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1 f zu{ } 18 í } Çf Chinese Journal of Tissue Engineering Research September 3, 2014 Vol.18, No.37 ƒ» Œ Âµ«Œq Î ŒÉä j 1 2 i 1 3 À 1 ( 1 }{ œ Þ Þ }{ œ Þ ) s 1 ñxœ Âµ«ŒqÉy» ¹ q Â ˆnƒzÉ 0.1% Œñ ä ˆŒ ä µé ü Îo ä i u ä Â ÂƒŒñ y Œq Ä ± qñx Œ 2 v È çq Œ ñx Œ Ñ g r ~Ž Œ ± gœ Âµ«Œ zî qu{ n o q ºÀ Œ Œ Œ Œ Âµ«Œ ŒÉä ÎÉä ŸÅ g gè q Œ Â«Œ ŒÉä «Ù ì «ÙÈr( ) Œ Âµ«Œº Ž Œ d ƒ g z º ýéä Œ g º q nð Œ Âµ«ŒqÉy»v Ä rq ƒ Éy» Œ Âµ«Œ º mg g ²Ÿ ¹ ºý Î ŒÉäq Œ É È çq Œ nœñ ä Éy Œ Âµ«Œ ²Ÿƒ» Œy ŒŸÅ g CCK-8 Œ Í ŒmÀ } 4 x Œ Âµ«Œý Œ Î Éäý²Ÿ O w w º Œ ÎÉä Œ Œ Âµ«Œ À «mà Œz ~xž} 10 x Œ Œy ùîÿ² CD29 CD90 ö CD44 CD34 CD45 Ÿ²±ƒ Œ ýq Œ Âµ«Œ O Â Î ýq Œ Âµ«Œw w Â x x Í ØÉy»Ç Œ Âµ«Œ q ŒmÀz ÎŸ²Âµ«ŒqŸÅ ñxöº ý Î Œ} ýéäq Œ gî z k q y Œ ì 1985 «}{ x nc u Þ Ôd š Þ doi: /j.issn [ f É ú:r394.2 h u:a ú: (2014) ÈÍ j i À. ƒ» Œ Âµ«Œq Î ŒÉä[J].f zu { (37): Osteogenic and adipogenic differentiation of rabbit adipose-derived mesenchymal stem cells in vitro Lee Soo-min 1, Wu Zi-zheng 2, Wang Zhe 1, Li Zhi 3, Zhang Jian 1 ( 1 Department of Orthopedics, Zhongshan Hospital of Fudan University, Shanghai , China; 2 Department of Orthopedics, Baoshan Branch, Shanghai First People s Hospital, Shanghai , China; 3 Department of Orthopedics, Qingpu Branch, Zhongshan Hospital of Fudan University, Shanghai , China) Abstract BACKGROUND: Adipose-derived mesenchymal stem cells have the ability to self-renew and have pluripotent potential under specific conditions in vitro, which have broad application prospects in clinical practice. However, isolation and culture of adipose-derived mesenchymal stem cells still appear to have many difficulties and shortcomings. OBJECTIVE: To isolate and culture rabbit adipose-derived mesenchymal stem cells in vitro in order to study their morphology, cell surface markers and biological properties as well as to investigate the osteogenic and adipogenic potentials of adipose-derived mesenchymal stem cells in vitro. METHODS: Primary adipose-derived mesenchymal stem cells were isolated from the subcutaneous adipose tissue of posterior cervical region from New Zealand white rabbits and digested by 0.1% collagenase I. The cells were passaged and amplified by the trypsin digestion. The passage 4 adipose-derived mesenchymal stem cells were induced to differentiate after exposure to adipogenic or osteogenic