JP 2008-545412 A 2008.12.18 (57)【要約】 造血
Contents
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9. C57 6 6 CD45 20 L CD45 1101 0 O O Jackson Laboratory I O National Cancer Ins titute O O Whitehead Institute animal facility 000000000 MI T Comittee on Animal Care 0000000000 FACS L I L L 8 100 O C57BL 6 045 20 000000000 SP Sca 1 CD45 O O II II I SPO O 8 9 C Lodi sh HF Blood 1030 25 13 21 20040 Zhang C C Lodi sh H F Bl ood 1050 4314 20 20059 000 LI II II II
10. L LI LI LI LI II II II II II II II II II II II II II II II II II II II II II II LI LI II II LI II II II II II II II II II II II 10 LI II II II O L LI II II II II II II L ICI 1 1 1 11 II II II I FG I LI LI LI TP I LI II II II II II II II II II II II II II II II II II IUL IU 0 Ing ni 500ng m 0 0000000000 IUL LI LI II 11 II II II II II II II
11. 1 0 08 20 0 0 83 1 8 1 8 0 0 0000000000000000 0 Angptl20000000000000000000000020 CL B O FLAG 1 LI II II Angpt 20 0 0 DNA Flag Angpt 20 gGlFc Angptl 211 Pro100 Lys330 IEGRMM LI II II LI II II II II 10 II DNA 00000 2 JU 1 1 1 cDNA3 10 II I II II gi 0 I pof ect am ne 20000 nvi trogen 2931 0 10 0 0 III 480 0 0 0 poo00000000001u 9 m0 2
12. LI LI LI LI II 29310 LI II II O Angpt 2 hFc II II II I II II II II II II II II II II 1 g m An 900 2 1111111101 0 I0C1 LL II II II II 12 80 Angptl 2 0000004000000 LI II 5 30 LI Angpt 20 LI LI LI 5 1040 2x10 04 5 11 I II II 0 CD45 10 4 5 I I II II II II II II U 3 II LI 40 II I II II II II II II II II 1 I I 045 27 00000 II I LI L
13. 0 0 I I I 70 140 O O 1 I I II II I 0 710 II I Od II II II II II II II II II II II II II II II 10000 Od O II II II II II II II II II II II II II II II I II II II II II II II II II II II II II II II II II U LI II II II Od II II II II II II II II II II II II II II U 0 10000 0 ing mg 0 200 0 mo 10ng m Y 100691 0000000000 sk gt a m a 80 0 0 1000 0000000000000000 Od LI LI II 0 II 1 II II I II II II II II II II II II II DNA IM II II IL III II III I I III II I III I I II II II S Inn 2 2 8 a Sca 1 I GF2 hFC 0 O CD31 0 II II II II II II II II II II II II II II 00
14. 30 JP 2008 545412 A 2008 12 18 vO II II II II II II II II II II II II II II II II II II 00000 ex vivo 11 II II II II U LI II II II II II II II II II II II II II II II II II II L L 1 II II II II II II II II II II II II II II II II II II II II II II II II II II II II II LI II II II II II II II II II II II II II II II II II II II II II II II II II II II II FLAG II II II II II II II II II 90 211 II II II II II II II II LI LI II pcDN3 00010000 FLAGA LI 2 pcDNA3 1 O OO 200000000000 II II 0
15. HQ 0 0 0 0 0 0 8 0808 88 8 8 8106 HMM 1 881 081 I WO 95 058430 VO 95 036930 O WO 95 08100 0 0 0 0 DD DB DD DB DB D D DB D D 0 1 8 81 81811 88 0 10000000 10 10 08888888 181020 0 O II 1x10 appen 11 000 I 0 000 0 1x10 0 0 0 0 11065 0 5 0 MO Ina SP 5 0 0 O 1x102 5 1030 O m J p 000 78 Ina E 11 10 20 000 m 0 50 0000 0 mo 0 D 0 10 50 000 10 010000 0 120 24 0 O 96 00 12 5 110 0 00 0 0 0 0 0 0 0 0 0880800 0 7 0 Falcon Corning Costar 0 0 0 10 LI I IDOO0OO0O0000000000000000000000 0D0000000000000000000000000000000000050
16. 88000000 Angpt 2 0000 00 0000 0 0 0 11 10 0 0 II O SDS PAGEO 0 hAngptl in vitro I 0 0 O O C5I LI 50 000300000 4 40 800005210 000000 90 31 00050000 58 0 0 0 0 0 0 0 5 0 00 0 DO 210 0 11 1 mAngpt 30 100691 00000000000 O 7 hAngpt 4 1006911 00000000000 30 800 GST hAngpt 5g 100691 00000000000 40 9 0 O O hAngpt 70 lu 9 mpg 0000000000 10 0 150 200 CD45 20 SP 5 8 45 0 I 0D 4 30 O 10 4 OB 000000 pcDNA3 70 10 FLAG hM ap4 11 0 0 0 O FLAG hFg 10 0 0 0 O 0003 10 30 1 293 480 0 log 0 0 0 FLAG O log log II 50 8 STIFO OO I 1 40 O FLAG 9 10 I O O O O pcDNA3 10 I 293 0000 STI F 102 50
17. I II LI LI II II II II II II LI II II II II LI LI O in vitro x 5 gt HM 2 e C 2 e
18. the National Institutes of Health O 10000000000 I I II II II II II II II II II II II II II II II II o I L HSQ 0 I II II II II II II II II II I I II LI II II II 1 II II LI LI LI I LI II II II L II HS I II II II II II II II II II II II II II II II LI II II II II II II LI LI LI 1 III LI 1 1 1 111 1 11 BU ex vi vol Od Od LI II HSC ex vi vo HSCO II II 0 I I II II Od I II II II II II II II II II 5 1 HsC LT HSC LI II II RE RUD DNE II MI Od Od L L L II II 5 II II II II II II II LI in vitro ut in vitro uu uu L II 10 LI LI II II II II I 1 II II II II II 111 II II II II II U 2000000 3 4 7 M 0 I 0 II LI LI II Od Od Od O
19. o o o o A N in moog oo 3 3 3 3 3 3 3 Ma lt G E 3 3 e R a e gt UNE oo oF 3 A CI g oo O 3 ES E ET g EF Y E ETC O oo 3 E Oo 3 LL Lr O m O m EE On 3 ES TET L I __ M 3 a 3 Ej E EE ES 3 8 WEE EE 3 CI CJ EJ EJ _ EJ 3 O g _ 3 a 2 SZ _ 2 m m 2 35 E lt ez O m 3 3 a CL ICI LI iE 3 O lt ET CI EJ E L gt I nam a a N EE LI E Ld 2 o o 0 E 2 E E 3 a a O 3 3 oo 0 E E El EE Ci ca C CI go 3 ED ooo m LL EJ 3 3 EJ LL L3 EJ a 3 C3 o EJ O 3 2 8 O e 1 3 3
20. II II II II II ep II II II II II II II II II II II LI II I LI II II II II II II II II LI II II II II II II II II II II II II II II LI II II II II II II II II II II 3 II II II U Brom M Cell 49 603 612 1987 0 tetR 0 Gossen MO Bujard H Proc Natl Acad Sci USA 890 5547 5551 1992 Yao F Human Gene Therapy 90 19 39 1950 1998 Shockelt P Proc Natl Acad Sci USA 920 6522 6526 1995 LI II II II LI II II II II II II II II II LI LI astradiol II 1 V P160 0 LI p650 RU4860 II II II II II II O di phenol mrisleroner II II II II log log log nvi trogen Cl ont ech Ariad O U U D I II II UU II II II II II II II II II II II II I II II II II II II I LI LI II II II II II II I II 3 cO II II II II II 800 LI 0 LI LI LI LI II LI UU M Brown Cell 49 603 612 1987 Gossenl
