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1 大韓獸醫學會誌 (2013) 第 53 卷第 1 號 Korean J Vet Res(2013) 53(1) : 37~42 < 원례보저 > 돼지지방조직및골수유래성체줄기세포의성상분석과다능성에관한연구 이아영 최경임 나진주 소병재 이경우 장기윤 송재영 차상호 * 농림수산검역검사본부 ( 접수 : 2012 년 9 월 14 일, 수정 : 2013 년 1 월 3 일, 게재승인 : 2013 년 1 월 3 일 ) Characterization of multipotent mesenchymal stem cells isolated from adipose tissue and bone marrow in pigs Ah-Young Lee, Gyeong-im Choe, Jin-Ju Nah, ByungJae So, Kyung-Woo Lee, Ki-Yoon Chang, Jae-Young Song, Sang-Ho Cha* Animal, Plant and Fisheries Quarantine and Inspection Agency, Anyang , Korea (Received: September 14, 2012; Revised: January 3, 2013; Accepted: January 3, 2013) Abstract : Mesenchymal stem cells (MSCs) have ability to differentiate into multi-lineage cells, which confer a great promise for regenerative medicine to the cells. The aim of this study was to establish a method for isolation and characterization of adipose tissue-derived MSC (pad-msc) and bone marrow-derived MSC (pbm-msc) in pigs. Isolated cells from all tissues were positive for CD29, CD44, CD90 and CD105, but negative for hematopoietic stem cell associated markers, CD45. In addition, the cells expressed the transcription factors, such as Oct4, Sox2, and Nanog by RT-PCR. pad-msc and pbm-msc at early passage successfully differentiated into chondrocytes, osteocytes and adipocytes. Collectively, pig AD-MSC and BM-MSC with multipotency were optimized in our study. Keywords : mesenchymal stem cells, isolation, pig, adipose tissue, bone marrow 서 성체줄기세포는섬유아세포모양으로부착해서자라고골, 지방, 연골세포로분화하는능력을가진세포이다. 이러한세포의존재는 1970 년골수에서처음보고되었으나 [7], 최근지방을포함한뇌, 비장, 간, 신장, 폐, 근육, 흉선, 췌장등다양한조직에존재한다고알려져있다 [6]. 지방조직은다른조직보다인체내에양이많고채취시통증이거의없으며재충전이가능하다는장점을가지고있다. 또한, 골수에비해지방조직에는 1g 당대략 100,000 여개의성체줄기세포가존재하기때문에 [20] 단기간에대량으로증식하여사용될수있다. 성체줄기세포는이미성장한신체조직에서추출한다는점에서윤리적인논쟁을피할수있으므로실용적인면에서배아줄기세포보다성체줄기세포연구가주를이루고있다. 최근생체조직공학및재생의학을이용한줄기세포치료방법에관한연구들이활발히이루어지고있다. 조직일부를채취하여필요로하는특정조직으로분화시켜이를이 론 식하는연구분야는종교및윤리적인부분에제한을받지않고면역거부반응과같은문제점을해결할수있기때문에조직공학분야에활용도가높다 [4]. 또한, 줄기세포를원하는특정세포로분화시킬수있다면, 손상된세포및조직의기능을근본적으로재생 (regeneration) 가능하다는점에서심근경색, 뇌경색, 퇴행성관절염, 골절및피부등전반적인의학영역에서세포치료제로서이용가능하며줄기세포를각종세포및조직으로분화시켜신약후보물질의독성및효능을빠르게검증할수있기때문에새로운항암제및진단기술에활용할수있다. 하지만, 정확한분화기전및기능조절에관한기초연구와줄기세포의고효율분리, 대량증식, 면역거부반응, 종양형성등극복해야할과제가남아있다. 돼지는생명의학연구에중요한동물모델로서본연구는돼지의지방, 골수에서성체줄기세포를분리및배양법을확립하고골세포, 지방세포, 연골세포로분화시켜조직유래성체줄기세포의성상을비교하고분화능을관찰하는것에목적이있다. *Corresponding author Tel: , Fax: virusmania@korea.kr 37