medium. The oil red O staining, alkaline phosphatase and alizarin red staining were used to detect the results. The cell viability was Lee Soo-min, Studying for doctorate, Department of Orthopedics, Zhongshan Hospital of Fudan University, Shanghai , China Corresponding author: Wu Zi-zheng, Master, Associate chief physician, Department of Orthopedics, Baoshan Branch, Shanghai First People s Hospital, Shanghai , China Accepted: P.O. Box 10002, Shenyang

2 detected by the cell counting kit 8 method to drawn the growth curve. Cell surface markers were examined using flow cytometry. RESULTS AND CONCLUSION: The adipose-derived mesenchymal stem cells isolated from the subcutaneous adipose tissue of rabbits exhibited a fusiform adherent growth in a vortex pattern, and had a strong capability of proliferation that could be passaged stably to the 10 th generation. Flow cytometry results showed that the cells highly expressed CD29, CD90, CD44, but lowly expressed CD45 and CD34. After adipogenic induction, the adipose-derived mesenchymal stem cells were positive for oil red O staining; after osteogenic induction, the cells were both positive for alkaline phosphatase and alizarin red staining. These findings suggest that the adipose-derived mesenchymal stem cells were successfully isolated and cultured from the subcutaneous adipose tissue of rabbits, and these cells are pluripotent with the potential to differentiate into adipocytes and osteoblasts, which are expected to be ideal seed cells for bone tissue engineering. Subject headings: subcutaneous fat; mesenchymal stem cells; rabbits; adipogenesis Funding: the grant from Shanghai Municipal Commission of Health and Family Planning, No Lee SM, Wu ZZ, Wang Z, Li Z, Zhang J. Osteogenic and adipogenic differentiation of rabbit adipose-derived mesenchymal stem cells in vitro. Zhongguo Zuzhi Gongcheng Yanjiu. 2014;18(37): Introduction Î z qº gë p}ñ ³ Î q o ºfy Œ Î z u{ ÃsÎÎÂµ«Œ(bone marrowderived mesenchymal stem cells BMSCs)qÉy Ø [1] pîîâµ«œqî z üƒq xö Íu{ oà q² u{öj pîîq Âµ«Œ r Â [2-4] Œ Ä ù vîî Œ³ q ç öo Œ Âµ«Œ(adipose-derived mesenchymal stem cells ADSCs) vœ féy qº Ž ö ýéä Œq y ƒ Œ [5-6] ƒ g q z ùx ý Î Œ ±Î Œ Œ Œ Œöx Œ} y Œ Éä ²ùxÉ y ž~m Ãr ùœ y Œ ¼m}²zf ö n [7-8] npœ Œ µ ƒ oñ xö Œ «z }g [9] ² g zy Œqu{ Íƒ Éy» Œ Âµ«Œ º mg g ²Ÿ gº np zî qu{ k 1 Materials and methods