕ࿶җа΢‫ঁٿ‬ჴᡍǴ‫ॺך‬᛾ჴΑલ਼ᕉნჹ‫ ܭ‬Oct4 ‫߄ޑ‬౜‫ޑ‬ዴࢂԖ‫܌‬ᔅշ‫ޑ‬Ƕ. ಃΒ࿯ǵճҔ Oct4, Sox2 ፦ᡏӧલ਼ᕉნΠғԋᇨᏤ‫܄‬ӭфૈ༸ಒझ ፦ᡏᙯࢉࢂ΋໨ёаஒ DNA ፦ᡏᙯࢉ຾Εಒझਡ‫ೌמޑ‬Ƕ‫ॺך‬ჴᡍ ࠻೛ीΑճҔ፦ᡏᙯࢉ‫ޑ‬Б‫ݤ‬ଛӝલ਼ᕉნ(Hypoxia, 3% O2)‫ޑ‬ᔈҔٰ ғԋᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ޑ‬ჴᡍБ‫ݤ‬Ƕ२ӃஒԴႵखजᠼᆢ҆ಒझ୻Ꭶ ӧ 10 Ϧϩ‫ޑ‬ಒझ୻Ꭶҝ΢Ǵऊ 24 λਔಒझಒझኧໆ‫୼ى‬ϐࡕǴߡёа ໒‫ۈ‬፦ᡏᙯࢉ‫ޑ‬ჴᡍǶஒಒझ୻Ꭶన‫׳‬ඤԋ Opti-MEM ࡕǴᅀΕς࿶‫ک‬ Fugene HD (Roche)ϸᔈ‫ޑ‬፦ᡏ DNAǴOct4 аϷ Sox2Ƕᙯࢉ࿶ၸϤλਔ аࡕǴஒಒझ୻Ꭶన‫׳‬ඤӣԴႵखजᠼᆢ҆ಒझ‫୻ޑ‬ᎦనǴ٠Ъஒ຾Չ ፦ᡏᙯࢉၸࡕ‫ޑ‬ಒझ‫ܫ‬Εલ਼ᕉნ‫୻ޑ‬Ꭶጃ྽ύ୻ᎦΒΜѤλਔǶಃΟ Ϻӆ଺а΢୏բǴ٠Ъख़ፄѤԛǶӧಃΐϺ‫ޑ‬ਔংǴஒ࿶ၸѤԛ፦ᡏᙯ. . . 19.

(27) . ࢉ‫ޑ‬ಒझ୻Ꭶӧ෷Ꭶಒझϐ΢Ǵ‫؂‬Ϻ‫׳‬ඤԴႵखज༸ಒझ୻Ꭶనǵ࡭ុ ᢀჸǴ‫ޔ‬ԿԖᜪ՟ԴႵखज༸ಒझ‫ޑ‬ಒझიр౜ࣁЗȐკ΋ȑǶ‫ॺך‬ჴ ᡍ࠻Ǵӧஒ࿶ၸ፦ᡏᙯࢉ‫ޑ‬ԴႵखजᠼᆢ҆ಒझ୻Ꭶӧ෷Ꭶಒझϐ΢ࡕ ‫ޑ‬ಃΜΒϺǴ‫ܭ‬ᡉ༾᜔Πᢀჸว౜ΑԖᜪ՟ԴႵखज༸ಒझ‫ޑ‬ಒझიр ౜ȐკѤȑǶ‫ॺך‬ჴᡍ࠻ஒ‫ ࣁڮځ‬iPS-OSHǶࣁΑዴᇡԜಒझიࢂցࣁ ‫ڀ‬Ԗ‫ک‬ԴႵखज༸ಒझфૈ‫࣬ޑ‬՟‫ޑ‬ಒझǴ‫଺ॺך‬Αᝩж୻Ꭶ٠ճҔჴ ᡍ᛾ჴ‫ځ‬ӭૈ‫܄‬Ƕ. ಃΟ࿯ǵiPS-OSH ಒझύ༸ಒझ੝౦୷Ӣ‫߄ޑ‬౜ ӧԋф‫ޑ‬ճҔ፦ᡏᙯࢉ‫ک‬લ਼ᕉნғԋΑ iPS-OSH ϐࡕǴࣁΑዴᇡ iPS-OSH ࢂց‫ک‬खज༸ಒझ΋ኬ‫ڀ‬Ԗ༸ಒझ੝౦୷Ӣ߄౜Ǵ‫ॺך‬ճҔΑ PCR ϩ‫݋‬᛾ჴΑаΠ೭٤ԴႵखज༸ಒझ኱૶‫ޑ‬ϣғ‫ ܄‬mRANǺES cell associated transcript 1 (Ecat1), ES cell-expressed Ras (ERas), Nanog, myelocytomatosis oncogene (c-Myc), embryonal stem cell specific gene 1 (Esg1), Kruppel-like factor 4 (Klf4), POU domain, class 5, transcription factor 1 (Pou5f1, Oct4), RNA exonuclease 1 homolog (Rex1), SRY-box containing gene 2 (Sox2)Ƕӧ iPS-OSH ಒझύ೭٤༸ಒझ኱૶‫ޑ‬ϣғ‫܄‬ mRNA ‫߄ޑ‬౜ໆ‫ک‬ԴႵखज༸ಒझ‫߄ޑޣٿ‬౜࣬՟ǴӕਔΨ‫ ک‬iPS J ߄ ౜ໆ࣬՟Ƕՠࢂ೭٤༸ಒझ኱૶‫ޑ‬ϣғ‫ ܄‬mRNA ‫߄ޑ‬౜ໆӧ iPS-OSH ‫߄ޑ‬౜ࠅόӕ‫ܭ‬ԴႵखजᠼᆢ҆ಒझ‫߄ޑ‬౜ໆȐკϖȑ Ƕ. . . 20.

