stem cells (NSC) from iPS-OSH
२ӃஒᎦΑᇨᏤ܄ӭфૈ༸ಒझޑಒझᎦҝҔ PBS ؑࢱٿ ԛǴௗуΕ 1c.c ޑ 0.1mM trypsin/1mM EDTA solution (GIBCO BRL) ӧ 37ʚΠᎦ 1 ϩដǶуΕ 10c.c ޑ EB formation medium ࡕǴஒಒझѺ
ᎦҝύǶ ಒझஏࡋࣁ well ύ֖Ԗ 3.2*10^6 ᗭಒझǶ ܫΕΒ਼
ϯᅹಒझᎦጃ(5% CO2, 37ʚ)ύᎦϖϺǴ٬ઓౚϩϯԋઓ༸
ಒझǶӧҁჴᡍύ٬Ҕޑ EB 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
ಃΟകǵ่݀ БᏀᗺݤ (Western Blot)ٰዴᇡલ਼ᕉნჹܭ Oct4, Sox2, c-Myc, Klf4 ೭ ѤঁᙯӢηޑೈқ߄ໆࢂցԖ܌ቹៜǶךॺวǴखजᠼᆢ҆ಒझ
ೀܭલ਼ᕉნΠޑਔংǴOct4, Sox2, c-Myc, Klf4 ೭ѤঁᙯӢηޑೈқ ߄ໆܴᡉଯܭֹӄؒԖၸલ਼ᕉნೀޑಒझȐკΒȑǶ
ௗǴךॺஒ Oct4, Sox2 ೭ٿঁᙯӢηճҔ፦ᡏᙯࢉ(Plasmid transfection)ޑБԄΕԴႵखजᠼᆢ҆ಒझύǴၸϤঁλਔޑᙯࢉϐ
ࢉΕ Oct4, Sox2 ೭ٿঁ୷ӢޑԴႵखजᠼᆢ҆ಒझϐ RNA ߄ໆӧ લ ਼ ᕉ ნ ύ ߄ ໆ ଯ ܭ ҅ த ਼ ᕉ ნ Π ޑ ಒ झ RNA ߄ ໆ Ƕ வ Real-time PCRޑኧᏵკ߄ȐკΟȑύёа࣮ډǴၸલ਼ᕉნೀၸ ࡕޑखजᠼᆢ҆ಒझϐ Oct4, c-Myc ೭ٿঁ୷Ӣ߄ໆܴᡉଯܭ҅த਼
ᕉნΠޑಒझ୷Ӣ߄ໆǶՠࢂǴSox2 ޑ୷Ӣ߄ӧၸલ਼ᕉნೀ
ϐࡕࠅؒԖܴᡉϲǶԶ Klf4 ୷Ӣ߄ӧ҅த਼ᕉნΠکલ਼ᕉნΠ
߾ؒԖϼεৡ౦ǶҗаٿঁჴᡍǴךॺჴΑલ਼ᕉნჹܭ Oct4 ޑ߄ޑዴࢂԖ܌ᔅշޑǶ
ಃΒǵճҔ Oct4, Sox2 ፦ᡏӧલ਼ᕉნΠғԋᇨᏤ܄ӭфૈ༸ಒझ
፦ᡏᙯࢉࢂёаஒ DNA ፦ᡏᙯࢉΕಒझਡޑמೌǶךॺჴᡍ
࠻ीΑճҔ፦ᡏᙯࢉޑБݤଛӝલ਼ᕉნ(Hypoxia, 3% O2)ޑᔈҔٰ
ғԋᇨᏤ܄ӭфૈ༸ಒझޑჴᡍБݤǶ२ӃஒԴႵखजᠼᆢ҆ಒझᎦ ӧ 10 ϦϩޑಒझᎦҝǴऊ 24 λਔಒझಒझኧໆىϐࡕǴߡёа ໒ۈ፦ᡏᙯࢉޑჴᡍǶஒಒझᎦన׳ඤԋ Opti-MEM ࡕǴᅀΕςک Fugene HD (Roche)ϸᔈޑ፦ᡏ DNAǴOct4 аϷ Sox2ǶᙯࢉၸϤλਔ аࡕǴஒಒझᎦన׳ඤӣԴႵखजᠼᆢ҆ಒझޑᎦనǴ٠ЪஒՉ