21. o o lt i Ol 3 Ooo oo ooo g r3 r3 EJ par xox c r3 KACH m LL x Soc za Ex LL 5 O O LI E 3 c oo A 9 EE S 52 1 EPI E oO EJ r3 E r3 ED m ES 3 og EZ m OO AS EJ 0 SERIE TE EE 92 m M ooo E r3 M i 2 AZ EX r3 ET EJ ix VIE r3 Lj sat zu 1 LI C oO oo E Ol ET EJ 8 EEE El r3 7 3 0 I GO Lr Ol 18121 r3 FEE r3 EJ sk L I oO m oOo I st 8 EE Ep El Om r3 PSS m r3 ET EJ Lr D __ 3 OO M ES m m a 7 Of a I LI 1 m 1 r3 r3 uem KES O O 5 gt gt 25 6px _ I n 2 r3 r3 r3 a EJ
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23. 11 JP 2008 545412 A 2008 12 18 Di 000000000 nn n 10 000 00 00 568 100 11 8 0 in vitro 00000 0 0 0000 0 0 0001070 0 0 0 0 1 LM O 00000 00000 0 0 0 0 0 0 00001010011010 7 0 0 0DDODexvivo000000000000000000000000000000050 01 718 118708 log 00 0 0 10 80 100000000000 O IDnumgsusogggggugguggguguguge u 40 140 0 0000 1 70000000 18000000000 100kg 1050 10 430 poo000000000000000 100 90 D O 45 5x10 t 0 0 0 0 18188 08 100k9g D O 1x10 0 895 101 LE 00 11 88 II 100 90 D D D 4x10 0 O O O 100 90 U O II 2x10 g 000000000 o 0 0 0 0 0 00000000000 000000 0 0 O 0 0 0 0 0 0 30 70 0 0 0 0 0 0 0 00000000 10 10 II uu 0 0 0 0 O uu 00000000 O 0 0 0 01 0 I 0 0 0 in vitro LI II LI II I 0100000000 0000000000 00000 0 0 000010100110107 10 20 30 40 50 JP 2008 545412 2008 12 18 32 inv I LI LI LI 10 20 LI
24. O LTO HS 10003400007 12 II 6 11 D 0 0 500 O D 100ng m FLAG Angpt 2 500 LT HSQ 8 00 1300 7 120 SC FO TPO GF 20 FGF 1j O O Angpt 2 100 II II 000 Hs Angpt 20 I II II II II II II LT HSC O O 10 150 90 LI LI 140 Angpt 20 ood 9200000000 38 880070 3 0 I 1 5 90 0 II II II II II II II II 100 ng m FLA G 90 2 000000000 30000000 51 11 II L 30 2 50 4 0 O HSQ 0 500 TPO 20 0 O O FGF 10000 LT HS O 80 1 II II II O O Zhang 8 Lodish HF Blood 105 4314 20 2005 O Angpti20 00000000 HSC 0 0 II II II II II I 0 Angpt 20 0000000 II 0 O O 9 2 0 0 0000 0 0O FLAG LI 20100 00 D 293 1I II II FLAG 0 dog 0 O FLAG 0 0 0 SDS PAGE O O
25. LI II II II II 10 L II LI SPSca 1 CD45 0 O 5 1 1 STI F 100ng m O Angptl 3 Angpt 4 Angptl 50 0 LI O lu 9 m Angptl 7 LI LI II II 104 Angpt 3 ST HS LT HS LI LI 0 10 0 00 20 7 12 I 11 6 L 131 5 1 O0 LT HS O O Anget SU LI LI LI LI 1 0 II II D Angpt 7 O 111 60 E 110 141 9 40 LI LI II II II 50 0 15 1 41 II LI 2 10000000 IM5CII LI O 9 100ng m Angpt 3 6 HCV I II II II II 100 0 0 3 80000 13 Angpt 40 Map400 Zhao Z Hum MI Genet 4 589 97 1995 0 0 10 Fol 1l Yamamoto Biochem Bi ophys Res Commun 193 681 7 1993 ud 00000000 FLAG O I 717 II II II II I II II II II II II II II 0 108L II II II II II 1 00ng m FLAG M ap40 O FLAG Fgl 1 STI F HSC 1080 0000000000 Map4g 5000000 BM SP Sca 1 CD45 LT 501 ex vivo II II II 0 9 10 0000000 108 1 5
26. JP 2008 545412 2008 12 18 13 10 LI LI LI LI LI LI 20 LI 30 40 LI LI LI 50 LILV I LI I II HTL LI LI
27. D U LI LI I LI II II I II II II II II becco s MEM MMM X Vivo 150 0 RPM 1640 Stenspan Stem Cell Tec hnol ogi es pogo I 0 1 0 001 1 800 2708 LI LI 9 3 0 U Stenspang Stem Cell Technol ogi est
28. Angppti U DU DU D DD D D HS Angptl 20 Angpti 30 8000000000 LI Y Yasunaga K Suda T Int J Hematol 80 21 8 2004 0 O50 0000000 0 0 0 0 0 08772 3 DOHC 71688 LI 000 M5CI ex vivo II II II II II II II 293T Angpt 3 Angpt 150 000 Angpt 70 0 Angptl 20 Angpt 4 II II II I 40 Map4 O II 00000 L II I II II II II II II II II HSC LI II II 9 gt LI LI Map40000000000000002o0 45 2 O SP 5 8 1 CD45 O 10ng m SCF 20ng mM TPO 20ng m 2 10ng m FGF 10 D 8 8 100080000000 Angpt 2 Angpt 2 0 II LI 1 30 100 O Angptl 20 40 LI 110 Angptl 40 5L 0 1200000 0 110 1600 00 0 II I 1000004000 II I 1X10 CD45
29. u 0 moo m EJ a soto 3 EE EJ I 3 lt oo EJ Coo 9 EJ oOo Ej EJ gt ul moo EJ EJ a e n a st ELE E O s gt n E lt a 5 8 m a I _ oO EJ EJ _ w 3 EJ 2 EJ oOo E i Sot ed 5 LL E E 1 0 LI Co mE c gt EL LE o gt _ ZE y A __ E m a _ lt e ZE gt 3 3 EJ 58 ET a ER m gt BEE e CE 3 3 ooo G deg G rr w O c z AGE o Ui GC EJ oo s EJ om ooo gt E ET 2 5 ZC r3 Det F Em ow oOo m oo 3855 LI LI gt gt gt
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33. 100000000 0 1000000000 08001801 86 2006 01 0000 8 gogo 7 00 2006 01 0000 700 0000 3704 2006 01 37 04 00080101 31 04 2006 01 31 04 gogo 8 2006 01 7 04 0000 706 2006 01 7 06 DUDU 8 2006 01 31 18 80 2700 200601 27 00 83 II AP BWGH CM KE LS NA SD SL SZ TZ UG ZM ZW EA AM AZ BY KG KZ M RU TM EP AT BE BG CH CY CZ LE LX EE ES Fl FR B G HJ I E IS I T LT LU LV M N PL PT RO SE SI SK TR OX BF B G 0 QM GA QN GQ GYM M NE SN TD TG AE AG AL AM AT AU AZ BA BB BG BR BWBY EZ CA CH CN O CR QJ CZ LL LX EM LX LC LL EG ES FI G Q HMR HJI DIL IN TS JP KE KG KM KN KP KR KZ LC LK L R LS LT LU LV LY M M M W WK MZ NA NG N NO CM PG PH PL PT RQ RU SG SD SE SG SK SL SM SY J IM TN TR TT TZ UN UG UZ G W YU ZA ZMAN 2000 III III II 1 000000000000000000000 0 11 11 1 1 2000 III III II II II 1 IIIIIIII 000000000000000000000000 IIIIIIIIII I LI 1 1 LI 48024 BA80 C04 DOS HMM GALL 3
34. DU in vi vo LI I LI LI 30 1L 10 2 O EPO SCF 40 LI LI I LI LI M CSF 13 8 7 LI LI LI LI LI I LI LI LI I LI LI LI 50 I LI LI I LI LI o LI LI GM CSF CSF 6 5 4 3 i vo
35. 40 I LI LI LI PIO I LI LI LI LI 11 50 LI LI oo CS L3 r3 oo D wet L3 r3 EJ 5 ax LI oo oo 5 SNS 0 E gt gt I EJ O 5 2 CN LJ I 8 x oo m 6 EB 2 E O m pj I on lt b PE oc T m i B 10 JP 2008 545412 2008 12 18
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37. 48063 QN8 Qe Q9 MB R6 532 2 48065 80 00 ABO1 04 BAo2 8225 8819 8832 8834 CAM CM6 40081 AB11 2 8 01 84 DAS 1 EA1 2 40087 0 CADA M21 M66 NA4 2251 25 2809 2833 2825 25
38. FLAG 0 3 10 O O D 0 II 2937 0 5 0 O 0 200 CD45 2 SP Sca 1 CD45 4D 0 0 3 20 0 0 LAO 0 0 0 O O Angpt 20 LI 30 0 LI mRNA I II II IL III ZE 8 II II I An Maa 11 II I 0 0 8 Angpt13 rRNA O JU 11 D D 0 0 0 0 0 0 0 0 0 0 0 0245 2 0000000030 40 Angptl 2 I II II II II II II II II II II II I U U HSQ Angpt 10 0 10 128 5 0 30 FGF 10 0 0 D 0 40 I 10 0 O CD45 2 BM 4 10000000000 20 0 O 0 0 01 4000