(28) . ಃѤ࿯ǵiPS-OSH ಒझӧᡏѦǵᡏϣ‫ޑ‬ӭૈ‫܄‬ ᝩжϐࡕ‫ޑ‬ಒझǴ΋೽ҽ‫ॺך‬ճҔΑᡵ‫܄‬ᕗለ䁙ࢉՅ‫ޑ‬Б‫ٰݤ‬ዴ ᇡǴ‫ޑॺך‬ಒझࢂցԖଯໆ߄౜ᡵ‫܄‬ᕗለ䁙Ƕᡵ‫܄‬ᕗለ䁙཮ଯໆ߄౜ӧ ‫ז‬ೲϩ຋ቚғ‫ޑ‬ಒझ྽ύǴӢԜёаҔٰᔠෳಒझࢂցࣁ༸ಒझǶҗკ Ϥ‫ॺך‬ёа࣮‫ډ‬ǴiPS-OSH ‫ޑ‬ᡵ‫܄‬ᕗለ䁙߄౜࣬྽ܴᡉǴࢂ‫ک‬ԴႵखज ༸ಒझ࣬՟‫ޑ‬ȐკϤȑ ǶࣁΑ‫׳‬຾΋‫ޑ؁‬ዴᇡ iPS-OSH ‫ޑ‬༸ಒझ੝‫܄‬Ǵ ‫ॺך‬٬ҔΑаΠ‫ޑ‬༸ಒझ኱૶ٰբխࣝᑻӀࢉՅǶх֖Α Oct4ǵSox2ǵ NanogǵSSEA1ǵTra-1-60ǵTra-1-81ǶճҔխࣝᑻӀࢉՅ‫ޑ‬Б‫ݤ‬Ǵ‫ॺך‬ ёаዴᇡ iPS-OSH ಒझ‫ک‬ԴႵखज༸ಒझ‫߄ޑ‬౜࣬՟Ǵ೿Ԗ߄౜೭٤༸ ಒझ኱૶ȐკΎȑ Ƕ ྽ iPS-OSH ёаᙖҗᝌੌ୻ᎦԶ‫׎‬ԋखजౚ(embryonic body, EB) ȐკΖ(a)ȑǴж߄೭٤ಒझܰ‫ܭ‬໒‫ۈ‬ϩϯԋΟঁखቫ‫ޑ‬ӚᅿಒझǶӧᝌ ੌ୻ᎦΟϺϐࡕǴஒ೭٤ᜪ՟खजౚ‫ޑ‬ಒझი୻Ꭶ‫ܭ‬ႣӃӧ୻Ꭶҝۭ೽ Ҕ 0.1%‫ ޑ‬Gelatin ଺༡ቫ‫ ޑ‬24-well ಒझ୻Ꭶҝϐ΢ǴεऊӧΟ‫ډ‬ϖϺϐ ࡕǴ೭٤ᜪ՟खजౚ‫ޑ‬ಒझიߡ཮ϩϯԋӚᅿ‫׎‬ᄊ‫ޑ‬ಒझǴх֖ઓ࿶ಒ झǵԼԺಒझǵϣखቫಒझ฻฻ǶխࣝᑻӀࢉՅϩ‫݋‬᛾ჴ೭٤όӕᜪࠠ ‫ޑ‬ಒझӧҔаΠ‫ޑ‬኱૶ࢉՅࡕԖ߄౜ǶTuj1 ȐѦखቫ኱૶ȑ ǴASMȐύ खቫ኱૶ȑǴGata4Ȑϣखቫ኱૶ȑȐკΖ(b)ȑ Ƕ ќѦǴ྾जዦ‫׎ޑ‬ԋࢂஒ iPS-OSH ճҔҜΠ‫ݙ‬৔‫ޑ‬БԄ‫ݙ‬৔‫ډ‬ᇘႵ . . 21.

