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1 대한골다공증학회지 : 제 3 권제 1 호 pp 1~6, 2005 종설 성인중간엽줄기세포의계대배양에따른특성변화 연세대학교의과대학정형외과학교실 1, 연세대학교 BK21 의과학과 2 김윤희 2 선현진 2 한승환 1 이진우 1,2 Characterization of Human Mesenchymal Stem Cells during Serial Subculture Yun Hee Kim 2, Hyun Jin Sun 2, Seung Whan Han 1, Jin Woo Lee 1,2 Department of Orthopaedic Surgery 1, Brain Korea 21 Project for Medical Science 2, Yonsei University College of Medicine, Seoul, Korea 1) 성체줄기세포 야할과제로여전히남아있다. 줄기세포는사람의배아를이용해만들수있는배아줄기세포 (embryonic stem cell) 와제대혈및성체의골수와혈액등에서추출한성체줄기세포 (adult stem cell) 로나누어볼수있다. 배아줄기세포는장차인체를이루는모든세포와조직으로분화할수있는세포로알려져있고, 골수에다량존재하는성체줄기세포 (adult mesenchymal stem cell) 는크게조혈모줄기세포 (hematopoietic stem cell) 와중간엽줄기세포 (mesenchymal stem cell) 로나눌수있는데, 조혈모줄기세포는적혈구, 면역세포혈소판등의혈구세포로, 중간엽줄기세포는골아세포, 연골세포, 지방세포, 근육세포, 신경세포등으로분화할수있다. 성체줄기세포는면역학적문제및추출의용이등의장점을가지고있으며, 자가이식을통한조직재생의이용가치를가지고있으며현재다양한임상시험평가가진행중이다 (Table 1). 그러나조직재생이나치료에적용할만큼의충분한양의중간엽줄기세포를확보해야하는점, 시험관배양중의증식및분화효율등은앞으로도해결해 책임저자 : 이진우, 연세대학교의과대학정형외과학교실 Tel: 02) , Fax: 02) ljwos@yumc.yonsei.ac.kr * 위연구는 21세기프론티어연구개발사업단인세포응용연구사업단의연구비지원 (SC3020) 에의해수행되었음. 중간엽줄기세포의특성 1. 세포의형태 중간엽줄기세포는골수, 제대혈, 말초혈액, 간및윤활액등에존재하며, 골수의단핵세포중 0.001~0.1% 비율로존재한다. 중간엽줄기세포는시험관에서배양되면서빠른속도로자가증식을하고, 크기가좀더큰세포일수록 50 계대배양에서증식의감소를보이는것으로보고되었으며, 분화되지않은세포는 spindleshape의섬유아세포와유사한형태를지니고있다 (Fig. 1). 2. 표면항원말초혈액과달리중간엽줄기세포는여러종류의비특이적표면항원 (surface marker) 을발현하며, 아직도대변할수있는단일표면항원이밝혀지지않은상태이다 (Table 2). 일반적으로중간엽줄기세포는혈구세포 (hematopoietic cell) 의특이적항원인 CD34, CD45, CD14 또는내피세포 (endothelial cell) 의 CD34, CD31, VWF 등을발현하지않는다. 골수에서추출한줄기세포는시험관배양시 CD34의표면 1

대한골다공증학회지 : 제 3 권제 1 호 Table 1. Case studies and phase I clinical trials in a variety of conditions that employed mesenchymal stem cells (Kassem M. Colning Stem Cells.

2 대한골다공증학회지 : 제 3 권제 1 호 Table 1. Case studies and phase I clinical trials in a variety of conditions that employed mesenchymal stem cells (Kassem M. Colning Stem Cells 논문에서발췌 ) Induction Number of Patients Large bone defect 3 Autologuse MSC mixed with hydroxyapatite scaffold Osteogenesis imperfecta Cells used and route of decolvery Resuls Reference 3 Mixed allogenic bone marrow transplantation Myocardial infarct 60 Autologous mononuclear bone marrow cells (mixed hematopoietic and stromal cells) Peripheral ischemia in lower extremities Metchromatic leukodystrophy (MLD) and Hurler syndrome Severe idiopathic aplastic anemia Bone marrow transplantation of Breast cancer patients Chronic nonhealed skin wound 22 Autologous mononuclear bone marrow cells (mixed hematopoietic and stromal cells) 4/6 Allogenic MSC transplntation of HLAidentical sibling Improved bone repair New bone formation and some clinical improvement Improved global left ventricle function in Improved clinical parameters related to vascular supply Well tolerated and some improvement in neurological functions 1 Allogenic MSC transplantation Improved stromal functions 28 Allogenic MSC together with peripheral blood progenitor cells infusion 3 Autologous bone marrow (mixed hematopoietic and stromal cells) implantation Well tolerated and improved haematological recovery Dermal rebuilding and closure of nonhealed wounds (Quarto, et al., 2001) (Horwitz, et al., 1999) (Wollert, et al., 2004) (TateishiYuyam a, et al., 2002) (Koc, et al., 2002) (Fouillard, et al., 2003) (Koc, et al., 2000) (Badiavas, et al., 2003) Fig. 1. Differentiation paradigm and related signals and phase contrast photomicrograph showing a confluent layer of cultured MSCs (magnification 200). 항원은여전히존재하지않으나, 간에서추출한줄기세포의경우계대배양중 CD34+/ CD50의표면항원을가지며, 이러한세포가중간엽줄기세포와 유사한분화기능을보였다. 일반적으로, 중간엽줄기세포는 CD44, CD29, CD90 ( 부착관련인자 ) 과 SH2, SH3, SH4, inter 2