Œ ƒ» Í Âö Íp Ž Â f çã Íu{f Í ÍÙg Ÿ º q 2ù 2 Ú ƒ«(2.0±0.2) kg ÄÄ Â «Ž Â f çãù g Íf Ï ƒ DMEM fž 0.25%Œ Å Ä (ˆ Gibco) Œñ ž βm w ¾ fœ ˆ± IMBX pn qu O(ˆ Sigma) («d) w w Ïr CCK-8 Ïr(êsÆ ) Anti-rabbit CD90-FITC( Abcam) Anti-human CD34-FITC CD29-FITC CD44-PE Mouse IgG1 K Isotype control FITC Rat IgG2b K Isotype Control PE Flow Cytometry staining buffer(ebioscience) Anti-human CD45-FITC( ˆ BioLegend) Í ÍnŒ q Š ÅŒ 10 ml{ Ùg ˆÙgp ú çó È µ féø4 cm q Œ ( 1) ²³±zŽÿ 1%Å Ä qpbsf Œ Âµ«ŒqÉy» ú ÇŒ Éy sù qž~ } ( 1C) n PBSÀ 3³ nsyó Ó g( 1D) Ø 3 ƒz g0.1%q Œñ 37 q ä40 min r/miny 10 min Ç Œ ö nÿ ƒzé g10% fž 1%Å Ä qƒ DMEM» Œ n200r }ˆ² ôã äq vf yp»mf ˆp37 ƒzé g5%co 2 q»~²ÿñx»»48 hýë ôã «Œ iý e 1 ŒmÀ üž90% n0.25%œ q (ÿ0.02%edta) ä Œx1 3²Ÿ~x» ˆ À Œ ö ü q Œm À À ŒŸÅ gq }4x Œ ä Ø PBS m Œ z 15 mly ~f p1 500 r/miny 5 min ØPBS Œ ¼ p1 500 r/miny 5 min PBS Œ Œ Ž L ml/ ~( f Œ)É ŒPBS Ž~A B C D E F ~¼ ØFlow Cytometry staining buffer 100 μl ~A B C DÉÌ Ø5 μlqfitc anti-human CD34 FITC anti-human CD45 FITC anti-human CD29 FITC ISSN CN /R CODEN: ZLKHAH 6009

3 anti-rabbit CD90 ~C¼ Ø5 μlqpe anti-human CD44 ~E Ø5 μlqrat IgG2b K Isotype Control PE ~F Ø5 μlqfitc mouse IgG1 ˆp4 qà~ 30 miný ~Ø1 ml PBS p1 500 r/min y 5 min ² Œp500 μlqpbsí y ŒŸ q nflowjo±zé CCK-8 Œ ÉÌ }3x Œ }7x Œ }10x Œ L 1 Œ yp24 24 héìø 100 μl CCK-8 4 hý vü ¼ Ç ü 100 μl± 96 ¼ n Š o y 450 nm q Í ŒmÀ Œ Âµ«Œq Œ }4x Œ Ø Œ» (ÿƒzé g10% fž 1 μmol/l 10 mg/lœ 0.5 mmol/l IBMX 0.2 mmol/l ˆ± [6] )²Ÿ Œ» Œ 14 d ý²ÿ O m Œ ù ˆ d Ø Œ Ï» ˆ À e Œ ä Œ Âµ«Œq Î }4x Œ Ø Î» (ÿƒzé g10% fž 0.1 μmol/l 50 mg/l ž 10 mmol/l βm w ¾ [6] )²Ÿ Î» Î dýéì ²Ÿw w m Œ ù ˆ d Ø Î Ï» ˆ À e Œ ä g Œ Âµ«Œ ŸÅ g Î ŒÉäŒ 2 Results 2.1 Åp h ñx Œ 46 h «24 h¼ «Œ u ö 710 dý Œù²80%90%ê ü Œ À ù Œ«{ Ê g ²Ÿ~x ~xý Œ ³ Ø Œ ² ² ~x Œ² ä }4x Œ ±Î À g Ê Œv rq Œ ( 2) 2.2 Åp ½ ŒmÀ S ~xýq3 d¼ Œ Âµ«Œ pmà² 47 dg mà 7 d² ú ŒmÀ Ã üx iâq À ( 3) xƒ»q Œ Âµ«Œ º z q Œ 2.3 n Œy öj Œ Âµ«ŒŸÅCD29(99.54%) CD44(99.62%)ö CD90(81.12%) Â Ÿ² ƒÿ²cd34(2.58%)ö CD45(4.98%)( 4) } 3 x } 7 x } 10 x Â(d) 3 Œ Âµ«ŒqmÀ Figure 3 The growth curves of rabbit adipose-derived mesenchymal stem cells at passages 3, 7, Åp n Î 2 ý Œ öm ä Ê Œƒzn ( 5A) Î 4 ý uä ( 5B) ²Ÿ¾¾ Œ¼w w q ŒŒ«f j Ø È g g ( 5C) 2.5 Åp n Œ Âµ«Œ Œ ý Œƒz² Œ¼rm Œ ( 5D) Œ 2 ý O x ŒŒ«¼ q Œ È ( 5E) 3 Discussion ÍqŒ Âµ«Œvq Œ fé y u{ q Œ qâµ«œ ³i p Œ ˆ Œ qâµ«œ d q Œ qñx Œ ±Î [10-11] Ñu{ö j q Œ ± È ç çq Œ r ± np ç p ç d Œ ž~ } r ü q Œ o ¼q ž Œ} «[12] ±È ç Ž È çq Œ ŽvZuk} [5] Ë v Œ qœ féy Í Œ Œx q ÉyŒ Âµ«Œq ²Ÿ ºp ôã q Œ ùx³²ønh 4 Cl Œ q fqnh 4 Cl Œ Âµ«Œ È³²ö y ôã Lee} [13] ³² w ôãž~ ö À xö Ã q Œ ùrôønh 4 Clq Î n [14] u{öj Œ v q n ŽŒ Âµ«ŒýŒ ŒÉä }ˆ ² Œ ôãœ v ñx Œ«â x±î q Œ x ÍÉyñxŒ Âµ«Œ k ˆn}ˆ² ²y P.O. Box 10002, Shenyang