(29) . ‫ޑ‬ङ೽ҜΠϐύǶ࿶ၸϤ‫ډ‬ΖຼϐࡕǴ཮ว౜྾जዦ‫׎‬ԋӧᇘႵ‫ޑ‬ङ ೽Ƕ೭٤྾जዦх֖ΑΟखቫ‫ޑ‬ಔᙃǴх֖ǺϣखቫԖ‫֎ڥ‬ၰ΢Ҝಒझ (Respiratory epithelium)‫ک‬ဉ΢Ҝಒझ(Gut epithelium)ǴύखቫԖԼԺಒ झ(Muscle)‫ک‬િެಒझ(Adipose)ǴѦखቫԖ߄Ҝ(Epidermis)‫ک‬ઓ࿶ಔᙃ (Neural tissue)Ȑკΐȑ Ƕ᛾ჴΑ iPS-OSH ಒझӧᡏϣаϷᡏѦࣣ‫ڀ‬Ԗӭ ૈ‫܄‬Ƕ. ಃϖ࿯ǵճҔ Microarray ϩ‫ ݋‬iPS-OSH ‫ޑ‬ӄ୷Ӣ߄౜ ‫ॺך‬ΨճҔΑ Microarray ϩ‫݋‬ΑԴႵखज༸ಒझ(ES)ǵԴႵखजᠼ ᆢ҆ಒझ(MEF)ǵВҁ‫ޑ‬ᇨᏤ‫܄‬ӭфૈ༸ಒझ(iPS J)аϷ iPS-OSH ೭Ѥ ᅿಒझӧ୷Ӣ߄౜΢ԖՖ࣬՟‫࣬ک‬౦‫܄‬ǶҗკΜ‫ॺך‬ёа࣮‫ډ‬ǴԴႵख ज༸ಒझǵiPS J аϷ‫ॺך‬ჴᡍ࠻ғԋ‫ ޑ‬iPS-OSHǴ೭Οᅿಒझӧ୷Ӣ‫ޑ‬ ߄౜΢ࢂКၨ࣬՟‫ޑ‬ǴԶԴႵखजᠼᆢ҆ಒझ߾‫ځک‬дΟ‫୷ޑޣ‬Ӣ߄౜ Ԗၨε‫ޑ‬ৡ౦‫܄‬ǶЀ‫ࢂځ‬ǴOct4, Sox2, Nanog ೭Οঁ୷ӢǴӧԴႵखज ༸ಒझǵВҁ‫ޑ‬ᇨᏤ‫܄‬ӭфૈ༸ಒझаϷ iPS-OSH ೭Οᅿಒझύ‫߄ޑ‬౜ ൳Яࢂ࣬ӕ‫ޑ‬ȐკΜȑ ǶӧკΜ΋྽ύ‫ॺך‬ёа࣮‫ ډ‬iPS-OSH ‫ ک‬iPS J ӧӄ୷Ӣ‫߄ޑ‬౜΢ࢂКၨ࣬߈‫ޑ‬ǴԶ‫ޣٿ‬ΞၟԴႵखज༸ಒझ߄౜ໆ࣬ ߈ǴԴႵखजᠼᆢ҆ಒझ‫ޑ‬ӄ୷Ӣ߄౜߾‫ک‬ќΟ‫ޣ‬ಒझၨࣁ࣬౦ȐკΜ ΋ȑ Ƕ!. . . 22.