፦ᡏᙯࢉၸࡕޑಒझܫΕલ਼ᕉნޑᎦጃύᎦΒΜѤλਔǶಃΟ ϺӆаբǴ٠Ъख़ፄѤԛǶӧಃΐϺޑਔংǴஒၸѤԛ፦ᡏᙯ
ࢉޑಒझᎦӧᎦಒझϐǴϺ׳ඤԴႵखज༸ಒझᎦనǵុ 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༸ಒझޑϣғ܄
mRNAޑ߄ໆکԴႵखज༸ಒझٿޣޑ߄࣬՟ǴӕਔΨک iPS J ߄
ໆ࣬՟Ƕՠࢂ೭٤༸ಒझޑϣғ܄ mRNA ޑ߄ໆӧ iPS-OSH ޑ߄ࠅόӕܭԴႵखजᠼᆢ҆ಒझޑ߄ໆȐკϖȑǶ
ಃѤǵiPS-OSH ಒझӧᡏѦǵᡏϣޑӭૈ܄
iPS-OSH ёаᙖҗᝌੌᎦԶԋखजౚ(embryonic body, EB) ȐკΖ(a)ȑǴж߄೭٤ಒझܰܭ໒ۈϩϯԋΟঁखቫޑӚᅿಒझǶӧᝌ
ޑङҜΠϐύǶၸϤډΖຼϐࡕǴว྾जዦԋӧᇘႵޑङ
Ƕ೭٤྾जዦх֖ΑΟखቫޑಔᙃǴх֖ǺϣखቫԖڥ֎ၰҜಒझ (Respiratory epithelium)کဉҜಒझ(Gut epithelium)ǴύखቫԖԼԺಒ झ(Muscle)کિެಒझ(Adipose)ǴѦखቫԖ߄Ҝ(Epidermis)کઓಔᙃ (Neural tissue)ȐკΐȑǶჴΑ iPS-OSH ಒझӧᡏϣаϷᡏѦࣣڀԖӭ
ૈ܄Ƕ
ಃϖǵճҔ Microarray ϩ iPS-OSH ޑӄ୷Ӣ߄
ךॺΨճҔΑ Microarray ϩΑԴႵखज༸ಒझ(ES)ǵԴႵखजᠼ ᆢ҆ಒझ(MEF)ǵВҁޑᇨᏤ܄ӭфૈ༸ಒझ(iPS J)аϷ iPS-OSH ೭Ѥ ᅿಒझӧ୷Ӣ߄ԖՖ࣬՟ک࣬౦܄ǶҗკΜךॺёа࣮ډǴԴႵख ज༸ಒझǵiPS J аϷךॺჴᡍ࠻ғԋޑ iPS-OSHǴ೭Οᅿಒझӧ୷Ӣޑ ߄ࢂКၨ࣬՟ޑǴԶԴႵखजᠼᆢ҆ಒझ߾کځдΟޣޑ୷Ӣ߄
Ԗၨεޑৡ౦܄ǶЀځࢂǴOct4, Sox2, Nanog ೭Οঁ୷ӢǴӧԴႵखज
༸ಒझǵВҁޑᇨᏤ܄ӭфૈ༸ಒझаϷ iPS-OSH ೭Οᅿಒझύޑ߄
൳Яࢂ࣬ӕޑȐკΜȑǶӧკΜύךॺёа࣮ډ iPS-OSH ک iPS J ӧӄ୷Ӣޑ߄ࢂКၨ࣬߈ޑǴԶٿޣΞၟԴႵखज༸ಒझ߄ໆ࣬
߈ǴԴႵखजᠼᆢ҆ಒझޑӄ୷Ӣ߄߾کќΟޣಒझၨࣁ࣬౦ȐკΜ
ȑǶ!