4 rð nq Œ ä Œ «³ Ìn Œ±ºw Œº «ØÂ qg ³²» q ä Œ [15] u {k n ~xq Œ24 hë ô «q Œ }4x Œ~xý24 h¼ Œ ¹«Í q Œ d}4x Œ²Ÿ Œy ŒŸÅ g Âµ«Œg gâ i± Î(CD % CD %xöCD %) Ÿ Œ~xŽ}4xý Œ ±Î go²ÿz»ö ä Œ ²ŒÌn ŒŸÅ ñqg É² Œ g wjé² Œy É² wjé²ñk g qê Ã ƒ owj ² Œ üýˆpw q² º Â ô w ý Í äq Œ [16-17] ŒyÉ² qñk Ì n É² Œ ü ƒœ q Ìn «q u{ ncd105 gÿå ŒÉ²Ç Î ± xöý ÎÉ äœ qœ Âµ«Œ [18] rð² ¹v Œ Âµ«Œqg ŸÅ u{ ³² ŒyÉ² ÉyqŒ Âµ«Œ É²qŒ Âµ«ŒÂq ÎÉäŒ i [19] dnp n ŒÉ² Œ ± ÂÍoº n ³²üy Éy qœ Âµ«Œ Œ Œ Œ«¼ ÍŒ Œ qœ u{f³ ˆnÿƒzÉ g10% fž qƒ DMEM Œ» ²Ÿ» Â Œ» m º«mÀý ²nŒ ä ~x [20] u{ Œ Âµ«Œº ýé ä Œ [6,21-23] ƒ g q z ùxý Î Œ ±Î Œ Œ Œ Œöx Œ} y Œ Éä Ÿ g ÎÎÂµ«Œ Ð²qÎ zqy Œ Í n Œ ¹» fø ƒ ž βm w ¾ L Œ q ü Œ Âµ«Œý Î ŒÉ ä qœ Âµ«Œ ŸjÇ Î Œ qg d nw w xö ¹ xºº Î Œq g Gronthos} [24] u{öj Œ Âµ«Œ ÎÎÂµ«ŒiÂ Å r qÿå ñ ÉäqŒ Âµ«Œ ÎÎÂµ«Œ ŒŸ²CD105 CD29 CD90 CD73 CD166 CD44 CD49e CD49b Ÿ²CD14 CD34 CD45 CD133 CD31 CD38 HLA-DR ù CD106 ey ŒŸÅŸ² Àrö ÎÎÂµ«Œ CD106Â Œ Âµ«Œ Â ºf CD29 ž ~ö ²zf n CD44 m Œ «m ²zf n Œ Âµ«Œ Ÿ² ƒ Ÿ²HLA-DR xœ Âµ«Œº ùnp ƒz q Œ [13,25] ³ qu{f n yÿå Š ü¹ q ¹ Âµ«Œ Í nœñ äö«}² Œ Âµ«Œ Œ y CD29 CD34 CD44 CD45 CD90 º x Œ Âµ«ŒÎŸ²Âµ«ŒqŸÅ CD29 CD44 CD90 ƒÿ²³ž ŒqŸÅ CD34 q ŒŸÅ ñcd45 Zuk} [5] ³qt Œ Âµ«ŒŸÅ Ž Ù g}º wùgqœ Âµ«ŒŸÅ k Ž [26-29] x Í qœ Âµ«Œ ±Î yçìp³ž Œqù ˆ p Éäg qå ý Œ Åž jfq Œ ŸÅ ñ q ¹ Œº Å q n rðu{ y ¹ Âµ«Œqg ŸÅ ñ g ³² Œ Ÿ ö ýéä Œ ²Ÿ ü¹ Íƒ Éy»q Œ Âµ«Œ«m À ÎŸ²Âµ«ŒqŸÅ xöº ýéä q Œ }ü Â Œ oé~è qâµ«œ i Â [30] u{ƒ ØÉy» Œ Âµ«Œ Œ d vo ÎÉä q z º q Î ²q}ü y Œq [31-32] v gî z k q y Œ p È çq Œ r d ¼ Œ µ ù~êƒ Éy» Œ Âµ«Œ ƒ ³ Œ z ~x Ž}10x º Éä Î Œ} ýéäq Œ zîk qy Œ «h Â cì ì c cì ì c ò À ì dì n c Ç d ÌrÀ{ À À í q d k «n t 2009 wethical issues in animal experimentationx üì íd Åp Ã n æ g g Œ dåp r Œ c o y Ð c à ƒ d Åp ½ È ß c c c¾ c¾ c c Òc c ¾c Œ î t ëˆ z Œ ß ì é d u k À í À d ISSN CN /R CODEN: ZLKHAH 6011