39. HOWE IL OI Vy 6 LI II II II II I LI 100ng m An 4 65 3 O 150 LI 100ng ni Angpt 20 LI 11 II I II II II 100 9 4 LI LI 0 HSC II 60 II II II II 200 1111 1 1 II II II II II II II II II II II II II II II II II II II II II Angpt 2 ex viv gt 2 2 Angpt 2 200 BM SP CD45 Sca 1 100 II II O n3p LI LI II II II LI 1 1 oa a oa a 6 L E EJ LI II II II Angpt 2 6L II II II II II II II II II II
40. LI LI I LI LI LI I LI LI I LI I LI LI LI LI I LI LI LI I LI LI LI 40 myc FLAG Cy5 Cy3 356 in vitro 32 125 ex vi vol 50 in vitrol JP 2008 545412 2008 12 18 8 LI LI LI LI LI I LI LI I LI LI LI
41. II II II II III O PCR RNAI 1 01 1 1800000000010 CCMMI O SuperScript I RTO I nvitrogen 0 O D 100000000 SyBR Geen PCR UU DDD 180000000000 Applied BiosystensO D O D a RT PCRO O D O 254 I0 3000M 00000001254 100000 0 0 0 D Gageng Angptl 2 0 1511790 O I O Angpt 30 GT001108240 0 D L II PCR Applied Bi osystens Prism 70000 DD Angpt 21 Angpt 30 000000000 I I I II 18S RN 40 MI I 0 I LI LI FLAG hAngpt 20 9 20 LI LI LI LI II II II U hAngpti 2 FLAG II U II II 0 LI 0 II FLAG hAngpt 49 FLAG hFgl 10 L LI FLAG hM 8040 1 0 LI I II U Lipofectanine2000 Invi 1 09680 H 00000 L II 100 FBSU U MM D II II 0 0 MOM 14000000000 Stenspang O Sten ell Technol
42. O 6 5 96 5 3 Hx Ha Co m 20 7 20 7 13 8 13 8 co XE N 09 A x lt AN gt 5 Y 7 M A x 11 eo eo w e P 00 GI eo 18 7 4 2 4 5 3 7 3 2 7 5 2 D3 co d Hg 00 00 N N I A PM N Co m pm RIKEN cDNA o 4931414P19 1 4328494 NP 062373 208 5 TERA 9625035 062523 201 1 9 1 2 6 e E 2 d m gem os 24 N e d gt J I c 05 Co F co o D3 37 1 FEAT 15 5 NP 038941 171 6 NP 705757 L 8 e Co 3 9 eo EE P e co w co P m r a AI ct o He O E O gt W e e D3 00 1
43. 000000 00000000020 99065 20 000000000030 30 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 0 0 0 8 0 8 0 0 O 0 10 0 1140 0 00000000000 0 0 SPCD45 5 1 0 0 0 0 0 0 0 0 0 000000000 IBM 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 1180 0 116 0 0 0 0 68 0 0 0 0 6 0 0 0 0 1100 0 0 3 8 0 0 0 0 0 8 3 0 0 0 0 000000 1000 4 0 HSC Angpti2j D D 196 0 0 0 0 0 0000000000000 0 20 10 20 30 40 50 26 JP 2008 545412 2008 12 18 20 90 0 0 II II I Angpti2 I I II II II II II II I II 8 8 0 O BM 5 0 14 000000 4000000 01280 O II I II I II II II I 1 1 nn 20 II II 1 1 1 1 291111111 Hoq
44. LI II II L 100000000 45 170 CD45 2 LI II Zh ang C C Lodi sh HF Blood 103 2513 21 2004 Zhang CC Lodi sh H F Blood 105 4314 20 2005 LU Ul LI II II CD45 2 FI T CD45 1 PE 0 000 3 2 PE T O B220 PE 8 Mac 1 PED O G 1 Mac 11 Gr 1 Ter 119 PE BDPhar ni ngen II II II 1CII 0 L r epopul ati on 10 CD45 2 FI TQ CD45 1 PE II FACS II CRU LI LI LI L Cal CL II II II II Stencell Technol ogi es L Od Zhang C C Lodi sh H F Blood 105 4314 20 2005 0 0 Canbrex Poietics O 000000000000 Catf 2C 150 0 160 10 D M 104 9 m0 O H D Signal 10 90 m 0 0 D 500 2 000 II TPO 2089 1 GF 2H O O RED System O 1069 1 FGF 10 Invitrogen 100ng m Angpt 30 O 0 Angpt 50 0 0 O O StensSpang 0 20 St enCel Technologies II II LI LI II II II II II II II II II II II II II II 0 50 80 12 151 O 000 0000 00 00 00000 00 II II 5x1050 1 51 0 Imp II II II II eS NOD SCI D II II II II II II II II 3501 800 NOD SCI D O I O Jackson 0o li LI II II II LI II II II II II 21I II II II II
45. LI LI LI LI LI LI LI LI LI LI LI 16 JP 2008 545412 2008 12 18 II II II II II II DNA LI LI II II II II II II II II II II II II II II II II II II II II II II II II II I IMVI II II II II II II II II RNA II II II L U L L LI II II II I L Od Od O Od Od LI neo puro LI ll MDR II II II II BFPO L U U O LacZ III II II II II II II II II
46. MENS c ODO oc _ EJ L m _ I I E 21 I C I C 3 a a E m m 8 m O I 86 0 m EJ I dos E Z m ES 0 E O O a E oov Ol L3 c Y O L I st m L Ol LL Edid E _ i E lt c Q m I I 0 a Oo L lt JP 2008 545412 2008 12 18 7 10 LI LI I LI LI LI LI I LI LI I LI LI I LI LI I LI LI LI LI I LI LI I LI LI LI I LI LI LI LI LI I LI LI I LI LI 20 LI 30
47. II II 1 1 1 IUL Sca 1 0 CD45 I GF2 CD31 O O O Kit 0 I PrP Li n CL62LL 1 LI II II II II II II II II II II II II II II II II LI LI II II II II 30 40 50 JP 2008 545412 2008 12 18 3
48. JP 2008 545412 2008 12 18 19 55918581 JP PT 2 SR A 1 SEA 28 5282008 545412 P2008 5454124 43 20 12 18 2008 12 18 51 Int Cl 3 C12N 2006 01 5 00 ZNAE 48024 C12N 2006 01 15 00 4BO63 C12N 2006 01 5 00 B 4BO65 0 2006 01 1 02 40081 A61K 35 28 X 200 01 AGIK 35 28 4 087 FABRIK RER 2 42 B BERCA HEBES 2008 513668 P2008 513668 71 HERA 505291480 22 6 FE 185557 238 2006 5 23 RDA YAFA Fir 7 20 1 168 2008 1 16 4 4 UFF PCT US2006 020078 yAUZGRE vHF21 kbwW O2 7 V02006 127809 142 gt gt FAY 18 11 30 2006 11 30 YV EXA 15 8 95 25 60 684 147 74 KHE A 6 2 FHA 17 5 24 2005 5 24 FET FX EEE US 74 R38 A 100096183 1 60 753 212 FET AF RR 32 48 3E 74125 22H 2005 12 22 74 3 33 1512 3 RH US FET RA M 74 KEA 100111741 REL Ah EX R
49. FLAG hA ngptl 40 TBS LI LI LI O 0 1mg FLAG N DYKDDDDK C 0 LI LI LI 2 LI II II II II L LI 60 90 80 100 p C57BL 6 CD45 20 0000000 200 BM SP Sca 1 45 I II 100 of mq Si L II 10ng m SCF 20ng m TPQ 20ng ni 8 I GF 2 0 R amp D System 10ng m L FGF 1 Invitrogen O St enSpan St Technol ogi es 100 160p 96 3799 Corning LI II II II II II II II II II II II LI L II I II II U 29317 I II II II II I LI 30 100 II II LI II LI LI II II II II II II II II II II 0 10u 9 m0 0 0 O 10ng m SCF 20ng m TPO 20ng m LI II 20 0 O O 1089 1 y FGF 111 00000 STI LI II O 2930 O II I II II II II II II L II I 111 II II 5IILI 1 II II II II II II I II II II II II II 6L II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II CD45 20 00000 11070 0 0 2x10 0 045 11 00000 L II LI II LI II II II II II II II II II II II II II I II II II II 10CVII II II II II II II II 24 JP 2008 545412 2008 12 18