(30) . ಃϤ࿯ǵ ճҔᇨᏤ‫܄‬ӭфૈ༸ಒझғԋઓ࿶༸ಒझ Generation of neural stem cells (NSC) from iPS-OSH ᇨᏤ‫܄‬ӭфૈ༸ಒझࣁ‫ڀ‬Ԗӭૈ‫ޑ܄‬༸ಒझǴѬ‫ڀ‬ԖϩϯࣁΟঁख ቫ‫ޑ‬ӚᅿಒझϐૈΚǶӧ೭ᜐǴ‫ॺך‬གྷाճҔ‫ॺך‬ғԋ‫ ޑ‬iPS-OSH ٰ຾ ՉϩϯჴᡍǴ‫׆‬ఈૈճҔ iPS-OSH ϩϯԋઓ࿶༸ಒझǴ٠Ъૈ୼ӧ҂ٰ ᔈҔӧύ॥‫ݯޑ‬ᕍ΢Ƕ‫ॺך‬ճҔᝌੌ୻ᎦӃᡣ iPS-OSH ‫׎‬ԋखजౚǴӆ ճҔғߏӢη B27 ᡣखजౚғߏԋᖿӛઓ࿶‫ޑ‬ઓ࿶ౚǶനࡕǴᡣ೭٤ઓ ࿶ౚϩϯࣁઓ࿶༸ಒझǶ‫ॺך‬ճҔ PrP ‫ ک‬Tuj1 ೭‫ঁٿ‬ઓ࿶༸ಒझ‫ޑ‬኱૶ ٰዴᇡ‫ॺך‬ϩϯрٰ‫ޑ‬ಒझࢂցࣁઓ࿶༸ಒझǴҗკΜΒ่݀ᡉҢǴ೭ ٤җ iPS-OSH ϩϯғԋ‫ޑ‬ಒझዴჴԖ߄౜ PrP ‫ ک‬Tuj1 ೭‫ঁٿ‬ઓ࿶༸ಒ झ‫ޑ‬኱૶ǴӢԜёа᛾ჴ‫ࣁځ‬ઓ࿶༸ಒझ (კΜΒ)Ƕ. . . 23.

