博士論文 ヒト多能性幹細胞を用いた赤血球分化モデルの構築 越智 清純
Contents
1. 1 ES iPS 2 iPS 31 ES iPS O RhD ipPS D Fy a b Di b Jr a 4 12. c MYC BCL XL c MYC BCL XL c MYC 18 21 39 39
3. iPS t iPS iPS
4. 41 1 1 0 2 0 x 10 6 A423 BT HEC do s x T i CD34 35 c MYC BCL XL 43 441
5. iPS 3 3 1 BE c MYC BCL XL 2 Lo
6. 2 2 1 2S p 2 2
7. X 5 A BCLIIA L ES iPS hESC H1 BCL11A L GFP hiPSC 8 BCL11A L GFP GFP DOX BCL11A L 5 B 5 C SSBA 4 K U GFP BCL11A L y BCLIIA L 6 A 20 BCL11A_L 3 6 B 0 DOX OFF Protocol BCLILA L HBG1 HBG2
8. SSC FSC P1 Pacific Blue conjugated anti CD235a CD235a P3 P2 Pacific Blue conjugated anti CD235a 350 nm FITC e 488 nm TRI COLOR y 633 nm PE 8 561 nm IgG normal mouse IgG1 FITC normal mouse IgG1 PE Cy5 and normal mouse IgG1 PE x li g vih x fue B vi pieh y ES H 18 3 D Control
9. ESAPS 2 3 2 3 1 Sigma Aldrich ES KhES 3 ES H1 WiCell Madison WI iPS iPS 4 OCT3 4 SOX2 KLF4 c MYC 13 iPS 2
10. GFP A DOX DOX ON 8 C A 57 6 2 1 B 46 9 1 8 C 99 144 5 B NN 9 A DOX DOX ON 9 A EB A N C B A A
11. 0 5 saponin TRAUE 2 0 x 10 30 10 1 ug Q0 uL PE conjugated anti human hemoglobin D mouse monoclonal IgG sc 21757 PE Santa Cruz Biotechnology Santa Clara CA 5 uL TRI COLOR conjugated tandem R PE Cy5 conjugated anti human fetal hemoglobin HbF mouse monoclonal IgG HFH 06 Invitrogen Carlsbad CA 1 ug 0 1 uL fluorescein isothionate FITC conjugated anti human hemoglobin amp mouse monoclonal IgG kappa 61 C CR8008MIF Fitzgerald MA Pacific Blue conjugated anti human CD235a 349108 BioLegend mouse IgG2a k 30 CD235 IgG I
12. Shandon Cytospin 4 Cytocentrifuge ThermoFisher Scientific HOTARIT PAT AES BER SAE Hemacolor Merck Whitehouse Station NJ BZ 9000 microscope Keyence Osaka Japan BZ Analyzer software Keyence E NIC meantSD 3 3 7 2 3 9 30 3 4 3 3 1 ES ES ES C3H10T1 2 ES KhES 3 H1 14 0 A 0 c MYC BCL XL 2 7 A SCE EPO DOX 12 H ERE Z T gt 7a HHL O
13. Bp HBB 30 Oy 8 EH CX HT EH SHER SE EK ZR LER C358 V Cli FEFLL OVA Zu w A CNN HBE 1 HBG1 HBG2 C HBB HBB K562 EPO UT 7 EPO 3 3 3 c MYC BCL XL ES c MYC BCL XL 2
14. Total c MYC Total BCL XL Endogenous BCL XL 11 A DOX ON Total c MYC A Total BCL XL A 96 P DOX OFF Total c MYC Total BCL XL B C 3 BCL XL Exogenous BCL XL BCL XL Endogenous BCL XL BCL XL 37 38 DOX OFF BCL XL DOX ON
15. 2 3 75 c MYC BCL XL mOKS 3 3 3 ES 2 3 2 5 0 x 10 C3H10T1 2 28 6 well plate 1well 1 5 x 10 c MYC BCL XL Deb 6 1 multiplicity of infection MOI 20 0 12 HAE 50 ng mL SCF 5 IU mL EPO 1 0 ug mL DOX 12 1 0 ugmL DOX MethoCult 4434 Veritas Tokyo Japan 12
16. 3 2 26 KS62 32 33 UT 7 EPO 34 6 c MYC BCL XL 2 ES DO X DOX 35 CD235a Glycophorin A 6 DOX
17. 40 1 3 jPS iPS
18. GCN5 OFFE n 3 mean SD 61 10 A Clone A Clone B Clone C ON OFF ON OFF ON OFF 10 B ALAS2 TFRC J E g 5 10 4 EE Clone A x it Ga Clone B 5 gt EH Clone C S 4 0 0 amp e amp eG FA 3 amp Ry e amp HBE1 HBG1 HBG2 HBB 8000 in L9 E 5 E 6000 d 60 a c 4000 uj 40 if s S gt amp 2000 20 E 0 10 8 A ALIS X y NE A B C DOX ON ON DOX OFF 72 OFF ON OFF B C B DOX ON OFF ALAS2 TFRC PCR
19. 141 2 ow oe 146 2 B B B 11 B B hke globin gene cluster 70kb B g Gy Ay 6 B B 3 g HBE1 Gy Ay HBGI HBG B HBB s bp
20. EEN OS ES iPS CD34 B g y Bp 16 3 A 4 100 0 5 CD235a B g y h FITC TRLCOLOR PE conjugated anti human globin subtype antibodies 3 B ES
21. sy y HBG1 HBG2 B HBB 30 BIL in vitro BSZPS 22 B 7 LEX 1 3 ES iPS
22. gs HBE1 y HBG1 HBG2 B HBB CD34 iPS hiPS 5 8 B HBB 3 D 3 E ESAPS 23 B CD34 y y BCL11A
23. DOX OFF DOX OFF GCNS 3 3 2 B 10 A Z A DOX ON 94 f amp z L DOX OFF B C B C DOX ON DOX OFE
24. 6 18 BP 6 g g d ct Mis do CD34 y B g 6 18 B 3 D ES H1 iPS 18 H B IC 0X mRNA HBB1 e 3 E ES iPS