50. CC O CI 3 3 CI C3 3 EJ EJ a U 3 3 3 a 3 3 e E m 77 2 eum ac Ec E CIE EJ ES px CI EJ 5 E CI EJ _ LI L I COLE Au im vix 5 mE oon I I E n n Eee zam 1 ES ES S EE il 0 a 0 lt EJ I I ou 4 JP 2008 545412 A 2008 12 18 x o L I EI LI DI LI L LI o LI LI LI LI LI HM 2 P 500ng nf
51. D D 10 II FLAG 000000000000 20 6 9 2 00000000000000 000 000000 0 0 0 0 u hAngpt 20 D l 91 2490 0 O O Arg276 Hi 54930 0 0 O D 1 76 I LI II II II II II LI CI FLAG III II I 0 9 108 II II 1 0 IC 0 FLAG II II II II 293 L 000000000 FLAG 20 II II I II II I II II II II II 20 L L LI LI LI 0 II II II II II I 40 120 NuPage Bis Tris TODO IMVIL0 0 II 08 888800 000 88 110880 0 II MO II Signal ir 50000 I 0 0 0 0 0 20 0 01 1 0880 8880108 8088810 9 m0 0 239829 1587 D D OD lu g m0 239809 119 239813 1110 239816 1110 239819 1110 239829 11 10 239830 119 239833 110 239834 110 0 O O 239835 1110 O RSD 5 5 0 0 D 0 D 08 8 8 8 I I RD 80 II L 000000 10 20000 000000000000000 LI II O CL II Amersha 30 m Arlington Heights II
52. LI LI I LI I LI LI LI I LI LI LI LI 25nM II II Lin O BU LI LI II 20L B220 CD48 CD10 19 JP 2008 545412 2008 12 18 9 10 20 Hoechst 33342 DNA 1 rho123 30
53. 10 LI I LI LI LI I LI LI I LI LI ex vi vol 20 14 HS LI LI 1 8 in vitro LI LI LI ex vi vol 1995 367 369 Med 3321 I C5 N Engl I ssaragri sh 30 LI LI LI LI LI I LI LI I LI LI LI LI 40 50 80 FCSI LI LI I LI LI LI O HEPESI LI I LI LI LI I LI LI I LI LI I I LI II I LI LI CD2 30 4 Mac 1 Gr 10 CD14 15 LI LI I LI LI I LI LI I 1 LI II LI I LI LI
54. O TPG 1 nig 00000000 ml TPOJ LI II LI LI II II II II II II II II II TPA 0 0 0 0 0 0 0 rliag Sci ence 2760 1696 1997 00000000 LI in vitro ut utu II LI II LI LI I LI LI II LI II II II II II II II II II II II II II II II II II II II II II II II II LI II Bg HEPES Dul becco MEM MM RPM 16400 0 Stentpan Stem Cell Technol ogi es 33 39 C II II 37 HCH II II II II II II II II II II II II II II II II II II II II II Ci poll eschi 1993 Blood 820 2031 77 0 000000 10 I LI II II II II II II II II II II II II II II II II II II 5 II II II II LI II II II MC I I II
55. 0000000000 LI 13 OO 5L II LI LI 5II LI II II II LI II II II II II II II II II II II II II SCI D 300000000 U U L HSC LI O HS HSC ex vi vol 0000 0 D O LT HS 0 4400000000 6 Angpt 2 Angpti 20 0 0 0 HS 00 1601 LI 0 HSQ 2020 00000 II II II II 20 L II O O Angptl 10000 Hs II LI SCF TPO 16 20 00 111 00000 HQ 000 U LI LI L HSC 100 0 II II O LT H8Q 00 80 L Zhang C C Lodi sh HF Blood 105 4314 20 2005 SCFO GF 2 FGF 1 LI II LI II II II II II II II II II II II II O TA II LI 1 31051 LI II I II II U II III III D D I I II I II II 0 LI I 000000 Angpti L Angpt 211 Angpt 3 Angpt 40 Angptl 5 Angpt 10 M ap4 HCI II II II II II II II II II II II II II U SCF TPO I GF 2 0 O FGF 1 Angpt log LI II II Angpt 2 LI 3 Hs 3 0 II IL II II II I II II II Angptl 20 opti 30 OO LI D in vi vo LI LI II II II L LI 0 138 II II Angpt 50 HS Angpti HS U 0 O O II HSC ex vi
56. H entrez LI II II II II II II II II Si gaal P 1 1 590 ttp ww cbs dtu dk servi ces Si gnal 00000 Neural networks Hi dden Markov LI I 10000 Nielsen H Engelbrecht J Brunak S von Heijne G Protein Eng 10 1 6 1997 0 O I Bendtsen J D Nielsen H v on Heijne Brunak S Mol Biol 340 783 95 2004 03 0 00000 22 JP 2008 545412 2008 12 18
57. II II Sca 1 PEJ O 45 II B II II 0 I LI LI II II II II II II II II II II II II Angpti 2 1 410 1 71 0 100000000 1p g m FLAG Angpt 20 O FLAG hFc 90 1 10 08 LI LI II 4C0 30000000000 0 FLAG MI 0 O O 1 II II II II II II II II II II II II II II II II II II II II II II II II II II DNAJ O LI LI B LI RNAI cRNA LI LI LI LI II II II II II II II II II II II II O Affymetri x U74Bv20 U7 4CV2 II II II II II II LI LI 1 RNAV TRIzol 0 B II 15u gl O RNAI 10 000 20 cDNA Ambi on T7 MegaScri pt in vitro CRNA LI Affymetrix U74Bv29 LI U74Cv20 0 LI LI LI LI 45 L 160 I II II II II II II II II II II II M Sui tel EE 03 I LI II II U Mcroarray Suite L L L I LI II I filter out DD 0 raw value 500 1 LI O 0370 00000 6r 1 U L I L L L LI II II II II 50 LI LI II L II II 0 hRNA L II 0 00 00 DD normal zel 2 00 000 00 0 A II I LI ID Aff ymetri x data center lO Ref seq J DI transfer 00000000 NCBI Entrez
58. Bujard 1992 M Gossen Natl Acad Sci USA 890 554 7 5551 1992 tet R L O 8 tetR 161 d ud tetR I II II III II 161 OO O tetR tetO 0 II II II II II II tet 0 0000000 utu Yao I H LI II F Yao Human Gene Therapy Hu Hu CWI CL LI TATA tetR II II II II II II II II II II II LI ID L O tet RI O trans LI II LI II II II II II II I II II I II II II II II II I L II LI L II LI II II II II II 1 II II II II II I M Gossen Natl Acad Sci USA 890 5547 5551 1992 P Shockett D P roc Natl Acad Sci USA 920 6522 6526 1995 II II II II II L II I TetR IRESO pj II DB II II 11 1 1 1 IL II 3 LI LI LI II II LI LI II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II L L L U U 0 11010 0 1 100 0 0 0 D I I II I II L II wodchuk I d D VPRE Loeb V E Human Gene Therapy 100 2295 2305 1999 Zuffe rey R O J of Virol 730 28 5085 5092 1998 U 6 2892 1999 Donello J E J of Virol 720
59. O uu FLAG 7732 60 0980 1 0 0 0 08 4800003 L L O FLAG Angpt 2 ut Angpt 20 2700020000000 Angpt 2 0 ER Kim 1 0 J Biol Chem 2740 2 3 8 1999 FLAG LE TYT FLAG 000000000 48 Angpt 20 1 4 O FLAG 6 20 8 11 0 0 O 900 20 0000000000 200 CD45 2 SP Sca 1 CD45 O I O 10ng m 50 0 20ng mi TPO 20ng m GF 2 10ng m 10 O 10600000 100 Fl ag Angpt 20 0 p1000 00000041 0000000000000 rao CD45 1 0 O 045 11 00 50 30 2000000000000000 71 I 18000000000 Angpt 29 O O Angpti 30 HSC OO 20 I 1I I 8 58 D45 Sca 1 RU 0 00 300 230 10 950 I 0 II D 1 150 1 350 n 250 0 7A00 00000000000 60 0 39 0 11 II II II II II II II II II 95000 0 II 1 240 1