ಃϤǵ ճҔᇨᏤ܄ӭфૈ༸ಒझғԋઓ༸ಒझ Generation of neural stem cells (NSC) from iPS-OSH
ᇨᏤ܄ӭфૈ༸ಒझࣁڀԖӭૈ܄ޑ༸ಒझǴѬڀԖϩϯࣁΟঁख ቫޑӚᅿಒझϐૈΚǶӧ೭ᜐǴךॺགྷाճҔךॺғԋޑ iPS-OSH ٰ
ՉϩϯჴᡍǴ׆ఈૈճҔ iPS-OSH ϩϯԋઓ༸ಒझǴ٠Ъૈӧ҂ٰ
ᔈҔӧύ॥ޑݯᕍǶךॺճҔᝌੌᎦӃᡣ iPS-OSH ԋखजౚǴӆ ճҔғߏӢη B27 ᡣखजౚғߏԋᖿӛઓޑઓౚǶനࡕǴᡣ೭٤ઓ
ౚϩϯࣁઓ༸ಒझǶךॺճҔ PrP ک Tuj1 ೭ٿঁઓ༸ಒझޑ
ٰዴᇡךॺϩϯрٰޑಒझࢂցࣁઓ༸ಒझǴҗკΜΒ่݀ᡉҢǴ೭ ٤җ iPS-OSH ϩϯғԋޑಒझዴჴԖ߄ PrP ک Tuj1 ೭ٿঁઓ༸ಒ झޑǴӢԜёаჴځࣁઓ༸ಒझ (კΜΒ)Ƕ
ಃѤകǵፕ
ᇨᏤ܄ӭфૈ༸ಒझനԐࢂӧ 2006 ԃҗВҁޑঁი໗ࣴزԋф ޑǶдॺճҔΑѤঁᙯᒵӢηǺOct4, Sox2, c-Myc, Klf4 کੰࢥၩᡏޑБ Ԅԋфޑஒԋᡏಒझᡂӣӭфૈ༸ಒझ(9)ǶҞǴࣴزวΑӭᇙ
ѝԖӧֹ፦ᡏᙯࢉϐࡕᡣԋᡏಒझೀܭલ਼ᕉნϐΠ 24 λਔǴೀܭ લ਼ᕉნΠޑਔ໔ᕴӅࢂ 96 λਔǶԶϐޑჴᡍ߾ࢂᡣԋᡏಒझೀܭ લ਼ᕉნΠԿϿ 12 Ϻ(14)Ƕךॺჴᡍ࠻ғԋᇨᏤ܄ӭфૈ༸ಒझޑБݤ КϐޑჴᡍࣴزၨࣁӼӄǶ
ךॺჴᡍ࠻ࢂ२ԛѝճҔٿঁᙯᒵӢη٠Ъᗉ໒ठᕎ୷ӢаϷੰࢥ
ၩᡏǴߡёаԋфޑғԋᇨᏤ܄ӭфૈ༸ಒझǶӧғԋᇨᏤ܄ӭфૈ༸
ಒझޑၸำύǴᗉխ٬Ҕठᕎ୷ӢаϷੰࢥၩᡏǴ܈ёаගٮӧᇨᏤ
܄ӭфૈ༸ಒझޑࣴزঁԖਏޑᡏѦࣴزኳࠠǴаϷගٮ٤ёૈ
ޑݯᕍՉࣁঁཥޑᇨᏤ܄ӭфૈ༸ಒझٰྍǶ҂ٰǴךॺஒݙख़ӧϩ ϯჴᡍǴ٠ЪճҔ೭٤ಒझಒझݯᕍǶ
ୖԵЎǺ
1. Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., Scholer, H., and Smith, A. (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.