(31) . ಃѤകǵ૸ፕ ᇨᏤ‫܄‬ӭфૈ༸ಒझനԐࢂӧ 2006 ԃҗВҁ‫ޑ‬΋ঁი໗ࣴ‫ز‬ԋф ‫ޑ‬ǶдॺճҔΑѤঁᙯᒵӢηǺOct4, Sox2, c-Myc, Klf4 ‫ࢥੰک‬ၩᡏ‫ޑ‬Б Ԅԋф‫ޑ‬ஒԋᡏಒझᡂӣӭфૈ༸ಒझ(9)ǶҞ߻Ǵࣴ‫ز‬ว౜Α೚ӭᇙ೷ ᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ޑ‬БԄǴх֖Ǻ٬Ҕϸᙯᒵੰࢥ(9, 36)ǵᄌ‫ࢥੰ܄‬ (37)ǵဏੰࢥ(38)ǵख़ಔೈқ(39)ǵᙯӢη(40)ǵ༾ࠠਡᑗਡለ(41)аϷ፦ ᡏᙯࢉ(13, 14)ǶᏃᆅ፦ᡏᙯࢉς࿶ᗉ໒Α٬Ҕੰࢥ‫ޑ‬БԄٰᇙ೷ᇨᏤ‫܄‬ ӭфૈ༸ಒझǴՠࢂᗋࢂค‫ݤ‬ᗉ໒٬Ҕठᕎ୷ӢǶᇨᏤ‫܄‬ӭфૈ༸ಒझ നЬा‫ޑ‬Ҟ‫ࢂޑ‬Ҕٰ଺ᖏ‫ݯ׉‬ᕍǴӢԜሡा΋ঁ࣬྽Ӽӄ‫ޑ‬Б‫ٰݤ‬ғԋ ᇨᏤ‫܄‬ӭфૈ༸ಒझǶԶӧ‫ॺך‬ჴᡍ೛ी྽ύǴ‫ॺך‬၂๱ѐᗉ໒٬Ҕੰ ࢥаϷठᕎ୷Ӣٰ٬ளԋᡏಒझᡂӣӭфૈ༸ಒझǶ‫ॺך‬ԋф‫ޑ‬ள‫ډ‬΋ ঁၨࣁӼӄ‫ޑ‬Б‫ٰݤ‬ғԋᇨᏤ‫܄‬ӭфૈ༸ಒझǴ೭ঁБ‫ݤ‬ჹ‫ܭ‬҂ٰӧᖏ ‫ݯ׉‬ᕍ΢Ԗၨε‫ޑ‬ᔅշǶ ӧ 2009 ԃǴВҁ‫ ޑ‬Yoshida ࣴ‫ز‬ი໗ว౜Αલ਼ᕉნԖշ‫ܭ‬ғԋᇨ Ꮴ‫܄‬ӭфૈ༸ಒझ (14)Ƕ‫ޑॺך‬ჴᡍ೛ी‫ک‬дॺϐ໔Ԗ‫ঁٿ‬Ьा‫ޑ‬όӕ ᗺ (14)ǶനЬा‫ޑ‬όӕᗺӧ‫ॺךܭ‬ჴᡍ࠻ѝ٬ҔΑ‫ঁٿ‬ᙯᒵӢηٰғԋ ᇨᏤ‫܄‬ӭфૈ༸ಒझǴ٠Ъᗉ໒٬Ҕठᕎ୷ӢǶ೭΋ᗺჹ‫ܭ‬҂ٰӧᖏ‫׉‬ ‫ݯ‬ᕍ΢ࢂΜϩख़ा‫ޑ‬ǴԶЪ٬ளӧ٬ҔᇨᏤ‫܄‬ӭфૈ༸ಒझ଺ಒझ‫ݯ‬ᕍ ‫ޑ‬ၸำၨࣁӼӄǶќѦǴჹ‫ܭ‬લ਼ᕉნ‫ޑ‬٬ҔΨֹӄόӕǶ‫ॺך‬ჴᡍ࠻ . . 24.

(32) . ѝԖӧ଺ֹ፦ᡏᙯࢉϐࡕᡣԋᡏಒझೀ‫ܭ‬લ਼ᕉნϐΠ 24 λਔǴೀ‫ܭ‬ લ਼ᕉნΠ‫ޑ‬ਔ໔ᕴӅࢂ 96 λਔǶԶϐ߻‫ޑ‬ჴᡍ߾ࢂᡣԋᡏಒझೀ‫ܭ‬ લ਼ᕉნΠԿϿ 12 Ϻ(14)Ƕ‫ॺך‬ჴᡍ࠻ғԋᇨᏤ‫܄‬ӭфૈ༸ಒझ‫ޑ‬Б‫ݤ‬ Кϐ߻‫ޑ‬ჴᡍࣴ‫ز‬ၨࣁӼӄǶ ‫ॺך‬ჴᡍ࠻ࢂ२ԛѝճҔ‫ঁٿ‬ᙯᒵӢη٠Ъᗉ໒ठᕎ୷ӢаϷੰࢥ ၩᡏǴߡёаԋф‫ޑ‬ғԋᇨᏤ‫܄‬ӭфૈ༸ಒझǶӧғԋᇨᏤ‫܄‬ӭфૈ༸ ಒझ‫ޑ‬ၸำύǴᗉխ٬Ҕठᕎ୷ӢаϷੰࢥၩᡏǴ‫܈‬೚ёаග‫ٮ‬ӧᇨᏤ ‫܄‬ӭфૈ༸ಒझ‫زࣴޑ‬΢΋ঁԖਏ‫ޑ‬ᡏѦࣴ‫ز‬ኳࠠǴаϷග‫ٮ‬΋٤ёૈ ‫ݯޑ‬ᕍՉࣁ΋ঁཥ‫ޑ‬ᇨᏤ‫܄‬ӭфૈ༸ಒझٰྍǶ҂ٰǴ‫ॺך‬ஒ‫ݙ‬ख़ӧϩ ϯჴᡍ΢Ǵ٠ЪճҔ೭٤ಒझ଺ಒझ‫ݯ‬ᕍǶ. . . 25.