3 성인중간엽줄기세포의계대배양에따른특성변화 Table 2. 골수에서분리한중간엽줄기세포의표면항원 CD locus CD locus Cytokine and growth factor receptors Adhesion molecules/integrins IL1R (CD121) IL3R (CD123) ICAM1 (CD54) Endoglin (CD105) IL4R (CD124) IL6R (CD126) ICAM2 (CD102) Lselection (CD62L) IL7R (CD127) GCSFR (CD114) ICAM3 (CD50) Pselection (CD62P) TNFα1R (CD120a) TNFα2R (CD120b) VCAM (CD106) Eselection (CD62E) PDGFR (CD140a) Interferon R (CD119) NCAM (CD44) Extracellular matrix proteins Others β1 integrin (CD29) VLAα1 (CD49a) SH2 (CD105) STRO1 β3 integrin (CD61) VLAα5 (CD49e) SH3 (CD73) Thy1 (CD90) Fibronectin Hyaleronan SH4 (CD73) leukin1r, TNFαR 등을발현하며, 이러한발견은중간엽줄기세포를고효율적으로분리하는데에이용되어지고있으나, CD44, CD29 및 CD90은중간엽줄기세포에만국한되지않고여러종류의세포에서도발현하는것으로특이적항원이라고할수없다. 최근에혈구세포에는발현하지않고골수의중간엽줄기세포에서관찰된 STRO1 의경우, STRO1에양성인세포들에서 CFUF (fibroblastcolony forming units) 가 10~20배증가하였고, CD106은골수의중간엽줄기세포에서는발현하나골아세포의분화가진행되면발현하지않는것으로보고되었다. 기술한바와같이여러세포군에서중간엽줄기세포만을특이적으로분리하기위한특이적표면항원의개발연구가요구되며, 이를보완하고자여러표면항원을복합적으로사용하여세포를분리하고있으나, 여전히순수분리에대한한계를극복하지못하는단점이있다. 계대배양에의한특성변화 중간엽줄기세포의증식및분화능은연령, 성별및계대배양정도등의다양한인자에의해영향을받는것으로알려지고있으며, 특히성별보다는연령에의해좌우되는경향을보이고있으나, 이에대한연구결과도상반된결과를보이고있다. 동물의경우연령에의해성장동역학과골아세포로의화능 3 등의차이를보이지만, 사람에있어서는연령에의해나타나는결과가상반되어보고되고있다. 그중에서도세포의크기가작은세포가증식이빠르고, 골아세포, 지방세포및연골세포로의분화능력도높다고보고되었다. 중간엽줄기세포를고농도로분주할경우계대배양이여러번반복되면서세포의형태는동일하나, 세포자체의다분화능력이떨어짐이보고되었으며, 특정세포로의분화능에대해서만감소한다는결과도보고되고있다. 계대배양뿐만아니라, 여러요인에의해나타날수있는중간엽줄기세포의제한된증식력과분화능을향상시키고자 telomerase 형질도입, 세포외기질및성장인자의처리등다양한방법이시도되고있으나, 확실한기전은보고되지않았다. 뿐만아니라, 골수에서분리한중간엽줄기세포의경우, 다양한종류의세포군으로형성되어있기때문에, 줄기세포혹은하나의단일줄기세포만을분리하는방법이필요하며, 이러한방법은분화의효율을최대화할수있을것이다. 프로테오믹스를이용한계대배양에따른단백질변화 본연구에의해골수에서채취한중간엽줄기세포로부터계대배양과정중의전체단백질의변화양상을알아보고자하였다. 세포를 7 계대까지 1: 3의비율로배양하였으며, 배양시간이지날수록크

대한골다공증학회지 : 제 3 권제 1 호 8 day 8 day Primary culture (1day) passage 1 passage 2 passage 7 (70day) MSC characterization Osteogenic differentiation 2dimensional electrophoresis Fig. 2. 배양모식도및계대배양에따른세포의형태변화및세포의비대화 Fig.