Cell 95, 379-391
2. Scholer, H. R., Ruppert, S., Suzuki, N., Chowdhury, K., and Gruss, P. (1990) New type of POU domain in germ line-specific protein Oct-4. Nature 344, 435-439 3. Okita, K., Ichisaka, T., and Yamanaka, S. (2007) Generation of
germline-competent induced pluripotent stem cells. Nature 448, 313-317
4. Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B. E., and Jaenisch, R. (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318-324
5. Maherali, N., Sridharan, R., Xie, W., Utikal, J., Eminli, S., Arnold, K., Stadtfeld, M., Yachechko, R., Tchieu, J., Jaenisch, R., Plath, K., and Hochedlinger, K. (2007) Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1, 55-70
6. Wilmut, I., Schnieke, A. E., McWhir, J., Kind, A. J., and Campbell, K. H. (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810-813
7. Chung, Y., Bishop, C. E., Treff, N. R., Walker, S. J., Sandler, V. M., Becker, S., Klimanskaya, I., Wun, W. S., Dunn, R., Hall, R. M., Su, J., Lu, S. J., Maserati, M., Choi, Y. H., Scott, R., Atala, A., Dittman, R., and Lanza, R. (2009) Reprogramming of human somatic cells using human and animal oocytes.
Cloning Stem Cells 11, 213-223
8. French, A. J., Adams, C. A., Anderson, L. S., Kitchen, J. R., Hughes, M. R., and Wood, S. H. (2008) Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts. Stem Cells 26, 485-493
9. Takahashi, K., and Yamanaka, S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676
10. Hanna, J., Wernig, M., Markoulaki, S., Sun, C. W., Meissner, A., Cassady, J. P., Beard, C., Brambrink, T., Wu, L. C., Townes, T. M., and Jaenisch, R. (2007) Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318, 1920-1923
11. Wernig, M., Zhao, J. P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F., Broccoli, V., Constantine-Paton, M., Isacson, O., and Jaenisch, R. (2008) Neurons derived from
reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc Natl Acad Sci U S A 105, 5856-5861
12. Yamanaka, S. (2007) Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 1, 39-49
13. Okita, K., Nakagawa, M., Hyenjong, H., Ichisaka, T., and Yamanaka, S. (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science 322, 949-953
14. Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, II, and Thomson, J.
A. (2009) Human induced pluripotent stem cells free of vector and transgene sequences. Science 324, 797-801
15. Spradling, A., Drummond-Barbosa, D., and Kai, T. (2001) Stem cells find their niche. Nature 414, 98-104
16. Morrison, S. J., Csete, M., Groves, A. K., Melega, W., Wold, B., and Anderson, D.
J. (2000) Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells. J Neurosci 20, 7370-7376
17. Studer, L., Csete, M., Lee, S. H., Kabbani, N., Walikonis, J., Wold, B., and McKay, R. (2000) Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen. J Neurosci 20, 7377-7383
18. Danet, G. H., Pan, Y., Luongo, J. L., Bonnet, D. A., and Simon, M. C. (2003) Expansion of human SCID-repopulating cells under hypoxic conditions. J Clin Invest 112, 126-135
19. Ezashi, T., Das, P., and Roberts, R. M. (2005) Low O2 tensions and the prevention of differentiation of hES cells. Proc Natl Acad Sci U S A 102, 4783-4788
20. Harvey, A. J., Kind, K. L., Pantaleon, M., Armstrong, D. T., and Thompson, J. G.
(2004) Oxygen-regulated gene expression in bovine blastocysts. Biol Reprod 71, 1108-1119
21. Adelman, D. M., Maltepe, E., and Simon, M. C. (1999) Multilineage embryonic hematopoiesis requires hypoxic ARNT activity. Genes Dev 13, 2478-2483
22. Ramirez-Bergeron, D. L., Runge, A., Dahl, K. D., Fehling, H. J., Keller, G., and Simon, M. C. (2004) Hypoxia affects mesoderm and enhances hemangioblast specification during early development. Development 131, 4623-4634
23. Covello, K. L., Kehler, J., Yu, H., Gordan, J. D., Arsham, A. M., Hu, C. J., Labosky, P. A., Simon, M. C., and Keith, B. (2006) HIF-2alpha regulates Oct-4:
effects of hypoxia on stem cell function, embryonic development, and tumor growth. Genes Dev 20, 557-570