25. A E ES iPS BS iPS VEGF C3H10T1 2 hBS iPS sac CD34 B CD34 C CD235a CD43 CD71 ES H1 E CD34 CD34 day differentiation medium VEGF vascular endothelial growth factor EPO erythropoietin SCF stem cell factor TPO thrombopoietin C3H10T1 2 feeder C3H10T1 2 sac formation sac erythroid differentiation CB CD34 47 2 A Cell sources hiPSC line Neonatal dermal fi
26. 2 A gt B gt C 63 2 DOX Cell p um Nuclear um N C ratio ON 25 87 2 56 21 96 1 88 0 85 0 07 Clone A OFF 21 95 3 33 14 17 2 19 0 65 0 06 ON 17 78 1 96 12 43 1 45 0 70 0 03 Clone B OFF 15 82 0 94 10 43 0 82 0 64 0 06 ON 16 19 1 34 10 79 1 30 0 66 0 03 Clone C OFF 14 48 1 02 5 26 0 62 0 37 0 05 2 DOX ON OEF N C A B C DOX ON OFE um EVIE umk N C DOX ON OFF N C DOX ON OFF A gt B gt C 500 mean SD Gee J Sequence 9 ES Fw ACA CCA AGA GCT CCC ACC T Rv GTA GTG GCG GGT CAG CTC DATAS Fw AGC AGC TTC CTC CAC TGC Rv TGG GTA CAC CTG AAA GAC TGG Be Fw TCC TCA CTC ACT TCC CCA AA Rv TTG GAG AGT TTG CCC CAT AG re Fw AAG ATG TGA AGG CTT TCA AGAC Rv
27. ORZEC ERTO Beet Oe ais O7 TR B 18 CD34 jy B y ESAPS y 2 4 2 BCL11A ES iPS y ESAPS CILGERNZBEX 26 E44 BCL11A KLF1 GATA1 c MYB SOX6
28. C3H10T1 2 8 0 x 10 100 mm dish S IU mL EPO 12 6 18 3 3 6 12 18H H 2 3 3 CD34 CD34 CD34 C3H10T1 2 8 0 x 10 100 mm dish 30 ng ml SCF 50 ng mL TPO 5 IU mL EPO 0 6 0 6 6 C3H10T172 8 0 x 10 100 mm dish IU mL EPO 12 6 18 BRR 3 3 6 12 18 A 2 3
29. Total BCL XL A ON OFF B C OFF Exogenous BCL XL OFF n 3 mean SD B ON OFE c MYC BCL XL KLF1 GATAL ON c MYC BCL XL A gt B gt C ON KLF1 GATA1 OFF BCL XL KLF1 GATA1
30. c MYC 38 c MYC BCL XL 35 c MYC BCL XL BCL XL c MYC 37 DOX OFF BCL XL Total BCL XL BCL XL Endogenous BCL XL DOX ON DOX OFF KS62 UT 7 EPO
31. P DOX OFF A B C DOX ON DOX OFF KLF1 GATA1 GCNS 9 C KLF1 GATA1 33 GCNS KLPF1 GATA1 DOX ON DOX OFF DOX OFF A B C GCNS 36 DOX OFF DOX ON KLF1 GATA1
32. 12 24 HH 24 L4 50 ng mL SCF 5 IU mL EPO 1 ug mL DOX 12 24 24 24 H HA 3 3 4 2 3 4 Pacific Blue conjugated anti human CD235a 306612 BioLegend 29 PE conjugated anti human CD71 334106 BioLegend and APC conjugated anti human CD43 343206 BioLegend PBS 3 3 5 PCR 2 3 6 2 3 3 6
33. 10 B ALAS2 TFRC HBE1 HBG1 HBG2 HBB ALAS2 erythroid specific 5 aminolevulinate synthase ALAS2 TFRC DOX ON DOX OFE KLF1 GATA1 A lt B lt C 10 A HBE1 HBG1 HBG2 HBB g HBE1 y HBG1 HBG2
34. Locus control region LCR amp 5 B g gt Ay Gy gt B 0 globin switching ES IPS g y B 5
35. Lonza ipPS 8 6 CD34 Lonza iPS 7 8 16 17 18 C3H10T1 2 C3H10T1 2 cell line M3 16 17 18 2 3 2 ES iPS ES 16 17 18 C3H10T1 2 CGmitomyein C 0 25 1 mM 20 knock out serum replacement PBS ES 100 C3H10T1 2 8 0 x 10 100mm dish 20 ng mL VEGF ifr r vascular endothelial growth factor 14
36. O Rh D iPS 45 1 iPS ES iPS O Rh 41 4 1 K Miharada T Hiroyama K Sudo T Nagasawa Y Nakamura Efficient enucleation of erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells Nat Biotechnol 24 2006 1255 1256 2 A R Migliaccio C Whitsett G Migliaccio Erythroid cells in vitro from developmental biology to blood transf
37. 6 C 6 D B y 6 D ES iPS CD34 BCL11A L 6 A Protocol iii y CD34 3 BCL11A_ L 24 6 B y BCL11A ES iPS bin vitro a Oe es ES iPS B BCL11A L yy
38. DOX OFF A A B C DOX ON OFF A B C DOX ON ON DOX OFF 72 OFER ON A N C B A A o C OFF C OFF N C Biz f ORG A Bi ii
39. DOX BCLI1A L BCL11A L All in one inducible lentiviral vector Ai LV 19 mOKS BCL11A L zE U7 60 12 DOX 20 BCL11A L CD34 ES iPS 16 17 18 2 3 8 BZ 9000 microscope Keyence Osaka Japan BZ Analyzer software Keyence Lo 2 3 9 n 3 3 mean standard
40. Ay es ES KhES 3 H1 27 3 3 HE 3 3 1 Sigma Aldrich ES KhES 3 RHET ES H1 WiCell Madison WD iPS 3 3 2 c MYC BCL XL