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(35) . 25.. 26.. Cell Struct Funct 26, 137-148 Hu, T., Liu, S., Breiter, D. R., Wang, F., Tang, Y., and Sun, S. (2008) Octamer 4 small interfering RNA results in cancer stem cell-like cell apoptosis. Cancer Res 68, 6533-6540 Eminli, S., Utikal, J., Arnold, K., Jaenisch, R., and Hochedlinger, K. (2008) Reprogramming of neural progenitor cells into induced pluripotent stem cells in. 27.. the absence of exogenous Sox2 expression. Stem Cells 26, 2467-2474 Kim, J. B., Zaehres, H., Wu, G., Gentile, L., Ko, K., Sebastiano, V., Arauzo-Bravo, M. J., Ruau, D., Han, D. W., Zenke, M., and Scholer, H. R. (2008) Pluripotent stem cells induced from adult neural stem cells by reprogramming with two. 28.. factors. Nature 454, 646-650 Niwa, H., Miyazaki, J., and Smith, A. G. (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. 29.. Genet 24, 372-376 Shimozaki, K., Nakashima, K., Niwa, H., and Taga, T. (2003) Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in. 30.. neurogenesis-inducing cultures. Development 130, 2505-2512 Boyer, L. A., Lee, T. I., Cole, M. F., Johnstone, S. E., Levine, S. S., Zucker, J. P., Guenther, M. G., Kumar, R. M., Murray, H. L., Jenner, R. G., Gifford, D. K., Melton, D. A., Jaenisch, R., and Young, R. A. (2005) Core transcriptional. 31.. 32.. regulatory circuitry in human embryonic stem cells. Cell 122, 947-956 Boiani, M., Eckardt, S., Scholer, H. R., and McLaughlin, K. J. (2002) Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes Dev 16, 1209-1219 Jiang, Y., Jahagirdar, B. N., Reinhardt, R. L., Schwartz, R. E., Keene, C. D., Ortiz-Gonzalez, X. R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., Du, J., Aldrich, S., Lisberg, A., Low, W. C., Largaespada, D. A., and Verfaillie, C. M. (2002) Pluripotency of mesenchymal stem cells derived from adult marrow.. 33.. Nature 418, 41-49 Tai, M. H., Chang, C. C., Kiupel, M., Webster, J. D., Olson, L. K., and Trosko, J. E. (2005) Oct4 expression in adult human stem cells: evidence in support of the. 34. 35.. stem cell theory of carcinogenesis. Carcinogenesis 26, 495-502 Kiefer, J. C. (2007) Back to basics: Sox genes. Dev Dyn 236, 2356-2366 Avilion, A. A., Nicolis, S. K., Pevny, L. H., Perez, L., Vivian, N., and Lovell-Badge, R. (2003) Multipotent cell lineages in early mouse development. 36.. depend on SOX2 function. Genes Dev 17, 126-140 Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007) Induction of pluripotent stem cells from adult human. . . 28.

(36) . 37.. fibroblasts by defined factors. Cell 131, 861-872 Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, II, and Thomson, J. A. (2007) Induced pluripotent stem cell lines derived from human. 38.. somatic cells. Science 318, 1917-1920 Stadtfeld, M., Nagaya, M., Utikal, J., Weir, G., and Hochedlinger, K. (2008) Induced pluripotent stem cells generated without viral integration. Science 322, 945-949. 39.. Kim, D., Kim, C. H., Moon, J. I., Chung, Y. G., Chang, M. Y., Han, B. S., Ko, S., Yang, E., Cha, K. Y., Lanza, R., and Kim, K. S. (2009) Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell. 40.. 41.. Stem Cell 4, 472-476 Woltjen, K., Michael, I. P., Mohseni, P., Desai, R., Mileikovsky, M., Hamalainen, R., Cowling, R., Wang, W., Liu, P., Gertsenstein, M., Kaji, K., Sung, H. K., and Nagy, A. (2009) piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458, 766-770 Lin, S. L., Chang, D. C., Chang-Lin, S., Lin, C. H., Wu, D. T., Chen, D. T., and Ying, S. Y. (2008) Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state. RNA 14, 2115-2124. . . 29.