24. Niwa, H. (2001) Molecular mechanism to maintain stem cell renewal of ES cells.
Cell Struct Funct 26, 137-148
25. 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
26. Eminli, S., Utikal, J., Arnold, K., Jaenisch, R., and Hochedlinger, K. (2008) Reprogramming of neural progenitor cells into induced pluripotent stem cells in the absence of exogenous Sox2 expression. Stem Cells 26, 2467-2474
27. 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 factors. Nature 454, 646-650
28. 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 Genet 24, 372-376
29. Shimozaki, K., Nakashima, K., Niwa, H., and Taga, T. (2003) Involvement of Oct3/4 in the enhancement of neuronal differentiation of ES cells in neurogenesis-inducing cultures. Development 130, 2505-2512
30. 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 regulatory circuitry in human embryonic stem cells. Cell 122, 947-956
31. 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
32. 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.
Nature 418, 41-49
33. 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 stem cell theory of carcinogenesis. Carcinogenesis 26, 495-502
34. Kiefer, J. C. (2007) Back to basics: Sox genes. Dev Dyn 236, 2356-2366
35. 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 depend on SOX2 function. Genes Dev 17, 126-140
36. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007) Induction of pluripotent stem cells from adult human
fibroblasts by defined factors. Cell 131, 861-872
37. 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 somatic cells. Science 318, 1917-1920
38. 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 Stem Cell 4, 472-476
40. 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
41. 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
კ߄
߄ǵᆫӝ䁙ೱᙹϸᔈЇηׇӈំ߄
Gene Forward Primer Reverse Primer Taqman probe
Taqman64-mActin CTAAGGCCAACCGTGAAAAG ACCAGAGGCATACAGGGACA Roche Universal Probes 64 Taqman6-m
