In vitro directed differentiation of human embryonic stem cells

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1 Chinese Bulletin of Life Sciences Vol. 19, No. 5 Oct., (2007) ( ) (human embryonic stem cells, hescs) hescs ( ) hescs (mouse embryonic fibroblast, MEF) hescs hescs hescs Q813 In vitro directed differentiation of human embryonic stem cells LIU Weidong, REN Caiping*, YAO Kaitai* (Cancer Research Institute, Central South University, Changsha , China) Abstract: Human embryonic stem cells (hescs) can be derived from the inner cell mass of blastocyst embryo and propagate infinitely in an undifferentiated state. hescs display pluripotency and can be differentiated into derivatives of all three embryonic germ layers (ectoderm, mesoderm and endoderm) both in vitro and in vivo. Generally hescs culture requires mouse embryonic fibroblast (MEF) feeder cells to maintain the undifferentiated state. In order to optimize the culture conditions, human feeder cells and feeder-free, unconditioned culture systems have been developed. In vitro, hescs can be directed to differentiate into various cell types, which lays a foundation for cell transplantation therapy and clarification of the human embryonic development. hescs can be genetically modified in vitro, which will help to elucidate the specific genes' regulation and functions in embryonic development. Deeper studies of hescs will greatly further the progression of medical and life sciences, and will be finally applied in clinical medicine to benefit mankind. Key words: human embryonic stem cell; directed differentiation; genetic modification (embryonic stem cells, ESCs) hescs hescs [1-2] ( ) (1968 ) (1972 ) rencaiping@xysm.net (1931 ) ktyao@fimmu.com

2 5 519 RNA DNA hescs hescs [1] hesc (embryoid body, EB) EB hescs β hescs 1 hescs hescs γ- C MEF ESC ESC MEF [1-2] 7d IV MEF MEF hescs hescs [3] [4] [5] [6] hescs [7-8] hescs Ellerstrom [9] Tyrode s hesc SA611 hesc MEF hescs Xu [10] Matrigel laminin( ) hescs MEF Xu [11] bfgf BMP noggin hescs hescs hescs hescs 2 hescs 2.1 FGF-2 DMEM/ F12 EB, nestin Musashi-1 dispase FGF-2, hescs CNS γ- (GABA) (tyrosine hydrolase, TH) [12] Reubinoff [13] EGF bfgf hesc 99% NCAM 97% nestin hescs hescs MS5 MS5-Wnt( Wnt

3 520 MS5 ) Pax6 nestin NCAM Sox1 / SHH (sonic hedgehog) FGF8 N2 SHH FGF8 camp β3 N2 30% 50% Tuj1 64% 79% Pax2 Pax5 engrailed-1 10% 20% 2% 4% HPLC KCl 65% hescs [14] Yan [15] FGF8 SHH FGF8 SHH (isthmic organizer) [16-17] hescs [18] 6- ( ) 8 hescs TH hescs hescs (retinoic acid, RA) FGF2 RA SHH BDNF(brain-derived neurotrophic factor) GDNF(glial-derived neurotrophic factor) IGF-1(insulinlike growth factor-1), SHH [19] Lim [20] hesc EB RA SHH EB HB9 Wnt3A -2(bone morphogenetic protein-2, BMP-2) RA SHH hescs 2.2 cyclopamine hedgehog (Hh) cyclopamine hescs hescs (GFAP) nestin (GDNF) (BDNF) [21] 2.3 Fang [22] Wnt3A endothelin hesc 2.4 ( ) hescs Ahmad [23] hescs, hescs hescs Oct-4 Nanog SSEA4 p63 CK3 7d p H9 SCID 2 3T3 FAD p63 K14 20d

4 K14 hescs EB EB Oct-4 p63 K14 3T3 [24] Ji [25] EB K hescs EB 4 22d 8.1% EB Z EB α/β α I (ctni) (desmin) ANP (nebulin) [26] EB EB 70% DMSO [27] Norstrom [28] EB EB 30% Z Yoon [29] 5- hescs Pal Khanna [30] hesc EB, BMP-2, EB noggin BMP-2 hescs Mummery [31] hescs END-2 15% 20% [31-33] hescs Capi Gepstein [34] hesc 2.7 EB EB PECAM1(CD31) (VE-cad CD34) (VEGFR-2) Tie-1 GATA-2 GATA- 3 / AC133/ CD133 EB hescs PECAM1 FACS EB PECAM1 PECAM1 SCID PECAM1 CD34 [35] 2.8 (HSC) HSC 2/3 hesc H1 S17 C166 20% (FBS) 17d 1% 2% CD34 + CD38 - CFU 10 5 H1/ S H1/C CD34 + MACS CFU [36] EB BMP-4 (SCF) IL-2 IL-3 (GCSF) Flt-3 [37] BMP-4 A (VEGF-A) hescs [38] Bowles [39] HOXB4

5 522 hescs hescs Qiu [40] FH-B-hTERT hescs S17 S17 FH-B-hTERT ε mrna, γ mrna β mrna hesc OP9 CD34 + CD73 + (MSC) CD34 + CD73 + MSC MSC [41] 2.9 Hwang [42] EB hesc CD29 CD44 CD105 α (PDGFR-α) - - (RGD) TGF-β1 Aggrecan (link protein) EB EB hescs Col2 Col1 Col1 Col2a1 link protein Sox9 BMP-2 BMP-2 [43] 2.10 hescs EB 70 80% AAT CK8 CK18 70% 76% EROD H1 H9 hesc p450 trichostatin A hescs [44] Lavon [45] EB egfp ALB-eGFP hescs EB 20d egfp EB egfp FACS afgf bfgf BMP-4 HGF afgf 2.11 β 1 β hescs β EB 60% 70% hescs EB 1% 3% [46] EB β GLUT1 GK 1% 3% RT-PCR insulin-1 insulin-2 mrna C [47] Segev [48] hescs EB (ITSF) bfgf N2 B27 bfgf 225.8±78µu/ml/10 5 cell/hour 60min RT-PCR C

6 Chen [49] hesc c-kit STELLA VASA GDF9 hesc 2.13 BMP-4 hescs BMP BMP BMP noggin BMP-4 BMP-4 ESC cdna RT-PCR BMP-4 7d CG-α CGβ β GCM1 HLA- G1 CD9 BMP-4 H1 CG [50] 3 hesc hesc hescs hescs ( ) sirna Eiges [51] TK Lipofectamine(Life Technologies) FuGene(Boehringer Mannheim) ExGen 500(Fermentas) ExGen 500 ExGen 500 Rex1-EGFP hescs 10-5 hescs EB GFP Ren [52] EBNA1 hescs EBV orip orip orip shrna EBV hescs RNA hesc (mesc) ESCs PBS Zwaka Thomson [53] HPRT1 POU5F1 HPRT1 POU5F1 2% 27% hescs G (vesicular stomatitis virus G, VSV-G) VSV-G MOI (multiplicity of infection) egfp 20% 80% hescs egfp [54-55] VSV-G hescs MEF Jang [56] (GALV) (RD114) GALV RD114 hescs MEF hescs hescs sirna hescs Matin [57] Hay [58] Oct-4 β-2 hescs Zaehres [59] sirna hescs egfp Oct-4 Nanog Oct-4 Nanog hescs 4 hesc (1) hescs

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