Journal of Ex perimental Hem atology 2005 ; 13 (1) :54-58 MSC MSC

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1 54 Journal of Ex perimental Hem atology 2005 ; 13 (1) :54-58 (Article ID) : (2005) ,,,,,, ,, (mesenchymal stem cell, MSC), MSC ; MSC ;10m, : MSC, MSC,mMSC ; RS (rapidlymsc self2renewing cells) ; 99 %RS G 0 ΠG 1,mMSC G 0 ΠG 1 90 % (CD34 CD45 CD3 CD19 CD33 HLA2DR CD38 ),CD90 CD105 C166 CD29 CD44 CD49e CD54 CD13,RS mmsc,cd117 KDR mmsc 4-5 : MSC, RS, ; RS ; mmsc R39214 A Comparative Study on Various Subpopulations in Mesenchymal Stem Cells of Adult Bone Marro w ZHOU Zheng, J IA N G Er2L ie, W A N G Mei, L IU Qing2Guo, ZHA I Wen2Jing, HUA N G Yong, W A N G He2Hua, HA N M ing2zhe Hospital of Hematology, Instit ute of Hematology, Chinese Academy of Medical Sciences, Tianjing , China Abstract To explore the difference of biological characteristics between two subpopulations of adult bone marrow mesenchymal stem cells (MSC), this study was designed to observe the morphological feature and immunophenotype of the adult MSC in the ex vivo culture, the monouclear cells isolated from normal adult bone marrow were cultured in DMEM with 10 % fetal bovine serum. Cell morphology, immunophenotype and cell cycle of two different subgroups were investigated. Cells from 80 % confluence were passed through a 10m filter, then the fillered cells were cultured in the semisolid methylcellulose medium. The results showed that (1) two different subpopulations were observed in the ex vivo culture. The fibro2like cell was called mature MSC (mmsc) and the smaller round cell was defined rapidly as MSC self2 renewing cells (RS cells) ; (2) the average proportion of cells in G 0 ΠG 1 of RS cells was approximately 99 %, but that of mmscs was 90 % ; ( 3) both of the two populations were negative on the lineage2committed antigen ( such as CD34, CD45,CD3,CD19, CD33, HLA2DR, CD38), while positive on the expression of CD90, CD105, C166, CD29, CD44, CD49e,CD54,CD13. However, the expression of these antigens on RS cells was weaker than that on mmsc, but CD117 and KDR were higher expressed when compared with the mmsc ; ( 4 ) after 4 to 5 week semisolid culture, no hematopoietic progenitor cell colonies were observed. It is concluded that adult MSCs are heterogeneous in that distinct morphological populations exist. The RS cells appear to be the more primitive with greater potential for self2renewal and multilineage differentiation. Key words mesenchymal stem cells ; RS cell ; mmsc J Ex p Hem atol 2005 ; 13 (1) :54-58 :, MSC (mesenchymal stem cells, MSC) MSC, MSC, Pittenger :,. : (022) E2mail : com. cn MSC, ;

2 55, MSC 2, 1, 6 9 MSC, 0. 1 % BSA PBS 2 RS (rapid self2renewal cell),,, CD342F ITC, AC1332PE, CD1172PE, 3 KDR (, ) ; : CD32F ITC, CD42 F ITC, CD82PE, CD22F ITC, CD222PE, CD52PE,, CD72FITC, CD192PE, CD202FITC, CD102PE ;, MSC : CD142FITC, CD332FITC, CD152PE ; : CD542PE, CD802 FITC, CD132PE, CD862PE, CD562PE, CD1062PE, CD312PE, CD832FITC ; : CD49e2PE, CD11a2PE,CD292PE ; : CD402FITC, 6, CD442FITC ; : CD902F ITC, 8 ml,20 UΠml ( CD1052FITC, CD1662PE ; : ) CD382PE, HLA2DR2FITC mouse Ig G1 F ITC mouse Ig G1 PE, MSC, Cellquest DMEM ( Gibco BRL ) 2,,Ficoll ( , MSC ) Πcm 2 80 %, ( ), 10 % ( FCS, Gibco BRL ) DMEM mmsc RS 37 5 % CO 2 95 % MethoCult GF H4434 ( StemCell ), 4-5, 72, % - 90 %, 0125 % ( Sigma ), gx + s, t,, SPSS10. 0 mmsc RS Colter 5 mmsc m,rs 7 m, (,mmsc) 10m, < 10m RS (RS ), > 10 m mmsc, 2, 1-5,,, mmsc RS,70 % 4 24 mmsc RS (1),,PBS 2,, 5-7, 10 mgπml RNase 10 l 10 mgπml,, ( PI),4 30 ( FACS2 (2) Calibur) 2,ModFIT