60. 2931 0 0 48 FLAG II II II II II II II II II II II 9 2 000000000000 M 51 II II LI SP Sca 1 0 45 utu 10 0 LI 110 II II II II II II II II II 0 45 100 000 200 II II II II 045 21 O SP Sca 1 045 O00 3 LI 4 9 1111 11 38 Od 9 II 3 II 150 II II II 1 U L L U FASO II II II II II II II II II II II II II II II BAD LI LI 130 1511 II II II II II II II II II II II II L LI II II 3 Od LI II II 40 II II 1 FACS LI LI II II 3A0 10 8 0 U 0 4 1 II II II II Aj SDS PAGE II LI O Angpt 20 16ng I 2 LI II II LI II II II FLAG Angpt 2 2 5ng FLAG I II II II II 31 II II II II BO II FLAG Angpt 20 0000 LI II II 0301 Angpt 2 O D Angpt 2 85 O II LI LI III III II II II Angpt HSQ II II II II II II II II L II 6 II II II II hAngpti 20 000000000000 II
61. 630 1 Poi sson 10 II II II II II 23000 391 BM SP CD45 5 8 1 D D 1 NAN 630 e O Angpt 20 0 II 770 LI LI O 100 0 O 11 11 1141 10000 CRUD 0 30 0 1 1 1 78000000 95 0 50 1 2 30 300 0000 60 0 O 60 7B 0 01010000 II II I II 91 00000 1 1 10 1 2 30 0 1 1 10 30 40 50 28 JP 2008 545412 2008 12 18 10 LI LI BM SP CD45 5 8 1 63 LI LI II I LI LI LI II II II II II II II II 24 39 1 6 LI I LI II II II II II II II II II II II LT HS LI LI o L L Od Angpt 3 LI II 300 95 L 1 0 3 1 1 70 24 O 0 95 1 0 90 1 2 0 n 24 300000000 100 0 O0 BM SP CD45 Sca 1 1 3 L II LI II 6LI II II II II II LI II II DO II II Angpt 239 309 O LI LI
62. A 322 35 2 2 100 7 8 7 1013 40 20 Li ded T B Beat U U gt PE 191 a 51 lt 297 28 19 30 2 20 BE SAE 1 LES of 4 M 10 Ta 1 2 jA gt 123 x o 4 2 sam tog E E 5 s 8 a X L 3 S TH F S T l F CC Angpti2 55 S TH F FBN Angpti2 e S TH F Angptl2 STA HE Angpil2 co S T F 1557279070 Angpti2 N S T I F 18801 S T I F CC Angpti2 S T F FBN Angpil2 A a AL m N TAG 88868 1 9623742 727 a A 727 953742 0 9 XE BEBEASCE Y Y XE SER HAT AIO L 95 zt 0r68 419 Sk Eb ZL yk 8c 6 Ig L6 161 v9 E 61 gt Angptl7 GST Angpti5 Angptl4 3 I S TH F S T J F AngptiS S T l F Angptl4 S T 4F Angptls S T H F Angpti S TH F S T l F Angptl3 S T l F Angpti4 S T 1 F AngptiS S T H F Angptl7 S TH F S T I F Angpti3 5 T F Angpil4 S T i F Angpti5 S T itF Angptl gt 34 JP 2008 545412 2008 12 18 v E S 9 b i S T MF Angpti3 5 8 m S T H E Angptis S T F Angpti
63. EJ Ew c NEMPE oo EI T e NM 7 i lt i m ac gt gt ris 15 JP 2008 545412 2008 12 18
64. II II 10 9 0000000000 HSS II II II II II II II II II II O 0 1 O 50ng mp I II II II II II II II II I I LI II II II II II II II II II II II II II II 1 9 100ng LI II II II II II II II II 10ng m 50ng m 1 II II II II II II II II II II II II II II II II II II LI II II II II II II LI II UU t LI I HSC II LL II L L L L LI II II II II II II II II II L FAQ II II II FGF 0 FGOF 27 000000000 I GF 2 0 O GF 1 LI I II II II SCF II II 0 1ng ni 500ng ni 1ng nt 200ng ni LI II II 1009 mp 111112101 I II II II II L FGF 11 TPQ I GF 21 O I SCF SCF 10ng m 0 O TPO 20ng m O O GF 2 20ng m FGF 111 10ng LI II U L L DOHODU II II II II II G CSF GM CSF IL 1a 0 II O IL 11 LI II II LI II II II II II II I LI II II L LI
65. II II II 111111 II II I II II II II II II II II I 0o 445 I II II I I I I I II 0 10 csto I 1 II HCO 000000000101000010000 30 CD3 O O Angptl 2 0 O Angpt 30 D000 150 0 0 0 0 O POF 0 MASE 8 1 L LI 1 0 63 0 1I 1 I 1 1 7 0 II II II II II II II II II II II II II II II II II II II 1 25 JP 2008 545412 2008 12 18 LI I LI 1 SpnCD3 E FLCD3 GI 4E 1 2 claudin 13 10048432 ea fo RIKEN cDN 21313426 NP 084345 1774 3 A A 21515426 mes prz 8 123 Ws 7 4 gt C X C AY 12963823 NP 076274 1936 2 7 31560520 NP 036053 583 2 p 11612497 NP 071713 586 2528 417821 85492 31981988 NP 705768 0 RIKEN cDNA 2210020 01 34304038 NP 899082 875 1 1 2 7 2 23346595 NP 694792 RIKEN cDNA ilie 22122497 666132 277 8 RIKEN cDNA 1300010M03 28077025 7 1 28077031 FLGr 1 1 2 GRIE NP lt
66. 0 3 1 D II 10 LI FLAG hAngpt 20 0 20 LI B D FLAG hAngpt 20 O 1 3 29310 480 D II D O ID L LI LI LI II II II LI II II II 29310 0 STI LI II 111 oU II B Angptl 20 100 9 3 Y O 20 7 Angptl 20 100ng m 3 80 50 20 CD45 2 SP 5 8 1 045 0 D 3 LI oU TA BM SP CD45 Sca 1 780 O 100ng 0 O O Angpt 2 O00 STIF 10 BM SP CD45 Sca 1 0 0 7 II 100ng ni O O Angpt 3 STI F 10 DDD BM SP CD45 sca 1 LI II Angpt 3 Angpt 50 O Angpt 7 L I II II II II II II II II 10 20 30 40 50 31 JP 2008 545412 2008 12 18
67. 000 20 00000000 I I 0 70 00 0 88080 J D D D NOD 50 D 0 LI I 1 80 08 0 0 88 8 0 0 0 0 0 0 0 0 0 0 GARE JP 2008 545412 2008 12 18 32 12 S T I F pz S T I F A 100 10 10 3 5 1012 5 102 Jey zd 10 101 WMA z 10 1004 10033 109 101 102 109 104 109 101 102 403 104 109 101 102 403 404 100 40 40 408 10 FLA H FLT H FL1 H FL1 H 09M 588 56 SARI 72 N o 90 9 CD45 2 80 R 18IE3RHLT 70 60 50 40 30 20 10 0 T B SAR T B Bi USER SER USER 558 10858 S T F A u Lo E BE s fe o 28 5 OS mb vi m4 oe spo o mh EO 6 5 m 7 Um m m m Sm 2 c E m wo 4445 1213 11 91 78 6 45 JP 2008 545412 2008 12 18 33 191 100 12 3 Mac 1 Gr 1 10 10 Tr gt ER 102 101 101 ri O 1 10 8 10 101 102 103 0 100 10 10 109 10 100 101 402 403 104 109 10 102 103 404 FL1 H FL1 H FL1 H FL1 H 05452
68. 000000 1 II II LI U R amp D System II GST hAngpt 5 5 50 hAngpt 50 0 II II II II II II II in vitro J J Abnova Corporati onl Tai wan 100000000050 hAngpt 2 hAngpt 7 R amp D Syst ens II II II II II LI 20 LI LI LI II t Angpt 20 0 0 0 II O DNA Kim Biol Chem 274 26523 8 1999 II II II II I II II II LI O FLAG hAngpt 2 LI I CILCII E Pro100 LV5330L 0 00000 FLAG 00 II II O FLAG hAngpt 20 O O 8 DNA pcDNA3 1 I O C 1 08 293 0 O O II pof ectani ne 2000 Invi trogen U II II II II II U U LI 48L II 720 LI II II II II II II II II II II FLAG Angpt 20 0 FLAG Angpt 40 II II II II II II II 480 72 FLAG hAngpt 20 FLAG hAng L I 4 LI LI U 1 I D II Compl ete Protease nhi bi to r Cocktail Roche 50m 0 PMSF 54 XI II OO NaCl 1000 000000900000 LI 500m0 0 II O 5001 1 00000 FLAGI FLAG MO II II LI 5 9 8 0 II II II LI II II II II II II II 0 1000 0 TBS 5 Om Tris pH 7 40 150mM NaCl O O 110 FLAG hAngpt 20