OCT4 GAGGCTACAGGGACACCTTTC GTGCCAAAGTGGGGACCT Roche Universal Probes 6 Q-m Actin GTGCGTGACATCAAAGAGAAGC TGGATGCCACAGGATTCCATAC
Q-mMYC CATTCAAGCAGACGAGCA CGAGTTAGGTCAGTTTATGCAC Q-mKLF4 CCTTTCAGTGCCAGAAGT ACTACGTGGGATTTAAAAGTGC Q-mOct GCCAATCAGCTTGGGCTAGA TTCTGGCGCCGGTTACA Q-mSOX AGGGCTGGACTGCGAACTG TTTGCACCCCTCCCAATTC
Ecat1 TGTGGGGCCCTGAAAGGCGAGCTGAGAT ATGGGCCGCCATACGACGACGCTCAACT Esg1 GAAGTCTGGTTCCTTGGCAGGATG ACTCGATACACTGGCCTAGC
Nanog CAG GTG TTT GAG GGT AGC TC CGG TTC ATC ATG GTA CAG TC
Eras ACTGCCCCTCATCAGACTGCTACT CACTGCCTTGTACTCGGGTAGCTG Oct3/4 TCTTTCCACCAGGCCCCCGGCTC TGCGGGCGGACATGGGGAGATCC Rex1 ACGAGTGGCAGTTTCTTCTTGGGA TATGACTCACTTCCAGGGGGCACT Sox2 TAGAGCTAGACTCCGGGCGATGA TTGCCTTAAACAAGACCACGAAA Klf4 GCGAACTCACACAGGCGAGAAACC TCGCTTCCTCTTCCTCCGACACA
c-Myc TGACCTAACTCGAGGAGGAGCTGGAATC AAGTTTGAGGCAGTTAAAATTATGGCTGAAGC
͊actin GTGGGGCGCCCCAGGCACCA CTCCTTAATGTCACGCACGATTTC
კǵճҔ፦ᡏᙯࢉکલ਼ᕉნғԋᇨᏤ܄ӭфૈ༸ಒझǶ
კΒǵஒखजᠼᆢ҆ಒझܭલ਼ᕉნΠϩձᎦ 0, 4, 8, 24 λਔϐࡕǴ ճҔՋБᏀᗺݤϩखजᠼᆢ҆ಒझύ Oct4, Sox2, c-Myc, Klf4 ೭Ѥঁ
ೈқޑ߄ໆǶ
0 4 8 24 (hours)
Oct4
Sox2
cMyc
Klf4
Actin
კΟǵճҔ Real-time PCR ϩԴႵखजᠼᆢ҆ಒझҗ፦ᡏᙯࢉ Oct4, Sox2 ೭ٿঁ୷ӢϐࡕǴӧ҅த਼ރᄊΠکલ਼ᕉნύǴOct4, Sox2, c-Myc, Klf4 ೭ Ѥ ঁ ୷ Ӣ ޑ mRNA ߄ ໆ Ƕ Control: ፦ ᡏ ᙯ ࢉ Ε pCDNA3.1 ೭ঁޜၩᡏǶOS: ፦ᡏᙯࢉΕ Oct4 ک Sox2 ೭ٿঁᙯᒵӢηǶ Control H: ፦ᡏᙯࢉΕ pCDNA3.1 ೭ঁޜၩᡏࡕೀܭલ਼ᕉნΠ 24 λ ਔǶOSH: ፦ᡏᙯࢉΕ Oct4 ک Sox2 ೭ٿঁᙯᒵӢηࡕೀܭલ਼ᕉნΠ 24λਔǶɀ:߄Ң p<0.05Ƕ
კѤǵiPS-OSH ӧᡉ༾᜔ΠޑಒझᄊǶѰკࣁӧᡉ༾᜔Πวᅪ՟ख ज༸ಒझਔޑಒझࠠᄊǶѓკࣁஒѰკޑಒझᝩжϐࡕޑಒझࠠᄊǶ
კϖǵճҔ PCR ϩ iPS-OSH ಒझύ൳ঁϣғ܄ mRNA ޑ߄ໆǴ٠ ЪکԴႵखजᠼᆢ҆ಒझ(MEF)ǴԴႵखज༸ಒझ(ES)ǴВҁޑᇨᏤ܄
ӭфૈ༸ಒझ(iPS J)КၨǶ
კϤǵiPS-OSHǴճҔᡵ܄ᕗለ䁙ࢉՅϩǴჴځڀԖԴႵखज༸ಒ झϐ܄ Ƕ
კΎǵճҔ Nanog, Oct4, Sox2, SSEA1, Tra-1-60, Tra-1-81 ೭٤༸ಒझ marker ٰዴᇡ iPS-OSH ޑ༸ಒझ܄Ƕ
(a)
(b)
კΖǵ(a)ஒ iPS-OSH ճҔᝌੌᎦ܌ԋޑखजౚǶ(b) iPS-OSH ಒझ
ԋޑखजౚϩϯϐࡕғԋޑΟखቫಒझǶ
კΐǵஒ iPS-OSH ҜΠݙΕᇘႵङҜΠϤډΖຼ܌ԋޑ྾जዦϪ ТǴ֖ԖΟঁखቫޑಒझǶ
კΜǵMicroarray ϩखजᠼᆢ҆ಒझ(MEF)Ǵखजᠼᆢ༸ಒझ(ES)ǴВ ҁޑᇨᏤ܄ӭфૈ༸ಒझ(iPS J)аϷ iPS-OSH ӧ୷Ӣ߄ޑ࣬՟Ϸ࣬
౦܄Ƕ
კΜǵ Microarray ϩखजᠼᆢ҆ಒझ(MEF)Ǵखजᠼᆢ༸ಒझ(ES)Ǵ ВҁޑᇨᏤ܄ӭфૈ༸ಒझ(iPS J)аϷ iPS-OSH ӧ୷Ӣ߄ޑ࣬՟Ϸ
࣬౦܄Ƕ
კΜΒǵiPS-OSH ಒझԋޑઓౚϩϯϐࡕғԋޑઓ༸ಒझǶճҔ Prpک Tuj1 ೭ٿঁઓ༸ಒझޑ marker ٰዴᇡಒझࢂցڀԖઓ༸ಒझ ޑ܄Ƕ