5 A B C D 1 È çq Œ q öéy» Figure 1 Isolating and culturing the subcutaneous adipose tissue of posterior cervical region from New Zealand white rabbits A B C 2 Œ Âµ«Œ ( 100) Figure 2 Morphological characteristics of rabbit adipose-derived mesenchymal stem cells ( 100) f A gñx Œ» 24 h ý ù Œ«mÀ öà ù «fq Œ B gñx Œ» 7 d ý ŒxÀ gg ù ö Œ C g} 4 x Œ Œ ± À Ê g«mà A B C D E 4 Œy Œ Âµ«ŒŸÅ Figure 4 Surface markers of rabbit adipose-derived mesenchymal stem cells determined by flow cytometry f A x CD29 Ÿ²Â ig 99.54% B x CD44 Ÿ²Â ig 99.62% C x CD90 Ÿ²Â ig 81.12% D x CD34 Ÿ ²Â ig 2.58% E x CD45 Ÿ²Â ig 4.98% A B C D E 5 Œ Âµ«Œý Î Œ Éä( 100) Figure 5 Osteogenic and adipogenic differentiation of rabbit adipose-derived mesenchymal stem cells ( 100) f A g Î 14 d Œ Ê Œƒzn B g Î ý uä C g Î ý w w ŒŒ«f j Ø È g D g Œ 14 d Œƒz Œ¼rmŒ E g Œ ý O ŒŒ ¼ù q Œ È 6012 P.O. Box 10002, Shenyang

6 4 õ h References [1] Gregory CA, Prockop DJ, Spees JL.Non-hematopoietic bone marrow stem cells: molecular control of expansion and differentiation.exp Cell Res. 2005;306(2): [2] Dragoo JL, Choi JY, Lieberman JR,et al.bone induction by BMP-2 transduced stem cells derived from human fat.j Orthop Res. 2003;21(4): [3] Nakao N, Nakayama T, Yahata T,et al. Adipose tissue-derived mesenchymal stem cells facilitate hematopoiesis in vitro and in vivo: advantages over bone marrow-derived mesenchymal [4] stem cells.am J Pathol. 2010;177(2): iiö, ÄÖ,Ê,}. ÎÎ Œ Œ Œ É äœ q ±[J].jxmgç ², 2013,13(11): [5] Zuk PA, Zhu M, Mizuno H,et al. Multilineage cells from human adipose tissue: implications for cell-based therapies.tissue Eng. 2001;7(2): [6] Zuk PA, Zhu M, Ashjian P,et al.human adipose tissue is a source of multipotent stem cells.mol Biol Cell. 2002; 13(12): [7] Shigemura N, Okumura M, Mizuno S,et al. Autologous transplantation of adipose tissue-derived stromal cells ameliorates pulmonary emphysema.am J Transplant. 2006; 6(11): [8] Morizono K, De Ugarte DA, Zhu M,et al. Multilineage cells from adipose tissue as gene delivery vehicles.hum Gene Ther. 2003;14(1): [9],ëdl,,}.Œ Œ ÎÎ Œmg gq ±[J].fé Í y, 2009,26(3):396. [10] Âs, Ä, j,}. Ñµƒ Œ Œmg Ÿg ö ÎÉäŒ q [J].éfy (ç f), 2013, 42(5): [11] Toyoda M, Matsubara Y, Lin K,et al. Characterization and comparison of adipose tissue-derived cells from human subcutaneous and omental adipose tissues.cell Biochem Funct. 2009;27(7): [12] j, ³,Ú,}. Ù Œ ŒqÉy»ö Î[J].f zu{, 2013,17(23): [13] Lee RH, Kim B, Choi I,et al.characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.cell Physiol Biochem. 2004; 14(4-6): [14] n. ÙŒ ŒqÉy»ö ü º Œ g q [D].