69. 10 0 0 2 1111 11 251 D45 10000 8000000 29310 O Angpti2 00 000000000 5 NJ n 5 LI LI LI LI LI LI Angpt140 2931 00 0 0 0 0 0 0 0 0 0 0 FLAGI 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 01 0 0 0 0 0 0 0 0 0 0 Angpt130 0 0 0 0 0 0 8 8 8 6 0 8 0 sf210 5 0000000 65 0 Angpt 50 linvitrog 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7 0 0 0 0 29 JP 2008 545412 2008 12 18
70. 150 1 II I II II II II II II II II II II II II II II DOD 1 II II II II II II 1 II I II II 0 O D DNA cDNA O DNA O RNAJ J 0 0 III II II II II II II II II II II II II II 0 1 0 00 0 II I II II II II II II 0 933 I I II in vi vo ex vi vo in vitro 1000000 I I II III 1 I II II II II II II 21 JP 2008 545412 2008 12 18
71. 20ng m G 20 10ng m FGF 1 180000000000 Flag 2 000000000 0 29310 00000000000 2 100 9 4 Angpt 20 0000 5 10 9 m SCF 0 O _ O 100ng m Anget 2 0 H U U D D 0 0 5 10ng m SCF 20ng m TPO H O O 100ng m Angptl 2 1 0 O O S TH 10ng m SCF 20ng TPQ 20ng m GF 2 10ng m FG 19 0 O I O O 100ng 1 Angpt 20 1 1 30 II II II L L 3A O O Angpt 20 LT HSG ex vi vol Angpt 20 0 0 O FLAG Angpt 20 291 00400000000 U U U I LI I II II II II II II II II II 20 BM SP CD45 Sca 1 1000 HSCI Camargo F D Green R apetanaki Y Jackson K A Goodell MA Nat Med 9 1520 7 2003 SU 0 SCHOO LI II LI II II II II II II II II II II II II II II II II II 27 JP 2008 545412 2008 12 18
72. 314 4320 4308 XP002382752 ISSN 0006 4971 the whole document ZHANG CHENG CHENG ET AL Insulin like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells ian W B SAUNDERS COMPANY ORLANDO FL S vol 103 no 7 30 October 2003 2003 10 30 pages 2513 2521 XP002382851 ISSN 0006 4971 ihe whole document CHEN CHANG ZHENG ET AL Identification of endoglin as a functional marker that defines long term repopulating hematopoietic stem cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA vol 99 no 24 26 November 2002 2002 11 26 pages 15468 15473 XP002408121 ISSN 0027 8424 the whole document CHOONG M L ET AL novel role for proliferin 2 in the ex vivo expansion of hematopoietic stem cells FEBS LETTERS ELSEVIER AMSTERDAM NL vol 550 no 1 3 28 August 2003 2003 08 28 pages 155 162 XP004448385 ISSN 0014 5793 the whole document Form PCT ISA 210 continuation of second sheet April 2005 39 JP 2008 545412 2008 12 18 International application No PCT US2006 020078 Relevant to clalm No INTERNATIONAL SEARCH REPORT C Continuation DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document with indicatlon where appropriate of the relevant passages CAMENISCH G ET AL ANGPTL3 stimulates endothel
73. 7 S T I F i S T FrAngpii3 S T F Angpt5 S T 1 F Angpti7 LYSteci 151016 8 S T I F 6 0 2 S T F Angpti2 CC S T i F Angptli2 FBN S T HHF Angptl4 S TH F Fglt S T H F mfap4 S T I F S T HF Angptl2 S T F Angptiz CC S T l F Angpii2 FBN S TH F Angpild S T I F Fg 1 S T H F mfap4 N 1 S T F S T HF Fgi1 S T F Mfap4 S T F S TH F Fgl1 S T I F Mfap4 JP 2008 545412 2008 12 18 35 SERV SER n 3 FLCD3 88 CD3 FL Gr 1 FL CD3 8 CD3 FL Gr 1 351148 Xj HEB n 3 r 7824 3048213838 n 3 8 8 8 8 5 B 8 1 5 Ba Angptl2 Grit Ta M CHR 8 282 2 1 88 IL 3 M CSF 85 08 PERLA CREME DO EIS ELIT Epo SCF Epo Q GM CSF O sA 6 6 CRU E BFU E 53 SCF Tpo IL 3 GM CSF 2 6 GI 12 112 112 SORA ALT IM IL THF TCC 860 REROTE KUBAR LU LU 198 23 1E6 356 186 Angpti5 186 Angptl3 1 LI IESS 3 mrpicvtcww lgllaamgav agqedgfegt 61 paarvtgaic vnskepevil enrvhkgele 121 kllrkesrnm nsrv
74. Ea 3 EJ a 3 NO E CI CI 3 E 3 3 za O OE EJ EJ 3 L3 EJ p 3 EJ I o E r3 r3 ET EJ EJ 1 ni mo m oa 112 t 2 SN um a r3 us lt D a c LI oo 3 0 3 moo G moo 3 LI EJ L3 LI SM gt 3 EJ EJ EJ _ oc gt gt zz a El o ot EJ 3 r3 3 L I ro 3 3 CI 3 2 L3 EJ 3 EJ r3 lt 3 3 EJ 3 3 3 m o CI CI El EI i 8 LI E EP El r3 L I I 2 O I r3 38 ES r3 r3 EJ 0 Ol 3 EJ _ 1 1 C OD a r3 lt o EJ r3 1 CI gt 3 1 2 0 3 Yoo no EJ ca c 3 lt AN L3 55 r3 m r3 o 3 3 EJ lt r3 o X 2 o a EE 3 OE r3 O EJ a 85 lt ri EE L3 EJ 3 EJ El r3 3 3 00 2 __ foo L3 EJ 3 EB 3 EJ E E n CI o CI E OE 3 g onog 3 EE CI wo O CEE 3 3 3 EME CI 3 C
75. I TM 3 EJ 3 CIE o R CI O Oo CIE AE O a o 3 EJ 3 ET EJ 0 lt OF ET g OO ES Y ODO 3 CI 3 ND 3 c 2 r 3 a E c ELE lt N oOo EEE OO 3 o EXE 3 oo ma C mo cooo m OO 8 L3 EJ cC ET 00 ooo es EJ EF 1 EJ O I 3 a mon I L3 EJ o o _ lt Cv lt o E lt O 5 3 a c E o EJ o lt 1 3 1 ZE U gt EJ 3 3 L3 EJ E o m 5 3 ET EJ st x O LI O o c ET EJ N m I m Ev C 95 ol m c 0 Et SLE lt 5 0 lt mE O o lt x 0 on C EJ i N I EIT c o LI I I Ol LI o O I 2 o M 0 r a 18 JP 2008 545412 2008 12 18
76. II II II II II II II II II II II II II II 1 1 LI IL II II L I L L L I IULIU III LI Hse in I I II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II in vitro 1 t I LI II II II II 0 NOD SCI D SCI D hul 0 0 B U Kyoi zum 1992 Bl oo d 79 17040 Murray 1995 Blood 85 2 368 378 0 II utero II gt anjani 1992 J Clin Invest 899 11790 0 000 000000000000000 Srour 1992 J Hematother 19 143 1537 D 000000000 II II II II II II II II II II II vitro II II II II II II II II 50 II I II II II II II II II II II II I LI LI LI LI II II II II II II II II II II II II II II II II 1800000000 Sutherland 1990 Proc Nat Acad Sci 870 3584 3588L O0 LICICII II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II I cobblestone area form ng cell CAFCI 000000000 in vivo B Bt Breens 1994 Leukema 80 10950 II II II II II II 10 L II LI II II II II II L II HBO II II II II II II II II II II II II II II II II II II I II II II II II II II II II II L II uu II II II II II II II II II II I L II LI II II II II II L II L II II II II II II II II II II II II 5 II II II II II II II II II II II II II
77. II II II II II II II II II II II II O VO 94 294380 VO 97 21824 w 97 21825 7 0000 0 II pox ex vi vol U ex vivo in vitro G000000000 HS 20 30 40 50 JP 2008 545412 2008 12 18 17