3 56 J Ex p Hem atol 2005 ; 13 (1) mmscs( P < 0. 01) KDR mmscs( P < 0. 05) 4 Figure 1. Phase2contrast photomicrograph of mmscs and RS cells at day 3 after the cells were curtured ( 300 ). Arrows indicate one large mmscs ( thick arrow) and one small RS cells ( thin arrow) Figure 2. Phase2contrast photomicrograph of mmscs and RS cells at day 6 after the cells were curtured ( 60 ) Figure 3. Histogram of cell cycle of RS cells( A) and mmscs ( B) mmsc, G 0 ΠG 1 90 %, RS, 99 %, (3), 2 (4 ),2, ( ) 2,CD90 CD105 C166 CD29 CD44 CD49e CD54 CD13,RS mmscs Figure 4. FACS analysis of RS cells and mmscs for size and ( P < 0. 01),CD117 KDR granularity

4 57 Table. Surface epitopes and mean fluorescence intensity for mmscs and RS cells ( gx SD) Number Positive rate ( %) MFI of cases mmsc RS cell mmsc RS cell CD CD CD CD # CD49e # CD CD CD CD CD KDR # 3 P < 0. 01, compared with mmsc. P < 0. 01, # P < 0. 05, compared with mmsc - 5, mmsc RS,, CD34 CD45 CD38 CD133 HLA2DR 1970,Friedenstein 4 CD14 CD15 CD33 CD3 CD4 CD8 CD19 CD20 (CFU2F),CD22 CD2 CD31,, CD90 CD105 CD166 CD29 CD44 CD49e CD54 CD13, RS mmsc, RS mmsc ;RS, mmsc, RS (MSC) mmsc, RS mmsc, MSC RS CD117 KDR, mmscs CD117 KDR CD34 MSC,, RS Pittenger 1, Colter 5 MSC : RS, Colter 22 ( FL K21) ( TR K) 5, (3 cellsπcm 2 ) Annexin II, RS CD117 CD4 CD104, mmscs STRO21 CD10, MSC, CD147,CD90 RS,, CD34 CD45 CD117 KDR, RS, CD34 CD133 RS mmsc ( cellsπcm 2 ),MSC 2, 6, RS,,mMSC,RS MSC

5 58 J Ex p Hem atol 2005 ; 13 (1), - 147, RS 99 % G 0 ΠG 1, mmsc 90 % G 0 ΠG 1,, RS mmscs, vation. J Cell Biochem, 1997 ; 64 : ,4-5, RS,,MSC, RS 403, MSC, 2001 ; 98 : of adult human mesenchymal stem cells. Science, 1999 ; 284 : Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self2re2 newal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreser2 3 Colter DC, Class R, Di Girolamo CM, et al. Rapid expansion of re2 cycling stem cells in cultures of plastic2adherent cells from human bone marrow. Proc Natl Acad Sci USA, 2000 ; 97 : Friedenstein AJ, Chailakhjan RK, Lalykina KS, et al. The devel2 opment of fibroblast colonies in monolayer cultures of guinea2pig bone marrow and spleen cells. Cell Tissue Kinet, 1970 ; 3 : Colter DC, Sekiya I, Prockop DJ, et al. Identification of a subpop2 ulation of rapidly self2renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci USA, 6,,..,2002 ;24 : Pittenger MF, Meckay AM, Beck SC, et al. Multilineage potential

Growth Kinetics of Human Mesenchymal Stem Cells from Bone Marrow and Umbilical Cord Blood

Brief Communication Acta Haematol 2004;112:230 233 DOI: 10.1159/000081281 Received: December 16, 2002 Accepted after revision: May 12, 2004 Growth Kinetics of Human Mesenchymal Stem Cells from Bone Marrow