CHINESE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY. R34915 A DOI : / zgzzhx

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1 CHINESE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY Vol1 181 No1 3 J un ( ; 1, ), (embryonic body, EB), : A, EB ; B, noggin ITSFn, RT2PCR Nestin, RT2PCR MA P2 GFAP RT2PCR OCT4 nestin B nestin A, ( P < 0101), A,, MA P2 GFA P, EB,, EB ; ; R34915 A DOI : / zgzzhx DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS INTO NEURAL STEM CELLS IN VI TRO Jiao Shujie, Xu Huifang, Yang Huajing 1, Xu Jie, Zhan Yanqiang, Zhang Suming 3 ( Department of N eurolog y, 1 Dep artment of O p ht hal molog y, Ton g j i Hos pit al, H uaz hong Uni versit y of S cience and Technolog y, W uhan , Chi na) Abstract Objective To investigate the feasibility and efficiency of differentiation of human embry2 onic stem cells (h ESCs) into neural cells wit h or wit hout a period of embryonic body ( EB) cult ure i n vit ro1 Met hods The h ESCs were digested and divided into 2 group s : group A underwent EB cult ure, while cells in group B were induced by noggin and ITSFn directly wit hout EB cult ure1 Morp hologic changes were ob2 served under inverted micro scope1 The different marker s were detected by R T2PCR1 Nestin was detected by immunofluorescence and flow cytometry after h ESCs differentiated1 In the f urt her differentiation of neural stem cens, MA P2 and GFA P were observed by immunofluorescence and R T2PCR1 Result s In group B, t he positive rate of nestin was higher t han t hat in group A ( P < 0101) 1 The cycle time of group B was shorter t han t hat of gro up A1 MA P2 and GFA P were detected af ter t he differentiatio n of neural stem cells1 Conclusion The h ESCs can differentiate into neural cells wit hout EB cult ure i n vit ro1 This met hod is more efficient t han the traditional1 Key words Human embryonic stem cell ; Cell differentiation ; Neural stem cell, Parkinsonπs, : (embryonic bodies, EB) [1 ] ; ( PA6, MS5 ) [2 ] ; [3 ] EB, , (1976 ),, 3 ( To whom correspondence should be addressed) 11

2 (h ESCs) TJ MU1 TJ MU2, [4 ], h ESCs : Knock out DM EM ( Gibco) ( Gibco) L2 ( Hyclone) ( Hyclone) 2 ( Sigma) (basic2fibroblast growt h factor, bf GF) ( Sig2 ma) ( Hyclone) EB : h ESCs bf GF : DM EM/ F12 ( Hyclone) B27 ( Gibco) 500ng/ ml noggin ( R &D) bf GF L2 : DM EM/ F12 300ng/ ml noggin ITS ( Sigma) (fibronectin, Fn) ( Gibco) B27 bf GF L2 : DM EM/ F12 ITS ( Sigma) N2 ( Gibco) 80 % ( R &D) (R &D) B27 L2 :, ( R &D) 112 Nestin ( ) 2 2 (anti2microt ubule 2 associated protein 2, MA P22, ) (anti2glial fibrillary acidic protein, GFA P, ) ( Chemicon ) ; DA PI ( Sigma) FITC ( ) ; and R T2PCR ( Fermentas ) h ESCs Trizol h ESCs,, h ESCs, 37 5 %CO2, 627d, A B A EB, EB 6210d, 14d, B EB, h ESCs, 12214d, 223d Nestin, 3b A Nestin,, %, ( nestin MA P2 GFA P ) 4, FITC 37 1h, DA PI, 3, 10, nestin = / DA PI 213 R T2PCR RNA cdna, Fer2 mentas R T,, GA PD H 214, 2 %, , 2000 r / min 5 min, 80 %, 0125 % Triton2X100, ( nestin ) 4, FITC 30min, PBS, Nestin ( %) 3 31 Nestin ( %) gx s, SPSS 1110 t 11 h ESCs, h ESCs,,,,,, 1 h ESCs EB, 6d,,,,, 2 21 (1), B, 48h Nestin, 3a, 6913 % 3143 %, B Nestin % 6119 %,,