78. II II II II II II HG II II II II II II II II II II I 5 L I L 0 Goodell J Exp Med 183 1797 6 Apr 1 1996 0 D LI II LI II III III II II II II L I L I L O O 5 061 6200 I LI O O CD34 Thy 1 LI 1 20 CAF Murray 1995 O Lansdorp J Exp Med 177 1331 1993 5 681 5590 II II II II II II II II II II II II II II 19 JP 2008 545412 2008 12 18 L II L II L II II II II II II II II II II II II II II II II II I I LI LI II II II II II II II II II II II II II U Shi Q0 1998 Blood 92 362 367 L I I II II II I 19880 J Cell Science Supp 109 63 760 L 0 U D Pittenger rshak DR 2001 Marshak D Gardner D KG O Gottlieb pring Harbor New York Cold Spring Harbor Laboratory Press 349 374 Gussoni E 1999 Nature 401 390 3940 H 0 I LI II II II II II II II II II II II II II II II II II II II
79. LI 128L 4800000000000 CD45 20 0 00 O SC TPO IGF 2 000 FGF 1 L II L II II II II II II 4 II II Reya T Nature 423 409 14 2003 I LI II II II II II II II II II II 311 II II II II II II II II II 0 FI LI 2 LI II II II 1p g m F ag Angpti 211 000000000000530 000000000 FLAGI 0 II II 130 0I II II II II 80000 II II II II II II 2X105 CD45 10 O L LI LI L LI LI LI II II C5 0 0 00000 II II 300000 30 III 1 II II II II II II II II II II II 5 2 Angpt 2 0 Angpt 3 ex vi vog HSC 1 II 0003 1 1 0 0 11 II LI FLAG 00000000 Angpti 2000000000 II FLAG Angpt 20 O 0 II II I LI LI LI LI II II II II II II II II II II II II II II II oko 1 OOO FLAG LI 211 00000010000 I I II II II II II II II II II II II II II LT HS 4 a Y LT Asco 1 1 1 11 11 120 2000000 a 200 CD45 2 SP Sca 1 CD45 96 000 II LI II II II II II II II II II II II II II II 293 II II II II II II II II II II II II 5 FU SCF 10 ng m SCI II II LI LI 5 1 10ng m 50 01 l 20ng m TPO O00 0 S TH 10ng m 5 20ng m TPO
80. MENTS CONSIDERED TO BE RELEVANT Category Citation of document with indication where appropriate of the relevant passages Relevant to claim No EP 1 308 511 A MERMAID PHARMACEUTICALS 1 2 18 GMBH DE 7 May 2003 2003 05 07 19 39 the whole document 3 17 i 20 38 40 46 1 18 39 2 17 19 38 40 46 1 18 39 EP 962 530 A2 LILLY CO ELE US LILLY CO ELI 8 December 1999 1999 12 08 the whole document WO 00 52167 A1 REGENERON PHARMA US JONES PAMELA F GB VALENZUELA DAVID M US 8 September 2000 2000 09 08 the whole document 2 17 19 38 40 46 x Further documents are listed in the continuation of Box C IX See patent family annex i Speclalcategorles of cited documents T later document published after the international filing date or priority date and not in conflict with tha application but document defining tha general state of the art which is not considered to ba of particular relevance undormand the principle or theory underlying the 8 MANNA but published on or after the international xX document of particular relevance the claimed invention ng dal cannot be considered novel or cannot be considered to Lt mm 272 T priority olalm s Br Involve an inventlve step when the document taken alone m 0 establish the publication date of another Y document of particular retevance
81. O I II II II II II II II II II 440000000 5 ut 070000 4 U I L 5L I II II II II II II II II II I II II II II II II II II II ES E 3 ZE E ie E II HS L L 0 1H 10 I II II II II II II II II II LI 10 20 30 40 50 JP 2008 545412 2008 12 18 6
82. RAC 64 ROZM SRL ISMN SIA LO ART 79 57 0 000 10000 0 0 0 0000000000000000000 0 0 8 8 0 8 8 8 8 8 8 8 8 0 8 0 8 0 8 0 0 0 8 8 8 0 8 80 0 0 8 0 0 0 0 00 000000 0000 0000000000000000000 000020 0 0 030000000000000004000 000000 D n 50 0000000 000000000000 000000 0 Mapd D 0 0 0 8 8 0 8 0 8 0 0 0 0 0 0 0 0 0 00 10000 0 0 2 JP 2008 545412 2008 12 18 in vitro I I LI C3 C3 C3 HM 2 P Ul 5 P I LI LI II II II II II II II II II LI LI I LI II II II II II II LI LI LI II II II II II II II II LI
83. ial cell adhesion and migration via integrin alphavbeta3 and induces blood vessel formation in vivo JOURNAL OF BIOLOGICAL CHEMISTRY AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS BIRMINGHAM US vol 277 no 19 10 May 2002 2002 05 10 pages 17281 17290 XP002333996 ISSN 0021 9258 the whole document KIM ET AL Molecular cloning expression and characterization of angiopoietin related protein Angiopoietin related protein induces endothelial cell sprouting JOURNAL OF BIOLOGICAL CHEMISTRY AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS BIRMINGHAM US 10 September 1999 1999 09 10 pages 26523 26528 XP002146664 ISSN 0021 9258 the whole document OIKE YUICHI ET AL Angiopoietin related angiopoietin like proteins regulate angiogenesis INTERNATIONAL JOURNAL OF HEMATOLOGY vol 80 no 1 July 2004 2004 07 pages 21 28 XP009075061 ISSN 0925 5710 the whole document ZHANG CHENG CHENG ET AL Angiopoietin like proteins stimulate ex vivo expansion of hematopoietic stem cells NATURE MEDICINE vol 12 no 2 February 2006 2006 02 pages 240 245 XP002408122 ISSN 1078 8956 the whole document Form 18 210 continuation of second sheet April 2005 40 JP 2008 545412 2008 12 18 International application No PCT US2006 020078 INTERNATIONAL SEARCH REPORT Box H Observations where certain claims were found unsearchable Conti
84. keeekelr rttykiqvko eevknmslel napetpehpe vtslktfvek qdnsikdllq pteislsskp raprttpflg lneirnvkhd hvycdvisgs pwtliqhrid gsquinetwe lrieledwkd nkhyieysfy lgnhetnytl hfncpegvsg gwwwhdecge nnlngkynkp ihptdsesfe ksprfaswde mnvlahgllq Igaglrehae lapesrvdpe vlhsigtglk agnsrigalf hldhevakpa rrkrlpemaq pvdpahnvsr pflvnckmts dggwtvigrr hdgsvdfnrp srlavglrdw dgnaellgfs vhlggedtay qgdhdlrrdkn caksisggww Egtcshsnin qattmliqpm aaeaas st ssvvniv edgsnakdes ksndtvcked trstkkllrn mmdeqgasld 7 tdikdtigsv tktpsglyii hpegssypfe dvldgfgdll gefwigikki fyivngknts fkmhigrysg nagdafrglk kednqnampE tgwwinecgl aningihhfs gkliatgiqw 36 JP 2008 545412 A 2008 12 18 37 JP 2008 545412 2008 12 18 II INTERNATIONAL SEARCH REPORT International application No PCT US2006 020078 CLASSIFICATION OF SUBJECT robe INV 1285 06 K35 14 6 0 According to Intemational Patent Classification IPC or to both national classification and IPC B FIELDS SEARCHED Minimum documentation searched classification system followed by classification symbols C12N Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the International search name of data base and where practical search terms used EPO Internal BIOSIS EMBASE WPI Data C DOCU