41. BCL11A 23 zinc finger transcription factor Cd F HbE 29 BCL11A isoform variant BCL11A L yy 5 25 27 BCL11A L y 27 CD34 y BES iPS 3 E BCL11A L ES iPS 6 B
42. A c MYC BCL XP GFP DOX c MYC BCL XL B E ES C3H10T1 2 14 0 B 0 ES Sac HPCs 12 12 12 24 2 A 20 ng mL VEGF B 50 nm mL SCF 5 IU mL EPO 1 0 u g mLDOX B 1 0 rg mL MethoCult H4434
43. 22 CD71 CD235a CD71 CD233a CD71 1 C ES HIf KKOt Hei CD34 14 ESAPS CD34 iPS epigenetic memory 23 241 Sendai viruns 8 iPS
44. ES iPS CD34 g ES iPS e BS iPS y 2 ES iPS 25
45. Jurkat B MED n 3 mean SD 56 1 Gene Seqene B Fw GCA AGA AGG TGC TGA CTT CC globin HBE7 Rv ACC ATC ACG TTA CCC AGG AG Fw TGG ATG ATC TCA AGG GCAC y globin HBG1 HBG2 Ry TCA GTG GTA TCT GGA GGA CA Fw CTG AGG AGA AGT CTG CCGTTA B globin HBB Rv AGC ATC AGG AGT GGA CAG AT Si Fw CAG CTC AAA AGA GGG CAG AC Rv GAG CTT CCA TCC GAA AAC TG GATA1 Fw AGC AGC TTC CTC CAC TGC Rv TGG GTA CAC CTG AAA GAC TGG KLF1 Fw ACA CCA AGA GCT CCC ACC T Rv GTA GTG GCG GGT CAG CTC c MYB Fw AGC AAG GTG CAT GAT CGT C Rv GAT CAC ACC ATG ATG AAG AAT CAG SOX6 Fw ACA AAA GCA GCA TCC CTA GC Rv GGC AGG GGA GTT GAG ACT AGA Fw AGC CAC ATC GCT CAG ACA C Rv GCC CAA TAC GAC CAA ATC C 1 PCR 5 3 7 A B pox TRE BCL XL S UbC rtTA GFP LTR 7 B hESCs EJ e Transfected cells Cloning hES sacS e HPCs K e Gu C gt Sse 53 Viral transfection Clonal selection Clonal expansion in semisolid culture MM e e v day 14 day 0 day 12 day 24 Protocol A Protocol B Protocol C Protocol B C3H10T1 2 feeder cells 7
46. Mtr CD34 LC Bd 2 C HBE1 6 CD34 HBG1 HBG2 385 FICBVY TILE P Jie CD34 HBE1 HBB CD34 6 18 CD34 40 50 ESAPS 8 HBB
47. 12 AB 12 12 12 24 HH 24 Di SCE EPO DOX eerste 24 B 7 B CD235a 6 Em ES KhES 3 A B HL C l 8 A DOX ON GFP GFP CD235a CD71 8 B FSC SSC
48. E BAT 1 2 2 1 EE 2 2 2 3 2 4 2 5 3 3 1 BB 3 2 3 3 3 4 3 5 BB 4 5 P2 P2 P2 P3 PT P13 P21 P26 P26 P26 P28 P31 P37 P42 P46 P46 1 cE b FR PMERR Embryonic stem ES Gnduced pluripotent stem iPS
49. ALAS2 TEFRC HBE1 HBG1 HBG2 HBB ALAS2 TERC ON OFEF C 8 HBB n 3 mean SD 62 11 A Total c MYC Total BCL XL Exogenous BCL XL 1 5 Relative Expression Relative Expression Relative Expression 0 0 0 0 La eG KA e Ey e E e amp e amp 3 Ea e ee o E EM Clone A BI CloneB BJ Clone C c MYC Jb LP Total SERPENS unici eL PER BCL XL 2 RE Total BCL XL NEL cn Endogenous II EMT pe Expression Levels Expression Repression 11 c MYC BCL XL A PCR Total c MYC Total BCL XL Exogenous BCL XL Total c MYC ON A gt B gt C OFF
50. n 3 mean c SD C BS H1 iPS ES iPS HBE1 HBG1 HBG2 HBB HBB1 6 n 3 mean SD 46 3 A Wash Wash 4 Paraformaldehyde gt 100 Methanol gt 0 5 Saponin 60 min at RT 5 min at RT 10 min at RT Wash Wash Wash gt Anti human globin antibodies Anti CD235a antibod gt Flow cytometry 30 min on ice 30 min on ice Embryonic globin Fetal y globin Adult B globin Anti human globin antibody Anti human y globin antibody Anti human B globin antibody FITC conjugated TRI COLOR conjugated PE conjugated 3 C Control P2 P3 P2 P3 O E 1 PA alee 1 I 1d am 192 o 2 k m cmo mnd Oe gt NT EU ou at a as wow oa oon FSC S Anti human e globin c Si 1 z rs zl dq FSC Anti human B globin 3 A B KERZE RAE y 8 C
51. 14 0 3 0 ES sac 30 mL 40 um hematopoietic progenitor cells HPCs phycoerythrin PB Cy7 conjugated anti human CD34 antibody 343516 BioLegend San Diego CA 30 FACS Ariall Becton Dickinson Franklin Lakes NJ CD34 CD34 5 0 x 10 C3H10T1 2 2 0 x 10 well 6 well plate 30ng ml human stem cell factor SCF R amp D systems Minneapolis MN 50 ng mL human thrombopoietin TPO R amp D systems 5 IU mL human erythropoietin EPO Kyowa Kirin Tokyo Japan 6 0 6 6
