DR - FITC CD29 - PE CD34 - PE CD42 - FITC CD44 - FITC CD45 - PercP CD59 - PE CD80 - FITC CD86 - FITC CD105 - PE CD166 - PE(Pharmingin ) ;

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1 Chinese Journal of Pathophysiology 2004,20 (6) : [ ] (2004) * 1,2, 1, 1, 2, 2, 3, 3, 1, 1 ( 1, ; 2, ; 3, ) [ ] :, / ( IUGT) : L - DMEM (MSC) ; ; /,- / ; MSC, MSC ; F344 MSC, 6 MSC ;/ (NOD/ SCID) MSC,30 d : 3 ml MSC, CD29 CD44 CD59 CD105 CD166,CD34 CD45 CD80 CD86 CD42a HLA - DR, ; % 3128 % ;, MSC,NOD/ SCID : MSC IUGT [ ] ; ; ;, [ ] R363 [] A,(mesenchymal stem cells, MSCs),, [1-3 ], 20 90, DMEM/ L - DMEM),, ( FBS), / ( in utero gene transfer/ therapy, I2 UGT),, IUGT, IUGT [4 ] MSC, IUGT 1 5 : , ,; / (NOD/ SCID) :4,, [ ] [ ] * [ ] (No. 2001CB509904) ; (2001A ) Tel : ; E - mail edu. cn 6-8, ;F344, 2 / ( H -,: (10 7 U/ L) (10 7 g/ L) ( Gibco - BRL ) ;: HLA DR - FITC CD29 - PE CD34 - PE CD42- FITC CD44 - FITC CD45 - PercP CD59 - PE CD80 - FITC CD86 - FITC CD105 - PE CD166 - PE(Pharmingin ) ; ( IBMX) (Sigma ) ;293 ATCC,; (Ad - GFP) ; O ( ) 3 MSC 3-20 ml, Ficoll (), L - DMEM, / cm 2,37,5 %CO 2, ;3 d

2 1004,,5 d 1,,30 d, 80 % - 90 %,,,1 3 4 MSC P5 P10,, 2,,4 30 min,,1 % 100 % ;MSC,28-35 d ; 5 P / cm 2 6, 80 % MSC, ( : 1 mol/ L,5 mg/ L,015 mmol/ L ,60mol/ L ), CD29 ( : 10-7 mol/ L, 10 mmol/ L-,50 mg/ L ),3 d,,14 d 3,10 %, O, 6 MSC p5 24,80 %,, ( mutiplicity of infection, MOI) 1 000Ad - GFP 2h,2 % FBS L - DMEM 3 ml,37,5 %CO 2,5 d,,30 d,,48 h, GFP 7 MSC P5,, cells/ 10 L 1 d F344, 10 L (4 ),6, /, MSC, CD105 - PE/ CD166 - FITC MSC,, MSC 5, 8 P5, d,, cells/ 10 L NOD/ SCID, 10 L, 1 MSC 10 %FBS L - DMEM, MSC,, ; P5, 10 d ;, 15,, 1,3 ml 2 MSC P5 P10 CD44 CD59 CD90 CD105 CD166, CD34 CD45 CD14 CD80 CD86, 2 MSC, MSC,, O,,, 3-1,,,, MSC Ad - GFP,,5 d, 4-1A,B ; 30 d GFP, % 3128 %, MSC CD105 - PE/ CD166 - FITC 6 NOD/ SCID MSC,30 30 d,;, (2 ), 1

3 1005 IUGT,, IUGT,, IUGT, 1997,Larson [5 ] IUGT,,IUGT Fig 1 The P15 human fetal cord blood mesenchymal stem cells, ( 40). IUGT, 1 P15 MSC IUGT,HSC Fig 2 FACS analysis of the surface antigen on the human fetal MSC. 2 MSC Fig 3-1 Adipogenic induction of MSC for 14 d (oil red O staining, 100). 3-1 MSC 14 d Fig 3-2 Osteogenic induction of MSC for 21 d (calcium deposition staining, 100) d

4 1006 Fig 4-1 MSC expressed the GFP ( 100). A : infected at the 2th day ; B : infected at the 30th day. 4-1 MSC GFP Fig 4-2 FACS analysed the MSC (infected at the 30th day). 4-2 GFP MSC Fig 5 Human fetal cord blood MSC homing in different organs after injected into the newborn rats (direct immunofluorescence staining). Red arrow : positive for rat anti - human CD105 - PE, Green arrow : positive for rat anti - human CD166 - FITC. 5-1a, 5-1b : rat lung ; 5-2a, 5-2b : rat heart ; 5-3a, 5-3b : rat blood ; 5-4a, 5-4b : rat liver ; 5-5a, 5-5b : rat spleen. 5 MSC

