京都大学 iPS細胞研究所
Contents
1. 500 MS MiniMACS Separation Unit 15ml MS 500 15 ml MS 500 Il 3 15m MS 1000 Il 10 300 x g 4 C 10 32. FRBOMRBEAEAMC 3 ml 15 ml 1 2 3 ml e PBS 12 ml 200 x g 18 C 5 1500 x g T BD Pharm Lyse 10x 1 x e 1 x Pharm Lyse 1 ml 1 e PBS 12 ml 200 x g 5 min e X VIVO 10 3 ml WA THRA D 10 ll 4 1 3 e 15m X VIVO 10 8 ml 37 C
3. 15 m 1 1 ml 2 1 PBS 12 ml 200 x g 18 C 10 400 x g Center for iPS Cell Research and Application CiRA Kyoto University 5 13 CiRA M amp M 2013 1 10 e X VIVO 10 3 ml 10 gl 4 1 2 18C BD 8 ml 18C 1500 1800 x g 20 mn Go 2
4. C SNL Center for iPS Cell Research and Application CiRA Kyoto University 1 13 CiRA M amp M 2013 1 108 http www sanger ac uk Terms Term82 Dr Allan Bradley European Collection of Cell Cultures ECACC http www ecacc org uk EDTA ACD A Cellular Technology Limited CTL UP1 Sanguine Biosciences PBMC 001 1 2 Dulbecco s modified eagle medium DMEM Nacalai tesque 14247 15 Phosphate buffered saline Ca Mg free PBS Nacalai tesque 14249 95 Fetal bovine serum FBS Penicillin streptomycin Life Technologies 15140 122 Recombinant basic fibroblast growth factor human bFGF WAKO 064 04541 Bovine serum albumin BSA ICN 810 661 0 25 Trypsin 1 mM EDTA solution Life Technologies 25200 056 Ficoll Paque PREMIUM GE healthcare 17 5442 02 BD CPT Na 362761
5. 3 MEF 6 1 5 ml well 1 1 5 x 10 e 37 C 5 CO THETA lt 8 10 12 gt iPS 1 5 ml 14 gt iPS iPS 1 5 ml 2 1 lt 20 30 gt iPS 2mm 96 1 200 wl iPS P10 96
6. 2 1 g ul 4 5 1 2 1 WPRE p53 shRNA 1 pCXLE hOCT3 4 shp53 F 0 83 ug pCXLE hSK 0 83 ug pCXLE hUL 0 83 ug pCXWB EBNA1 05 we 2 pCE hOCT3 4 0 63 ug pCE hSK 0 63 ug pCE hUL 0 63 ug pCE mp53DD 0 63 Ug pCXB EBNA1 05 we 4 2 1 A iPS 1 TCR iPS 2 TCR immunoglobulin iPS 1 ml 2 ml 1 gt 6 MEF
7. 24 SNL C 300 wl iPS 37 C 5 CO 80 90 20 ug ml RetroNectin Takara T100A 0 5 u g cm Vitronectin Life Technologies A14701SA TeSR2 Stemcell Technologies ST 05860 Essential8 Life Technologies A14666SA Center for iPS Cell Research and Application CiRA Kyoto University 10 13 CiRA M amp M 2013 1 10 4 2 3 C vitro iPS iPS TCR immunoglobulin ml lt 0 HA gt 6 1 3 2 ml 5 PBS
8. C 3 x 10 cell well lt 0 gt Electroporation 1 2 x 100 15 ml 200xg 18C 10 Elepo Center for iPS Cell Research and Application CiRA Kyoto University 7 13 CiRA M amp M 2013 1 10 Human T Cell Nucleofector Solution 81 841 Supplement 18 2 ul Plasmid Sug Elepo e NucleofectorIIDevice V 024 MEF 6 1 5 ml well 1 1 1 30 x 10 2 3 10 x 10 e
9. 20 C IL 3 10 ug ml 4 95 ml PBS 50 10 BSA 0 1 BSA PBS 2 ug IL 3 200 0 1 BSA PBS 20 C IL 6 10 ug ml 4 95 ml PBS 50 uI O 10 BSA 0 1 BSA PBS 5 ug IL 6 500 0 1 BSA PBS 20 C IL 6 100 ug ml 4 95 ml PBS 50 10 BSA 0 1 BSA PBS 5 ug IL 6 50 wl 0 1 BSA PBS 20C G CSF 10 ug ml 4 95 ml PBS 50 10 BSA 0 1 BSA PBS 2 ug G CSF 200 ul 0 1 BSA PBS 20 C GM CSF 10 ug ml 4 95 ml PBS 50 10 BSA 0 1 BSA PBS 5 ug GM CSF 500 0 1 BSA PBS 20 C IL 2 10 we ml 4 95 ml PBS 50 10 BSA 0 1 BSA PBS 10 ug IL 2 1ml 0 1 BSA PBS 20