52. 8 BCL11A L n 3 mean SD 92 5 A Bl pox a 1T Transcription TRE BCL11A 2 UbC rtTA GFP LTR LTR Bd5 B 5 C hESC H1 BCL11A L GFP hESC H1 BCL11A L GFP GFP SSEA 4 g r TN 7 Ja i hiPSC 8 BCL11A L GFP ES it HS GFP SSEA 4 Phase contrast GFP Merge Fluorescence intensity Control Cell line E 5 BCL11A L ES iPS A BCL11A L B BCL11A L ES H1 hESC H1 BCL11A L GFP BCL11A L NPS hiPSC 8 BCL11A L GFP mouse embryonic feeder cells GFP Bar 100 um C BCL11A L ES iPS GFP SSEA 4 DOX doxycycline LTR long terminal repeat TRE tetracycline response element UbC C rtTA IRES internal ribosom
53. s TOO Tr hESC H1 Anti human y globin Yocom onus hes asos CO i O u i ald dues um Anti human B globin D ES H1 iPS 8 HERE IL CD3ABSTPEIRRUIRL EH SE ZIRSE EK 33803257 a 4h ES iPS y 8 g CD34 g y 8 y CD34 day 3 E day 6 day 6 0 4 day 3 2 5 day 6 0 0 day 18 0 0 eo ha e e a o co j j c uo c S j t S hs ln si e ha e Ja h e ho e ho co rx e in S o wo e o e o as o S gt ce 0 0 g o 0 0 0 0 9 6 0 0 1 1 eo xs o Q e e Q e o o E ES
54. c MYC GATA1 KLF1 BCL XL ALAS2 TFRO DOX OFF c MYC 11 B 3 5 ES KhES 3 H1 c MYC BCL XL 2903815 T X 3 7 AB 8 ABO oT 9 ABC Ed 10 AB C DOX OFF 9 A FRA ii DIZ TERO AMG B
55. BCLIIA variant form BCLIIA L CD34 ESAPS BCL11A variant form Shorter variant BCLIIA S Ji Longer variant BCL11A L 19 BCL11A L y 27 28 cMYB KLFI BCLIIA GATA1 SOX6 BCL11A 26 KLFI GATAI bim CD34 cMYB SOX6 ES iPS y BCL11A L
56. 0 DOX ON Protocolii HBG1 HBG2 6 DOX ON Protocolii y 6 C 6 D BCL11A y 29 27 BS iPS BCLIIA L y 2 5 ER B e Gy Ay 6 5 COLD
57. 32 C DOX 72 DOX OFF 9 A C DOX OFF N C 9 A 9 A 9 A um 4 AEE um N C 2 9 B DOX ON OFF 0 1 0 x 10 CD23Sa DOX ON DOX OFE 1 3 5 7 CD235a AB DOX OFF 3 C
58. 58 SSC CD235a 8 A hESCs Cell Lines hESC KhES 3 gt Clone A Clone B hESC H1 ps Clone C 8 B 8 C Clone A Clone B Clone C GFP 8 E CD235a A E A B BS KhES 3 C ES H1 B A B C FSC SSC CD235a CD71 GFP EFSC SSC CD235a CD71 GFP C A B C DOX ON CD235a en B gt AC n 3 mean SD 59 9 A Clone A Clone B Clone C eee i 9
59. GGA AAA TGG CTT CCT TAG GC p Fw TGG CAG TTC AGA ATG ATG GA Rv AGG CTG AAC CGG GTA TAT GA TOR ATO ETE TONS GER C Fw CCA AAC AGG AAC ACA TAG CAG A Total BCL XL Rv GAG CTC CAT GTG CAG AAC G Fw TTA GTG AAC CGT CAG ATC GC Exogenous BCL XL py GTC TCC ATC TCC GAT TCA GT GAPDH Fw AGC CAC ATC GCT CAG ACA C Rv GCC CAA TAC GAC CAA ATC C 3 PCR 5 3 65
60. HI 18 3 C forward scatter SSC amp U side scatter SSC P1 CD233a P3 mean fluorescence intensity MED Jurkat Jurkat cells hES H1 6 HH E I CD34 6 H H KUE hR IM 17 A B 5 25 ES H1 iPS 8 3 AE g y
61. iPS 1 8 iPS iPS er hiPS 1 2 human neonatal dermal fibroblasts 3 6 CD34 human CB CD34 cells 7 8 human adult peripheral blood cells Ed 2 A Bd 2 B 18 CD34 iPS hiPS 5 6 7 Ek 8 iPS hiPS 1 2 HBB HBB HES H1 hESC H1 hiPS 8 hiPSC 8 2 15 CD34
62. j f i ee w L Ts 1 i 1 Isotype control JU d Anti human globin antibody CB day 6 8 T Fresh RBCs E E B 20000 L 20000 Lr 40000 E E E Ea S 45000 Z 9 15000 2 9 20000 oc ot ot o9 8 PO c Q 10000 gt amp 10000 15000 veo o co T T Eg 2 5000 26 5000 ES uM ps HE TE o ae E o E E E T e D se A Q ce M e e ce c e ge d e D e qe e et o qe d e d jt i K 5 Ry v G J E v e 3 o 3 G E G hi hai h CJ Isotype control EE Anti human globin immunofluorescence A Jurkat urkat cells BS H1 6 A CD34 6 E C y 8 E ES H1 6 EPI LCD34 by ERU EH OR aR SF 6 E Pacific Blue conjugated anti human CD235a