(37) . კ߄ ߄΋ǵᆫӝ䁙ೱᙹϸᔈЇη‫ׇ‬ӈ΋ំ߄ Gene Taqman64mActin Taqman6-m OCT4. Forward Primer. Reverse Primer. Taqman probe. CTAAGGCCAACCGTGAAAAG. ACCAGAGGCATACAGGGACA. Roche Universal Probes 64 Roche Universal Probes 6. GAGGCTACAGGGACACCTTTC. GTGCCAAAGTGGGGACCT. GTGCGTGACATCAAAGAGAAGC. TGGATGCCACAGGATTCCATAC. Q-mMYC. CATTCAAGCAGACGAGCA. CGAGTTAGGTCAGTTTATGCAC. Q-mKLF4. CCTTTCAGTGCCAGAAGT. ACTACGTGGGATTTAAAAGTGC. Q-mOct. GCCAATCAGCTTGGGCTAGA. TTCTGGCGCCGGTTACA. Q-mSOX. AGGGCTGGACTGCGAACTG. TTTGCACCCCTCCCAATTC. Ecat1. TGTGGGGCCCTGAAAGGCGAGCTGAGAT. ATGGGCCGCCATACGACGACGCTCAACT. Esg1. GAAGTCTGGTTCCTTGGCAGGATG. ACTCGATACACTGGCCTAGC. CAG GTG TTT GAG GGT AGC TC. CGG TTC ATC ATG GTA CAG TC. Q-m Actin. Nanog Eras. ACTGCCCCTCATCAGACTGCTACT. CACTGCCTTGTACTCGGGTAGCTG. Oct3/4. TCTTTCCACCAGGCCCCCGGCTC. TGCGGGCGGACATGGGGAGATCC. Rex1. ACGAGTGGCAGTTTCTTCTTGGGA. TATGACTCACTTCCAGGGGGCACT. Sox2 Klf4. TAGAGCTAGACTCCGGGCGATGA GCGAACTCACACAGGCGAGAAACC. TTGCCTTAAACAAGACCACGAAA TCGCTTCCTCTTCCTCCGACACA. c-Myc. TGACCTAACTCGAGGAGGAGCTGGAATC. AAGTTTGAGGCAGTTAAAATTATGGCTGAAGC. ͊actin. GTGGGGCGCCCCAGGCACCA. CTCCTTAATGTCACGCACGATTTC. . . 30.

(38) . კ΋ǵճҔ፦ᡏᙯࢉ‫ک‬લ਼ᕉნғԋᇨᏤ‫܄‬ӭфૈ༸ಒझǶ. . . 31.

(39) . 0. 4. 8. 24. (hours) Oct4 Sox2 cMyc. Klf4 Actin. კΒǵஒखजᠼᆢ҆ಒझ‫ܭ‬લ਼ᕉნΠϩձ୻Ꭶ 0, 4, 8, 24 λਔϐࡕǴ ճҔՋБᏀᗺ‫ݤ‬ϩ‫݋‬खजᠼᆢ҆ಒझύ Oct4, Sox2, c-Myc, Klf4 ೭Ѥঁ ೈқ‫߄ޑ‬౜ໆǶ. . . 32.

(40) . კΟǵճҔ Real-time PCR ϩ‫݋‬ԴႵखजᠼᆢ҆ಒझ࿶җ፦ᡏᙯࢉ Oct4, Sox2 ೭‫୷ঁٿ‬ӢϐࡕǴӧ҅த਼਻‫ރ‬ᄊΠ‫ک‬લ਼ᕉნύǴOct4, Sox2, c-Myc, Klf4 ೭ Ѥ ঁ ୷ Ӣ ‫ ޑ‬mRNA ߄ ౜ ໆ Ƕ Control: ፦ ᡏ ᙯ ࢉ Ε pCDNA3.1 ೭ঁ‫ޜ‬ၩᡏǶOS: ፦ᡏᙯࢉΕ Oct4 ‫ ک‬Sox2 ೭‫ঁٿ‬ᙯᒵӢηǶ Control H: ፦ᡏᙯࢉΕ pCDNA3.1 ೭ঁ‫ޜ‬ၩᡏࡕೀ‫ܭ‬લ਼ᕉნΠ 24 λ ਔǶOSH: ፦ᡏᙯࢉΕ Oct4 ‫ ک‬Sox2 ೭‫ঁٿ‬ᙯᒵӢηࡕೀ‫ܭ‬લ਼ᕉნΠ 24 λਔǶɀ:߄Ң p<0.05Ƕ. . . 33.