10. X VIVO 10 10 4 2 iPS iPS 3 iPS A aBT iPS iPS T cell receptor TCR iPS Center for iPS Cell Research and Application CiRA Kyoto University 6 13 CiRA M amp M 2013 1 10 B CD34 iPS A C iPS B TCR
11. iPS 3 EK Cellular Technology Limited CTL UP1 Sanguine Biosciences PBMC 001 iPS 4 1 1 Ficoll 18 5ml EDTA Ficoll Paque PREMIUM 5 ml 15 ml 2 PBS5ml Ficoll 5 ml COR 400x g 18C 30
12. 37 C 5 CO C6 5 gt 6 MEF C 3 x 10 cell well lt 6 H E gt Electroporation 3 15 ml 10 200 x g 18C 10 Elepo Center for iPS Cell Research and Application CiRA Kyoto University 9 13 CiRA M amp M 2013 1 10 Human CD34 Cell Nucleofector Solution 81 8 Supplement 18 2 ul Plasmid 3 Ug 100 Elepo e Nucleofector II Device U 008
13. 80 90 4 2 2 B AMMO CD34 2 x 107 20ml 10 HE CD34 MicroBead Kit 10 ml lt 0 gt CD34 6 1 3 2 ml Center for iPS Cell Research and Application CiRA Kyoto University 8 13 CiRA M amp M 2013 1 10 5 PBS 2 ml 37C 2 x 107 15 ml 300 x g 4 C 10 300 gl FcR Blocking Reagent 100 Il CD34 MicroBeads 100 4 30min 10 ml 300 x g 4C 10
14. 0 ee 2 4 6 8 10 12 14 20 30 A 4 4 iPS li c iPS ES iPS SRAM MAHA O 3 4 5 integration episomal vector iPS PCR PCR 4 pEP4 SF1 TTC CA
15. Center for iPS Cell Research and Application CiRA Kyoto University 4 13 CiRA M amp M 2013 1 10 e Nucleofector II Device Lonza e BICELL DynaMag 2 Life Technologies 12321D MS Columns Miltenyi Biotec 130 042 201 MiniMACS Separation Unit Miltenyi Biotec 130 042 102 MACS MultiStand Miltenyi Biotec 130 042 102 4 iPS iPS http www cira kyoto u acjp j research images protocol pdf hiPS_Protocol_080703a pdf 4 1 2
16. TPO 300 ug ml 4 95 ml PBS 50 10 BSA 0 1 BSA PBS 10 ug TPO 33 3 0 1 BSA PBS 20C FIt3 Ligand 300 ug ml 4 95 ml PBS 50 uI 10 BSA 0 1 BSA PBS 10 ug FIt3 Ligand 33 3 1 0 1 BSA PBS 20 C Center for iPS Cell Research and Application CiRA Kyoto University 3 13 CiRA M amp M 2013 1 10 SCF 300 ug ml 4 95 ml PBS 50 ui 10 BSA 0 1 BSA PBS 10 ug SCF 33 3 0 1 BSA PBS 20 C 10 FBS MEF 50ml FBS 2 5ml penicillin streptomycin DMEM 500 ml 0 22 u m 4C 1 iPS 0 2 ml 10 ug ml bFGF 500ml Primate ES 1 a 15 ml X vivo 10 15 IL 2 10 ug m b 1 5 ml 1 ml X vivo 10 Dynabeads CD3 CD28 5
17. BD Pharm Lyse 555899 X VIVO 10 Lonza 04 743Q 0 59 Nacalai tesque 29853 34 3 BLC 3S Primate ES Primate ES medium ReproCELL RCHEMD001 Amaxa Human T Cell Nucleofector Kit Lonza VAPA 1002 Amaxa Human CD34 Cell Nucleofector Kit Lonza VAPA 1003 MEM Nacarai 21444 05 StemSpan H3000 StemCell Technologies 09800 500 mM EDTA Solution pH 8 0 Nacalai 06894 14 Recombinant human IL 3 PeproTech 200 03 2 ug Recombinant human IL 6 PeproTech 200 06 5 ug Recombinant human G CSF PeproTech 300 23 2 ug Recombinant human GM CSF PeproTech 300 03 5 ug Recombinant human IL 2 PeproTech 200 02 10 ug Recombinant human TPO PeproTech t 300 18 10 ug Recombinant human FIt3 Ligand PeproTech 300 19 10 ug Recombinant human SCF PeproTech t 300 07 10 ug Dynabeads Human T Activator CD3 CD28 Life Technologies 111 31D CD34 MicroBead Kit human Miltenyi Biotec 130 046 702 Center for iPS Cell Research and Application CiRA Kyoto University 2 13 CiRA M amp M 2013 1 10 2 bFGF 10 ug ml 4 95 ml PBS 50 10 BSA 0 1 BSA PBS 50 Ug bFGF 5 ml 0 1 BSA PBS