63. Scale Bar 30 u m 60 Number of CD235a cells 9 B Clone A Clone B Clone C 1 0x1005 10x10 2 10x10 o o 1 0x1007 to 1 0x1007 to 1 0x1007 N N 1 0x1006 Q 10x10 Q 10x10 1 0x1005 1 0x1005 1 0x1005 2 2 1 0x1004 1 0x1004 1 0x1004 A A ss g a aue ss a FA es g as aue ON OFF 9 C KLF1 GATA1 GCN5 e e on S S 5 4 2 1 0 a 3 Q A x lt x x Ww nT j dh 2 QS 1 5 S 0 0 0 0 EN Clione A BI CloneB B Clone C B DOX ON OFF CD235a 1 0 x 106 DOX ON DOX OFF 1 3 5 7 CD235a n 3 mean SD C DOX ON OFE KLF1 GATA1 GCN5 PCR KLEF1 GATA1 GCN5 KLF1 GATA1 ON OFF OFF A lt B lt C
64. 21 5 26 2 HBB1 y HBG1 HBG2 7 HBG1 HBG2 B HBB 8 HBD B 4 B sy BH1 B1 B2 BH1 y HBG1 HBG2 ancestral gene y HBG1 HBG2 7 5
65. 4 3 FBS fetal bovine serum PBS 30 Pacific Blue conjugated anti human CD235a 306612 BioLegend PE conjugated anti human CD71 334106 BioLegend and APC conjugated anti human CD43 343206 BioLegend PBS ESAPS SSEA 4 PE conjugated anti SSEA 4 antibody FAB1435P R amp D systems 2 3 5 1 0 x 10 mL 4 paraformaldehyde PEA Wako Pure Chemical Industries Ltd Tokyo Japan 60 fe Kim UL 710096 methanol MtOH 3
66. L C Andersson K Nilsson C G Gahmberg K562 a human erythroleukemic cell line Int J Cancer 23 1979 143 147 34 N Komatsu M Yamamoto H Fujita A Miwa K Hatake T Endo H Okano T Katsube Y Fukumaki S Sassa et al Establishment and characterization of an erythropoietin dependent subline UT 7 Epo derived from human leukemia cell line UT7 Blood 82 1993 456 464 35 S Hirose N Takayama S Nakamura K Nagasawa K Ochi S Hirata S Yamazaki T Yamaguchi M Otsu S Sano N Takahashi A Sawaguchi M Ito T Kato H Nakauchi K Eto Immortalization of Erythroblasts by c MYC and BCL XL Enables Large Scale Erythrocyte Production from Human Pluripotent Stem Cells Stem Cell Reports 1 2013 499 508 36 P Ji M Murata Hori H F Lodish Formation of mammalian erythrocytes chromatin condensation and enucleation Trends Cell Biol 21 2011 409 415 37 K Hafid Medheb Y Augery Bourget M N Minatchy N Hanania J Robert Lezenes Bcl XL is required for heme synthesis during the chemical induction of erythroid differentiation of murine erythroleukemia cells independently of its antiapoptotic function Blood 101 2003 25775 2583 38 L Haughn R G Hawley D K Morrison H von Boehmer D M Hockenbery BCL 2 and BCL XL restrict lineage choice during hematopoietic differentiation J Biol Chem 278 2003 25158 25165 39 S R Jayapal K L Lee P Ji P Kaldis B Lim H F Lodish Down regulation of Myc
67. adult hemoglobin Cold Spring Harb Perspect Med 3 2013 23011643 26 D E Bauer S C Kamran S H Orkin Reawakening fetal hemoglobin prospects for new therapies for the beta globin disorders Blood 120 2012 2945 2953 27 V G Sankaran J Xu T Ragoczy G C Ippolito C R Walkley S D Maika Y Fujiwara M Ito M Groudine M A Bender P W Tucker S H Orkin Developmental and species divergent globin switching are driven by BCL11A Nature 460 2009 1093 1097 28 D Zhou K Liu C W Sun K M Pawlik T M Townes KLF1 regulates BCL11A expression and gamma to beta globin gene switching Nat Genet 42 2010 742 744 29 V G Sankaran T F Menne J Xu T E Akie G Lettre B Van Handel H K Mikkola J N Hirschhorn A B Cantor S H Orkin Human fetal hemoglobin expression is regulated by the developmental stage specific repressor BCL11A Science 322 2008 1839 1842 44 30 J Palis J Malik R L Emerson T P Bushnell K E McGrath M Bulger P D Kingsley Maturational globin switching in primary primitive erythroid cells Blood 106 2005 1012a 1012a 31 A Hill S C Hardies S J Phillips M G Davis C A Hutchison 3rd M H Edgell Two mouse early embryonic beta globin gene sequences Evolution of the nonadult beta globins J Biol Chem 259 1984 3739 3747 32 C B Lozzio B B Lozzio Human chronic myelogenous leukemia cell line with positive Philadelphia chromosome Blood 45 1975 321 334 33