³i:³iç Ã, [15] É,. ä Œ Î Íf Œ»q [J]. f zu{ g, 2011,15(40): [16] Rada T, Gomes ME, Reis RL.A novel method for the isolation of subpopulations of rat adipose stem cells with different proliferation and osteogenic differentiation potentials.j Tissue Eng Regen Med. 2011;5(8): [17] Rada T, Reis RL, Gomes ME. Novel method for the isolation of adipose stem cells (ASCs).J Tissue Eng Regen Med. 2009; 3(2): [18],,Ÿ,}. ŒyÉ² ätœ Œƒ Î q Íu{[J]. z y, 2010,6(6): [19] Liu Y, Zhao Y, Zhang X,et al. Flow cytometric cell sorting and in vitro pre-osteoinduction are not requirements for in vivo bone formation by human adipose-derived stromal cells.plos One. 2013;8(2):e [20] Sterodimas A, de Faria J, Nicaretta B,et al.tissue engineering with adipose-derived stem cells (ADSCs): current and future applications.j Plast Reconstr Aesthet Surg. 2010;63(11): [21] De Ugarte DA, Morizono K, Elbarbary A,et al. Comparison of multi-lineage cells from human adipose tissue and bone marrow.cells Tissues Organs. 2003;174(3): [22] Miyazaki M, Zuk PA, Zou J,et al.comparison of human mesenchymal stem cells derived from adipose tissue and bone marrow for ex vivo gene therapy in rat spinal fusion model.spine (Phila Pa 1976). 2008;33(8): [23] Han C, Zhang L, Song L,et al.human adipose-derived mesenchymal stem cells: a better cell source for nervous system regeneration.chin Med J (Engl). 2014;127(2): [24] Gronthos S, Zannettino AC, Hay SJ,et al. Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow.j Cell Sci. 2003;116(Pt 9): [25] Zhu Y, Liu T, Song K,et al. Adipose-derived stem cell: a better stem cell than BMSC.Cell Biochem Funct. 2008;26(6): [26] É. ü Ú ü µƒ ÙŒ Âµ«Œmg g q u{[d]. : ç Ã, [27],Í,. Ù Œ Âµ«ŒqÉy» ¹ ö ƒ [J].f zu{ g, 2011,15(14): [28] Ä, ¾, Ë,}. ÙŒ ŒqÉy»ö¹ q Ë u{[j].f Í, 2011,15(8): [29] Vieira NM, Brandalise V, Zucconi E, et al. Isolation, characterization, and differentiation potential of canine adipose-derived stem cells.cell Transplant. 2010;19(3): [30] Dominici M, Le Blanc K, Mueller I,et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4): [31] Ï,.y Œ Î zfqu{² [J].fé¹ yç, 2012,10(4): [32] Zanetti AS, Sabliov C, Gimble JM,et al. Human adipose-derived stem cells and three-dimensional scaffold constructs: a review of the biomaterials and models currently used for bone regeneration.j Biomed Mater Res B Appl Biomater. 2013;101(1): ISSN CN /R CODEN: ZLKHAH 6013

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