4 대한골다공증학회지 : 제 3 권제 1 호 8 day 8 day Primary culture (1day) passage 1 passage 2 passage 7 (70day) MSC characterization Osteogenic differentiation 2dimensional electrophoresis Fig. 2. 배양모식도및계대배양에따른세포의형태변화및세포의비대화 Fig. 3. 계대배양에따른중간엽줄기세포의증식률기가크고넓적하며과립이많은세포의형태로변화하였다 (Fig. 2). 기존의연구결과에서계대배양에의한세포의형태비대화가보고되어왔으며, 이는계대배양이중간엽줄기세포의세포크기, 모양등의특성변화에영향을미치는것으로인식되고있다. 세포의모양변화뿐만아니라, 세포의양성표면항원 (CD 105 & CD29) 의변화관찰에서 7 계대에서급격히감소하였고, 음성표면항원 (CD34 & CD14) 는 1.5% 이하로변화가없었다. 기본배양액에서각계대별중간엽줄기세포를 6일간배양한후, DNA량을측정한결과, 계대배양에의해중간엽줄기세포의증식이통계적으로유의하게감소하였다 (Fig. 3). 뿐만아니라, 6 계대배양에의해 ALP (alkaline phosphatase) 활성, Alizarin red 염색및칼슘침착량이점차감소하였고, 제 5 및 7 계대에통계적으로감소를보였다 (Fig. 4). 계대배양에따라프로테오믹스를이용한단백질변화는세포주기, 증식, 사멸등과관련된단백질, annexin A1, chain B, Cathepsin D, Tcomplex protein (TCP) 1, α subunit, chaperonin containing TCP1 (CCT), subunit 3, 세포구조와관련된 capping protein (actin filament), gelsolin like 등의변화단백질을발굴하였다 (Table 3). 특히, TCP1은계대배양에의해점차감소하였으며, 특이적항체에의한단백질발현감소가확인되었다. 그러나분화조절인자와관련된단백질변화는관찰되지않았다. TCP1은 cytosolic chaperonin containing Tcomplex polypeptide 1 CCT) 의 8개 subunit 중의하나이며, 이러한단백질은 actin이나 tubulin 등의 cytosolic protein folding에관여한다고알려져있으며, CCT발현이세포증식과세포주기등에밀접하게관련된다고알려져있다. 또한 CCT subunit 중일부가분화과정에서감소한다고보고되기도하였다. 일반적으로중간엽줄기세포의골아세포로의분화과정동안세포의증식력이감소한다고알려져있다. 따라서, 이러한가설을증명하기위해초기배양세포를골아세포로분화시킨후, TCP1의발현을측정한결과감소하는것이확인되었다. 이는계대배양에따른중간엽줄기세포의증식및골아세포로의분화능의감소와관련된중요한인자로서 TCP1의역할가능성을추측할수있다. 종합해보면, 골수에서채취한중간엽줄기세포는계대배양에의해세포의비대화및과립의증가와함께증식률이점차감소한다. 또한골아세포의분화능 (Alkaline phosphatase activity & 칼슘침착 ) 이 4

5 성인중간엽줄기세포의계대배양에따른특성변화 P1 P3 P5 P7 OS Control P1 P3 P5 P7 OS Control Fig. 4. 계대배양에따른중간엽줄기세포의골아세포능분석 Table 3. 프로테오믹스를이용한계대배양중변화단백질 No. Protein identity Accession number Upregulated 1 Annexin A1 NP_ Pyruvate kinase (3, muscle) S Enolase 1: phosphopyruvate hydratase NP_ Heat shock 27kD protein 1 NP_ Annexin A2 AAH Protein disulfide isomeraserelated protein NP_ Proteasome 26S ATPase subunit 5 NP_ Chain B, Cathepsin D 1LYA Downregulated 1 Tcomplex protein 1, alpha subunit P Proteasome 26S ATPase subunit 2 NP_ Capping protein (actin filament), gelsolinlike NP_ Chaperonin containing TCP1, subunit 3 NP_ 현저히감소되며, 계대배양에의한특성변화와관련된단백질을발굴하고자프로테오믹스에서여러단백질이관찰되었다. 그러나이러한단백질과중간엽줄기세포에미치는영향및기능에대한연구가보고된바가없으며, 앞으로더많은연구가진행되어야할것이다. 중간엽줄기세포의계대배양중의특성변화및분화능감소는임상적용을위 한배양기준을설정하는데에매우중요한기초자료로요구되어질것이다. 참고문헌 1. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential 5

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388 The Korean Journal of Hepatology : Vol. 6. No COMMENT 1. (dysplastic nodule) (adenomatous hyperplasia, AH), (macroregenerative nodule, MR

388 The Korean Journal of Hepatology : Vol. 6. No COMMENT 1. (dysplastic nodule) (adenomatous hyperplasia, AH), (macroregenerative nodule, MR 6 3 2000 ; 387-392 (3) Dysplastic Nodule Young Nyun Park, M.D., Chanil Park, M.D. Department of Pathology, Yonsei University College of Medicine BRIEF HISTORY 56. AST/ALT 72/73 IU/L, total bilirubin 0.7