68. iPS CD34 day 18 0 0 globin Y qlobin 3 a as B globin globin Y qlobin globin B globin 0 0 y globin B globin ce globin y globin B globin HSER n 3 mean 51 bi BCL11A L 1 5 S 1 0 a ui E 0 5 a 5 5 0 0 y I P i hESC H1 Ji hiPSC 8 CB Relative Expression Relative Expression Hind o cn tn 0 0 1 5 GATA1 KLF1 25 c 2 2 0 aq E 15 ui o 1 0 E 0 5 0 0 5 e o J V we SOX6 Relative Expression a 4 ES iPS hESC H1 hiPS 88PK ROC ha fL CD34 BB EAM Hd EH Sea ER BS iPS hBSC H1 hiPS CD34 283 6 12 18 BCL11A L GATA1 KLF1 cMYB SOX6 e HE CD34 ES iPS hBSC H1 hiPS
69. 3 ESAPS 6 7 8 9 10 11 12 13 14 OUI qq ES iPS ESAPS CD34 CD34
70. 8 KLF1 GATAI 35 39 CD34 1 40 f CD34 CD34 41 ES iPS 42 CD34
71. G2 HBB Protocol iii HBG1 HBG2 n 3 mean SD 5 D 6 C hESC H1 BCL11A L GFP hiPSC 8 BCL11A L GFP EU 98 096 o v O o z p n c c hen x 8 8 S o o s 03489 93 1 o CN CN a o a ci 2 E E l unl c x0 10 4 c 058 96 lt x lt x Anti GFP Anti human B globin Anti GFP Anti human B globin 6 D lL 20000 LL c z D 1e000 ao Se o9 T O 42000 To ui eL ou cE EE O 8000 o ES ES 5s zu 4000 f 25 Ss i ii iii i ii iii CB C hESC H1 BCL11A L GFP hiPSC 8 BCL11A L GFP C BCL11A L ES iPS CD235 GFP 18 Protoco ii mean fluorescence intensity MFI n 3 mean SD 55 A Anti human globin Anti human y globin Anti human B globin Jurkat cells 3 _hESC H1 day 6 E j i E Di j po 1 i 1 PAN B
72. aki A Oda T Takenawa H Nakauchi The WAVE2 Abi1 complex differentially regulates megakaryocyte development and spreading implications for platelet biogenesis and spreading machinery Blood 110 2007 3637 3647 21 S Hirata N Takayama R Jono Ohnishi H Endo S Nakamura T Dohda M Nishi Y Hamazaki E Ishii S Kaneko M Otsu H Nakauchi S Kunishima K Eto Congenital amegakaryocytic thrombocytopenia iPS cells exhibit defective MPL mediated signaling J Clin Invest 123 2013 3802 3814 22 M A Vodyanik J A Thomson Slukvin II Leukosialin CD43 defines hematopoietic progenitors in human embryonic stem cell differentiation cultures Blood 108 2006 2095 2105 23 K Kim R Zhao A Doi K Ng J Unternaehrer P Cahan H Huo Y H Loh M J Aryee M W Lensch H Li J J Collins A P Feinberg G Q Daley Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells Nat Biotechnol 29 2011 1117 1119 24 K Kim A Doi B Wen K Ng R Zhao P Cahan J Kim M J Aryee H Ji L I Ehrlich A Yabuuchi A Takeuchi K C Cunniff H Hongguang S McKinney Freeman O Naveiras T J Yoon R A Irizarry N Jung J Seita J Hanna P Murakami R Jaenisch R Weissleder S H Orkin I L Weissman A P Feinberg G Q Daley Epigenetic memory in induced pluripotent stem cells Nature 467 2010 285 290 25 V G Sankaran S H Orkin The switch from fetal to
73. broblasts gt 1 2 Cord blood CD34 cells gt 3 4 5 6 Peripheral blood cells gt 7 8 HBE1 HBG1 HBG2 HBB 5000 c ss E T 7 7 S 20 9 AA ZA 3000 h h 5 15 a 2000 2 f 7 o 10 2 h a AAA z AVA s 1000 7 AA A A AA or on oa P abo e be dS Poe HEE PL ee er o PP Hw P POR e KS e hESC hiPSC hESC hiPSC hESC hiPSC 2 C HBE1 HBG1 HBG2 HBB s amp n hESC H1 JF hiPSC 8 k CB Relative Expression h e un o a 8 8 8 e e e Relative Expression PJ amp n m Relative Expression 0 0 s nb E fs RU S je s A Se S S SUO A 2 ES iPS CD34 A EhiPS NiPS iPS B ES KhES 3 H1 iPS 1 3 CD34 18 HBE1 HBG1 HBG2 HBB ES H1 iPS 8 HBB
74. deviation SD CR P lt 0 05 2 4 2 4 1 ES PS CD34 13 CUE ART O 7 55 OX OBXE RATED RIL ESAPS C3H10T1 2 16 17 18 21 ES H1 KhES 3 1 A hA CD34 1 B CD233a CD43 CD71 3 6 18 CD233a FAR MERI CFE ELAS DO DI CD43 O