18. iPS C in vitro iPS 1 1 1 OCT3 4 SOX2 KLF4 L MYC LIN28 p53 shRNA EBNA1 Addgene http www addgene org Shinya_Yamanaka pCXLE hOCT3 4 shp53 F pCXLE hSK pCXLE hUL pCXWB EBNA1 4 WPRE p53 shRNA 2012 12 pCE hOCT3 4 pCE hSK pCE hUL pCE mp53DD pCXB EBNA1 5 Amaxa kit pmaxGFP MEF 13 5 Manipulating the mouse embryo a laboratory manual second edition Cold Spring Harbor Press New York 1994 TLS ReproCell RCHEFC003 Lonza M FB 481
19. 37 C 5 CO lt 2 4 6 gt iPS 1 5 ml lt 8 HA gt iPS iPS 1 5 ml 2 1 lt 20 25 gt iPS 2mm 96 1 200 wl iPS P10 96 e 24 SNL C 300 iPS 37 C 5 CO
20. 0 rl 5 DynaMag 2 1 DynaMag 2 a 2 9 ml wMEM FBS 1 ml 1IL 3 amp IL 6 G CSF GM CSF 10 g m 10 wl 3 10 ml StemSpan H3000 IL 6 100 g m SCF 300 ug ml TPO 300 ug ml FIt3 ligand 300 ug ml IL 3 10 g m 10 I 20 ml PBS 500 mM EDTA 80 FBS 200 gl 3 6 24 96 FALCON 353046 353047 351172 15 50 ml FALCON 352196 352070 1 5 10 25 m FALCON 357520 357543 357551 357525 Pippette aid FALCON GILSON 1 5 ml WATSON CO Thermo
21. 2 ml 37C HARKER 3 x 10 15 ml 300x g 4C 10 3 e 37C 5 CO 6 5 B 6 1x10 1 5 20 x 10 20 ug ml RetroNectin Takara T100A 0 5 u g cm Vitronectin Life Technologies A14701SA TeSR2 Stemcell Technologies ST 05860 Essential8 Life Technologies A14666SA 4 3 Ficoll C iPS Center for iPS Cell Research and Application CiRA Kyoto University 11 7 13 CiRA M amp M 2013 1 108 1
22. C GAG GGT AGT GAA CC pEP4 SR1 TCG GGG GTG TTA GAG ACA AG 94 C 2 min 94 C 20 sec 64 C 20 sec 30 cycle 72 C 40 sec 72 C 3 min Center for iPS Cell Research and Application CiRA Kyoto University 12 13 CiRA M amp M 2013 1 10 5 1 Okita K et al An Efficient Non viral Method to Generate Integration Free Human iPS Cells from Cord Blood and Peripheral Blood Cells Stem Cells 2012 Nov 29 2 Okita K et al A more efficient method to generate integration free human iPS cells Nat Methods 8 409 12 2011 3 Fujioka T et al A simple and efficient cryopreservation method for primate embryonic stem cells Int J Dev Biol 48 1149 54 2004 4 Yu J et al Human induced pluripotent stem cells free of vector and transgene sequences Science 324 797 801 2009 5 Mack AA et al Generation of Induced Pluripotent Stem Cells from CD34 Cells across Blood Drawn from Multiple Donors with Non Integrating Episomal Vectors PLoS One 6 e27956 2011 Center for iPS Cell Research and Application CiRA Kyoto University 13 13
23. CiRA M amp M 2013 1 108 Ver 1 iPS iPS iPS ARAM LAR MER PFA AER Ficoll 2 iPS iPS 3 A T iPS B CD34
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