85. nuation of item 2 of first sheet This Intemational Search Report has not been established in respect of certain claims under Article 17 2 a for the following reasons 1 x Claims Nos because they relate to subject matter not required to be searched by this Authority namely Although claims 31 36 are directed to a method of treatment of the human animal body the search has been carried out and based on the alleged effects of the compound composition 2 L Claims Nos because they relate to parts of the Intemational Application that do not comply with the prescribed requirements to such an extent that no meaningful International Search can be carried out specifically Claims Nos because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6 4 a Box Ill Observations where unity of invention Is lacking Continuation of item 3 of first sheet This International Searching Authority found multiple inventions in this international application as follows L As all required additional search fees were timely paid by the applicant this International Search Report covers all searchable claims 2 L As all searchable claims could be searched without effort jusiifying an additional fee thls Authority did not Invite payment of any additional fee 8 As only some of the required additional search fees were timely paid by the applicant thi
86. ogy 0000240 0 II IL III 10 580 LI LI II II II LI II II II II II II II II II II 0 II II II LI II II II II II II II II II II II II LI II II II II II II 0 2 Angpt 30 50 23 JP 2008 545412 2008 12 18
87. s International Search Report covers only those claims for which fees were paid specifically claims Nos No required additional search fees were timely paid by the applicant Consequently thls International Search Report is restricted to the invention first mentioned in the clalms it is covered by claims Nos Remark on Protest The additional search fees were accompanied by the applicant s protest 1 No protest accompanied the payment of additional search fees Form PCT ISA 210 continuation of first sheet 2 January 2004 41 JP 2008 545412 A 2008 12 18 INTERNATIONAL SEARCH REPORT International application No information on patent family members PCT 52006 020078 cited in search report date member s date EP 1308511 A 07 05 2003 NONE EP 0962530 A2 08 12 1999 AU 4320499 A 20 12 1999 WO 9962925 A1 09 12 1999 WO 0052167 Al 08 09 2000 AU 3359600 A 21 09 2000 CA 2362547 1 08 09 2000 EP 1157111 Al 28 11 2001 JP 2002537813 A 12 11 2002 rm Form PCTASA 210 patent family annex April 2005 42 JP 2008 545412 2008 12 18
88. tglymg llheiirkrd 181 hkyghlatla hnqseiiaql eehcqrvpsa 241 eiqsdqnlkv lppplptmpt ltslpsstdk 301 rlmavwodar hdpggwtvig rrldgsvn 361 gnykllvtme dwsgrkv ae yasfrlepes 421 rdhdvytgnc ahygkogwwy nacahsning 481 vvmmirpnpn tfh 5374 1 m tiklllfi vpivissrid qdnssfdsls 61 vhktkgaind ifqkinifdq sfydlslats 121 nsklesllee kiliggkvky leegltnlig 181 tvedaykaln gahsgikeie agirrtsige 241 gipaecttiy nrgehtsgmy airpsnsqvf 301 nykygfgrld gefwlgleki ysivkasnyv 361 hlvaitgnvp naipenkdlv fstwdhkakg 421 rakskperrr glswksqugr lysikstkmi 5 1 msgaptagaa imlcaatavl lsaggapvas 61 xtrsqlsale rrisacgsac agtegstdlp 121 hkvaggarhl ekghlrighl asafglldhk 181 lhrlprdege lfavgerasg ifeiapagsp 241 weaykagfgd phgefwigle kvhsitgdrn 301 slaltapvag glgattvpps glsvpfstwd 361 gayfrsipgg rqkikkgifw ktwrgryypl 6 1 mmspsgasll finveificg eavqgncvhh 61 ceescdvktk itreekhfmc rnlgnsivsy 121 nrvlllttev frkqidpfoh rpvashgl c 181 vmcdmdyrgg gwtvigkrid giidfarlwc 241 fmlyvalese ddtlayasyd nfwledetrt 301 stsdvdndge rpaclvnggs vkscshlhnk 361 gtwtknnspv kiksvsmkir xmynpyfk o B 2008545412000001 xm eegsprefiy lnxykrages qdkctytfiv linnelikak rqietlaalv evdggivsev nalelsglen rilngtadml qlaskykdle rpvpapppaa pprvyappty nriinqistn psepwrdclg aledghdtss iylvkpentn rnwetykqgf gnidgeywlg leniywltnq eyyklrlgry hgnagdsftw hngkgfttld vwyrgghyrs ryqdgvywae frggsyslkk pepkerfaml ddvkilangi lglghglkdf ei
89. the clalmed Invention Citation or other special reason as specified cannot be considered to Involve an inventive step when the O document referring to an oral disclosure use exhibition or document is combined with one or more other such docu other means ments such combination being obvlous to a person skilled P document published prior to the Intemational filing date but in the art later than the priority date claimed document member of the same patent family Dats of the actual completion of the international search Date of mailing of the intemational search report 04 12 2006 Authorized officer 20 November 2006 Name and malling address of the ISA European Patent Office P B 5818 Patentlaan 2 NL 2280 HV Rijswlik Tel 31 70 340 2040 Tx 81 651 epo nl Fax 31 70 340 3018 Morawetz Renate Form PGTASA 210 second sheal April 2005 38 JP 2008 545412 2008 12 18 International application No PCT US2006 020078 C Continuation DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document with indication where appropriate of the relevant passages Relevant to claim No INTERNATIONAL SEARCH REPORT ZHANG CHENG CHENG ET AL hematopoietic stem cells change their surface phenotype during ex vivo expansion W B SAUNDERS COMPANY ORLANDO FL vol 105 no 11 8 February 2005 2005 02 08 pages 4
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