75. e entry site GFP green fluorescent protein 53 6 A day 0 day 6 day 18 Differentiation medium SCF TPO Protocol i OFF Protocol ii Protocol iii C3H10T1 2 feeder Erythroid differentiation BCL11A L HBE1 HBG1 HBG2 HBB h i on a Fession Relative Expression o a B Relative Expression hESC H1 BCL11A L GFP B3 gt Bo uan k e Relative Expression e un Relative Expression th ression Relative Expression k h un c h e an DL LL E T o mn e a un i 2 xk E m S nh gt n a NY i ui nk n gt De JP Se SU S je S S a ae S rd a JP A je Protocol i 48 Protocol ii 9 Protocol iii CB 6 ES iPS BCL11A L A BCL11A L ES iPS DOX DOX Protocol DOX ProtocoLii 6 18 DOX Protocol 0 18 DOX B BCL11A L ES iPS BCL11A L HBE1 HBG1 HB
76. er M Lako Efficient hematopoietic differentiation of human embryonic stem cells on 42 stromal cells derived from hematopoietic niches Cell Stem Cell 3 2008 85 98 11 F Ma Y Ebihara K Umeda H Sakai S Hanada H Zhang Y Zaike E Tsuchida T Nakahata H Nakauchi K Tsuji Generation of functional erythrocytes from human embryonic stem cell derived definitive hematopoiesis Proc Natl Acad Sci U S A 105 2008 13087 13092 12 A R Migliaccio C Whitsett T Papayannopoulou M Sadelain The potential of stem cells as an in vitro source of red blood cells for transfusion Cell Stem Cell 10 2012 115 119 13 E N Olivier C Qiu M Velho R E Hirsch E E Bouhassira Large scale production of embryonic red blood cells from human embryonic stem cells Exp Hematol 34 2006 1635 1642 14 T Peyrard L Bardiaux C Krause L Kobari H Lapillonne G Andreu L Douay Banking of pluripotent adult stem cells as an unlimited source for red blood cell production potential applications for alloimmunized patients and rare blood challenges Transfus Med Rev 25 2011 206 216 15 K Nishimura M Sano M Ohtaka B Furuta Y Umemura Y Nakajima Y Ikehara T Kobayashi H Segawa S Takayasu H Sato K Motomura E Uchida T Kanayasu Toyoda M Asashima H Nakauchi T Yamaguchi M Nakanishi Development of defective and persistent Sendai virus vector a unique gene delivery expression system ideal for cell repro
77. gG 20 uL normal mouse IgGi PE Cy5 sc 2877 Santa Cruz Biotechnology normal mouse IgG PE sc 2866 Santa Cruz Biotechnology normal mouse IgG FITC sc 2855 Santa Cruz Biotechnology 3 FBS PBS GFP Green Fluorescent Protein anti GFP Alexa Fluor 488 sc 9996 AF488 Santa Cruz 11 Biotechnology 2 3 6 PCR RNA RNeasy Micro kit Quiagen Tokyo Japan oligo dT primer Invitrogen Oregon USA RT PCR Thermo Fisher Scientific K K Kanagawa Japan DNA complementary DNA cDNA PCR SYBR Premix Dimer Eraser Takara Bio Shiga Japan ABI7900HT Real Time PCR System Life Technologies Corporation CA USA GAPDH AACt 18 H Hoo b iri CD34 fold change 1 2 3 7
78. gramming J Biol Chem 286 2011 4760 4771 16 T Nishimura S Kaneko A Kawana Tachikawa Y Tajima H Goto D Zhu K Nakayama Hosoya S Iriguchi Y Uemura T Shimizu N Takayama D Yamada K Nishimura M Ohtaka N Watanabe S Takahashi A Iwamoto H Koseki M Nakanishi K Eto H Nakauchi Generation of rejuvenated antigen specific T cells by reprogramming to pluripotency and redifferentiation Cell Stem Cell 12 2013 114 1206 17 N Takayama H Nishikii J Usui H Tsukui A Sawaguchi T Hiroyama K Eto H Nakauchi Generation of functional platelets from human embryonic stem cells in vitro via ES sacs VEGF promoted structures that concentrate hematopoietic progenitors Blood 111 2008 5298 5306 18 N Takayama S Nishimura S Nakamura T Shimizu R Ohnishi H Endo T Yamaguchi M Otsu K Nishimura M Nakanishi A Sawaguchi R Nagai K Takahashi S Yamanaka H Nakauchi K Eto Transient activation of ccMYC expression is critical for efficient platelet generation from human induced pluripotent stem cells J Exp Med 207 2010 2817 2830 19 T Kobayashi T Yamaguchi S Hamanaka M Kato Itoh Y Yamazaki M Ibata H 43 Sato Y S Lee J Usui A S Knisely M Hirabayashi H Nakauchi Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells Cell 142 2010 787 799 20 K Eto H Nishikii T Ogaeri S Suetsugu A Kamiya T Kobayashi D Yamaz