5 1007,, UGT HSC IUGT, MSC,IUGT, IUGT, MSC ,, IUGT,, MSCErices [6 ] MSC (7 3) / , MSC 24 %,100 %, 3 ml MSC, potential of adult human mesenchymal stem cells[j ]. Science, 1999, 284 (5411) : MSC Campagnoli [7 ] [2 ],,,. [J ]., MSC,, 2003,19 (2) : MSC [3 ] Van Damme A, Vanden Driessche T, Collen D, et al. Bone, marrow stromal cells as targets for gene therapy[j ]. Curr Gene 1,,,2 Ther, 2002, 2 (2) : , , [4 ] Esmail D, Zanjani, French Anderson W. Prospects for in utero IUGT human gene therapy[j ]. Science, 1999, 285 (5436) :2084 -,15,,,,,,, brosis phenotype in mice by gene therapy in utero[j ]. Lancet, 1997,349 (9052) : [6 ] Erices A, Conget P, Minguell J. Mesenchymal progenitor cells in human umbilical cord blood[j ]. Br J Haematol, 2000,109 Ad - GFP MSC,48 h (1) : GFP,,5 d [7 ] Campagnoli C, Roberts IAG, Kumar S, et al. Identification of,30 d mesenchymal stem/ progenitor cells in human first - trimester,gfp % 3128 %, [8 ] MSC, fetal blood, liver, and bone marrow [ J ]. 2001,98 (8) : Hematopoiesis, [8 ],,,. EGFP MSC NOD/ SCID,,, MSC IUGT, IUGT [ ] [1 ] Pittenger MF, Mackay AM, Jaiswai SC, et al. Multilineage,MSC [9 ],,,. [2,9,10 ] [J ]., ; 2001,22 (5) : MSC (1 d) [10 ],,,.,, [J ]. MSC I ,22 (5) : [ 5 ] Larson J E, Morrow SL, Happel L, et al. Reversal of cystic fi2 [J ]., 2003, 19 (3) :

6 1008 Biological characterics of human fetal cord blood mesenchymal stem cells ZHU Mei - ling 1,2, NA Xiao - dong 1, LEI Jun - xia 1, LIU Hua 2, HU Yan - fen 2, HUANGLing - hui 3, HUANG Wei - hua 3, ZHANG Xiu - ming 1, LI Shu - nong 1 ( 1 Department of Pathophysiology, Sun Yat - sen Medical College, Zhongshan University, Guangzhou , China ; 2 Shenzhen Baoan Blood Center and Cord Blood Bank, Shenzhen , China ; 3 Shenzhen Baoan Xixiang Hospital, Shenzhen , China) [ ABSTRACT] AIM : To investigate the biological characterics of human second - trimester fetal cord blood mes2 enchymal stem cells (MSC) and its application prospects in utero gene transfer/ therapy ( IUGT). METHODS : Nuclear cells separated from cord blood were cultured in DMEM medium. Surface antigens of the MSC were analyzed by the FAC2 Scan flow cytometry. Adipogenic and osteogenic mediums were used to assess the differentiation ability of the cells. Ade2 novirus vector deliver green fluorescent protein gene (Ad - GFP) was used to transfected the MSC and the expressing of GFP was detected by fluorescent microscope. The MSC were injected into the liver of newborn rat. The immunofluo2 rescence analysis was conducted to determine the presence of double - positive CD105 + / CD166 + cells in different organs of rats. MSC were subcutaneous injected into the human - nonobese diabetes/ severe combined immunodeficiency disease (NOD/ SCID) mice and carcinogenesises of the MSC in vivo were detected by pathological diagnosis. RESUL TS : MSC could be separated from fetal cord blood. These cells were uniformly positive for CD29, CD44, CD59, CD105, CD166 and negative for CD34, CD45, CD80, CD86, HLA - DR. The cells had the abilities to differentiate into adipogenic and osteogenic cells in vitro, expressed the GFP at high levels (56132 % 3128 %). The MSC were located at different or2 gans after injected into the newborn rats and didn t have carcinogenicity in vivo. CONCL USION : Human second - trimester fetal cord blood MSC is an promising target cells in fetal IUGT. [ KEY WORDS] Fetal blood ; Mesenchymal stem cells ; Gene therapy ; Gene transfer, horizontal