(41) . კѤǵiPS-OSH ӧᡉ༾᜔Π‫ޑ‬ಒझ‫׎‬ᄊǶѰკࣁӧᡉ༾᜔Πว౜ᅪ՟ख ज༸ಒझਔ‫ޑ‬ಒझࠠᄊǶѓკࣁஒѰკ‫ޑ‬ಒझ଺ᝩжϐࡕ‫ޑ‬ಒझࠠᄊǶ. . . 34.

(42) . კϖǵճҔ PCR ϩ‫ ݋‬iPS-OSH ಒझύ൳ঁϣғ‫ ܄‬mRNA ‫߄ޑ‬౜ໆǴ٠ Ъ‫ک‬ԴႵखजᠼᆢ҆ಒझ(MEF)ǴԴႵखज༸ಒझ(ES)ǴВҁ‫ޑ‬ᇨᏤ‫܄‬ ӭфૈ༸ಒझ(iPS J)଺КၨǶ. . . 35.

(43) . კϤǵiPS-OSHǴճҔᡵ‫܄‬ᕗለ䁙ࢉՅϩ‫݋‬Ǵ᛾ჴ‫ڀځ‬ԖԴႵखज༸ಒ झϐ੝‫ ܄‬Ƕ. . . 36.

(44) . კΎǵճҔ Nanog, Oct4, Sox2, SSEA1, Tra-1-60, Tra-1-81 ೭٤༸ಒझ marker ٰዴᇡ iPS-OSH ‫ޑ‬༸ಒझ੝‫܄‬Ƕ. . . 37.

(45) . (a). (b). კΖǵ(a)ஒ iPS-OSH ճҔᝌੌ୻Ꭶ‫׎܌‬ԋ‫ޑ‬खजౚǶ(b) iPS-OSH ಒझ ‫׎‬ԋ‫ޑ‬खजౚϩϯϐࡕғԋ‫ޑ‬ΟखቫಒझǶ. . . 38.

(46) . კΐǵஒ iPS-OSH ҜΠ‫ݙ‬৔ΕᇘႵङ೽ҜΠϤ‫ډ‬Ζຼ‫׎܌‬ԋ‫ޑ‬྾जዦϪ ТǴ֖ԖΟঁखቫ‫ޑ‬ಒझǶ. . . 39.

(47) . კΜǵMicroarray ϩ‫݋‬खजᠼᆢ҆ಒझ(MEF)Ǵखजᠼᆢ༸ಒझ(ES)ǴВ ҁ‫ޑ‬ᇨᏤ‫܄‬ӭфૈ༸ಒझ(iPS J)аϷ iPS-OSH ӧ୷Ӣ߄౜΢‫࣬ޑ‬՟Ϸ࣬ ౦‫܄‬Ƕ. . . 40.

(48) . კΜ΋ǵ Microarray ϩ‫݋‬खजᠼᆢ҆ಒझ(MEF)Ǵखजᠼᆢ༸ಒझ(ES)Ǵ Вҁ‫ޑ‬ᇨᏤ‫܄‬ӭфૈ༸ಒझ(iPS J)аϷ iPS-OSH ӧ୷Ӣ߄౜΢‫࣬ޑ‬՟Ϸ ࣬౦‫܄‬Ƕ. . . 41.

(49) . კΜΒǵiPS-OSH ಒझ‫׎‬ԋ‫ޑ‬ઓ࿶ౚϩϯϐࡕғԋ‫ޑ‬ઓ࿶༸ಒझǶճҔ Prp ‫ ک‬Tuj1 ೭‫ঁٿ‬ઓ࿶༸ಒझ‫ ޑ‬marker ٰዴᇡಒझࢂց‫ڀ‬Ԗઓ࿶༸ಒझ ‫ޑ‬੝‫܄‬Ƕ. . . 42.

(50)