79. is essential for terminal erythroid maturation J Biol Chem 285 2010 40252 40265 40 M C Giarratana L Kobari H Lapillonne D Chalmers L Kiger T Cynober M C Marden H Wajcman L Douay Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells Nat Biotechnol 23 2005 69 74 41 M C Giarratana H Rouard A Dumont L Kiger I Safeukui P Y Le Pennec S Francois G Trugnan T Peyrard T Marie S Jolly N Hebert C Mazurier N Mario L Harmand H Lapillonne J Y Devaux L Douay Proof of principle for transfusion of in vitro generated red blood cells Blood 118 2011 5071 5079 45 42 H Lapillonne L Kobari C Mazurier P Tropel M C Giarratana I Zanella Cleon L Kiger M Wattenhofer Donze H Puccio N Hebert A Francina G Andreu S Viville L Douay Red blood cell generation from human induced pluripotent stem cells perspectives for transfusion medicine Haematologica 95 2010 1651 1659 43 S Pelengaris M Khan G Evan c MYC more than just a matter of life and death Nat Rev Cancer 2 2002 764 776 44 F Tzifi C Economopoulou D Gourgiotis A Ardavanis S Papageorgiou A Scorilas The Role of BCL2 Family of Apoptosis Regulator Proteins in Acute and Chronic Leukemias Adv Hematol 2012 2012 524308 45 J Zou P Mali X Huang S N Dowey L Cheng Site specific gene correction of a point mutation in human iPS cells derived from an adult patient
80. usion products Curr Opin Hematol 16 2009 259 268 3 K Takahashi K Tanabe M Ohnuki M Narita T Ichisaka K Tomoda S Yamanaka Induction of pluripotent stem cells from adult human fibroblasts by defined factors Cell 131 2007 861 872 4 S T Nance How to find recruit and maintain rare blood donors Curr Opin Hematol 16 2009 503 508 5 V G Sankaran J Xu S H Orkin Advances in the understanding of haemoglobin switching Br J Haematol 149 2010 181 194 6 C Cerdan A Rouleau M Bhatia VEGF A165 augments erythropoietic development from human embryonic stem cells Blood 103 2004 2504 2512 7 J Dias M Gumenyuk H Kang M Vodyanik J Yu J A Thomson Slukvin II Generation of red blood cells from human induced pluripotent stem cells Stem Cells Dev 20 2011 1639 1647 8 D S Kaufman E T Hanson R L Lewis R Auerbach J A Thomson Hematopoietic colony forming cells derived from human embryonic stem cells Proc Natl Acad Sci U S A 98 2001 10716 10721 9 O Klimchenko M Mori A Distefano T Langlois F Larbret Y Lecluse O Feraud W Vainchenker F Norol N Debili A common bipotent progenitor generates the erythroid and megakaryocyte lineages in embryonic stem cell derived primitive hematopoiesis Blood 114 2009 1506 1517 10 M H Ledran A Krassowska L Armstrong I Dimmick J Renstrom R Lang S Yung M Santibanez Coref E Dzierzak M Stojkovic R A Oostendorp L Forrest
81. with sickle cell disease Blood 118 2011 4599 4608 5 uel Pee Bae PAA iPS 6 46 1 A 1 B day 14 day 0 day 6 day 18 day 0 day 6 day 18 Differentiation medium Differentiation medium VEGF O EPO _ EEPO _ C3H10T1 2 feeder C3H10T1 2 feeder i ee Sac formation Erythroid differentiation Erythroid differentiation 1 C day 3 day 6 day 18 day 3 day 6 day 18 n ia Ps w T lam s ga sm y nek P i 1 G m amem s aJ a7 RE sm 15 lose ms Jacou aama Jacon peew lense Aoma 7797 OT 3 CN e CD43 e Mae 230 os 1125 873 2 T1 f 37 35 a7 8 i GEG P Lm qi qp iz E ic co m i t f i a 1 isse max bs we Tam lore nsum locos sem lox omew loo signwl CD71 1
Download Pdf Manuals
Related Search