3 ( P < 0101) (2),, 14d, MA P2, 4 ;, 10d, GFA P, 5,,,, (bone 31 R T2PCR morp hogenetic protein, BMP) Noggin R T2PCR, h ESCs BMP, OCT4 nestin, 6a BMP, [9 ], MA P2 GFA P, 6b 41, 3, Nestin % (A ) 7615 % (B ) noggin ng/ ml,, ITSFn,, noggin ITSFn, noggin ( 1/ 2 ),, (79111 % 6119 %), EB (6913 % 3143 %), ( P < 0101), R T2PCR ( h ESCs 426 ),,, EB h ESCs,, 2, [5 ], EB [1 ], F GF Wnt,, [6 ], ;,,, EB, h ESCs, [2 ], EB,, EB,, h ESCs EB EB, Smukler [6 ] 10 / l, EB, ( ), h ESCs EB,, 4h,, > 90 % Nestin, 3d, EB,, 7d,,,,,,, 48h ( default mechanism) ( 30d), h ESCs EB, [ 7,8 ], EB,, camp,, [3 ],, 1 Zhang SC, Wernig M, Duncan ID, et al1 In vitro differ2

4 entiation of transplantable neural precursors f rom hu2 man embryonic stem cells 1 Nat Biotechnol, 2001, 19 : Zeng XM, Cai JL, Chen J, et al1dopaminergic neurons differentiation of human embryonic stem cells 1 Stem cells, 2004, 22 : Shin S, Mitalipova M, Noggle S, et al1long term pro2 liferation of human embryonic stem cell2derived neuro2 epithelial cells using defined adherent culture condi2 tions1 Stem Cells, 2006, 24 : 1252l38 4 Chen H, Qian K, Zhang SM, et al1 The derivation of two additional human embryonic stem cell lines f rom day 3 embryos with low morphological scores1 Human Repro, 2005, 20 (8) : Chunyu Cai and Laura Grabel1Directing the differentia2 tion of embryonic stem cells to neural stem cells1dev Dyna, 2007, 236 : Smukler SR, Runciman SB, Xu S, et al1 Embryonic stem cells assume a primitive neural stem cell fate in the absence of extrinsic influences1 Cell Biol, 2006, 172 : Schulz TC, Noggle SA, Palmarini GM, et al1differentiation of human embryonic stem cells to do2 paminergic neurons in serum2f ree suspension cul2 ture1 Stem Cells, 2004, 22 : Vallier L, Reynolds D, Pedersen RA1 Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway1dev Biol, 2004, 275 : Munoz2Sanjuan I, Brivanlou A H1 Neural induction, the default model and embryonic stem cells1 Nat Rev Neu2 rosci, 2002, 3 : , hescs ( 100) 2,, ( 100) 3 Nestin a : B 48h nestin ( 100) ; b : nestin ( 100) 4 MAP2, B MA P2 ( 100) 5 GFA P, B GFA P ( 100) 6 RT2PCR OCT4 nestin MA P2 GFA P, GA PD H EXPLANATION OF FIGURES Fig11 Using contrast phase microscope, hescs are like nest ( 100) 1 Fig12 Using contrast phase microscope, when EB formed, the cells are larger and transparent1 Fig13 Expression of nestin detected by immunofluorescence1a : Nestin positive cells of group B 48h ( 100) ; b : Nes2 tin flurescence stain of NSCs ( 100) 1 Fig14 MA P2 positive neurons of group B detected by im2 munofluorescence1 ( 100) 1 Fig15 GFA P positive astrocytes of group B detected by im2 munofluorescence1 ( 100) 1 Fig16 Expression of OCT4, nestin, MA P2 and GFA P de2 tected by RT2PCR1 The expression levels of each gene are normalized to that of GA PD H1

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