2 38 이아영 최경임 나진주 소병재 이경우 장기윤 송재영 차상호 재료및방법 세포분리및배양 24~26개월령모돈유래복부피하지방, 3~5일령신생자돈대퇴골로부터골수시료를채취하였다. 100 U/mL penicillin과 100 µg/ml streptomycin이함유된 dulbesco s phosphate buffered saline(dpbs; Gibco, USA) 로수차례세척한지방조직을가위로잘게자른후, 0.1% collagenase type I solution(gibco) 을 37 o C에서 1시간반응시켰다 [14, 22]. 300 g, 5분원심분리후상층액을제거하고 DPBS를넣고다시원심분리하였다. 상층액을제거하고 10% MSC fetal bovine serum(fbs; Gibco), 100 U/mL penicillin, 100 µg/ml streptomycin, glutamine 포함된 DMEM/F12 (Gibco) 배지를넣고골고루섞은후 100 mm dish에분주하여 37 o C, 5% CO 2, 20% O 2 조건에서배양하였다. 대퇴골절반을절단후, 총 30 ml DPBS로골수를채취하였다. Ficoll(GE Healthcare, Sweden) 3 ml당골수액 4mL씩분주하여 400 g, 30분원심분리하고중간의세포층을분리하였다 [2, 16]. DPBS를넣고 300 g, 10분원심분리하여상등액제거후지방조직과동일한조건으로세포를배양하였다. 이시기에는 24시간마다배지를교체하며세포가 70~80% 밀집하면 0.25% trypsin-edta(gibco) 을처리하여계대배양하였다. 세포증식률분석 (cumulative population doubling level analysis, CPDL) 다음과같은공식을이용하여세포증식률을확인하였다. CPDL = ln (Nf/Ni) ln2 Ni 는초기세포수, Nf 는마지막세포수, ln 는자연로그를나타내며 6-well plate 에세포를 ( ) 세배수로배양하고 5 일이지나면세포수를측정한후, 다시세포를 ( ) 배양하여 10 계대까지반복, 계산하였다 [23]. Reverse transcription polymerase chain reaction (RT-PCR) 을이용한초기전사표지인자 Oct4, Sox2, Nanog 발현검사지방조직, 골수유래세포의초기전사표지인자 Oct4, Sox2, Nanog의발현을확인하기위해 RNeasy Mini kit (QIAGEN, Germany) 를이용하여 3계대배양한세포의 total RNA를추출하고, cdna를합성하기위해 GoScript Reverse Transcription System(Promega, USA) 을사용하였다. PCR은 GoTaq DNA Polymerase(Promega) 를이용하였다. 실험에사용한 primer는 Table 1에제시하였다. PCR은 predenaturation 95 o C에서 5분, denaturation 72 o C에서 30초, annealing 30초, extension 72 o C에서 30초, final-extension 72 o C에서 5분, 이과정을 40 cycle 반복하였고 PCR product 를 2% agarose gel에전기영동으로분리한후, 사진촬영하였다. 면역염색법 (immunostaining) 을이용한초기전사표지인자 Oct4, Sox2, Nanog 발현검사 4% paraformaldehyde 로상온에서 15 분세포를고정시켰다. 고정된세포에 0.2% PBS-triton X-100 을상온에서 30 분처리하여 permeabilization 시킨후, 5% BSA 로상온에서 1 시간 blocking 하였다. Primary antibody 는모두 1:100 으로 4 o C 에서 24 시간처리하였고사용한 antibody 종류는다음과같다. Anti-Oct4(Abcam, USA), anti-nanog(cell Signaling, USA), anti-ssea-4(millipore, USA) 를사용하였다. PBS 로세척후 secondary antibody 를 1:400 으로희석하여상온에서 1 시간처리하였다. DAPI(Invitrogen, USA) 를상온에서 10 분처리하고간단히세척후, mounting solution 을처리하고 sealing 하여형광현미경으로촬영하였다. 유세포분석 (flow cytometric analysis) 을통한세포표면특이마커발현검사 0.25% trypsin-edta 를처리하여떼어낸세포를 PBS 로 3 회세척한후, 성체줄기세포표면특이마커의발현정도를 Table 1. Primer sequence of pluripotency markers for reverse transcription-polymerase chain reaction Gene Primer sequence (5-3 ) PCR product size (bp) Annealing Tm ( o C) Oct4 Sox2 Nanog GAPDH F-AGGTGTTCAGCCAAACGACC R-TGATCGTTTGCCCTTCTGGC F-GCCCTGCAGTACAACTCCAT R-GCTGATCATGTCCCGTAGGT F-ATCCAGCTTGTCCCCAAAG R-ATTTCATTCGCTGGTTCTGG F-TCGGAGTGAACGGATTTG R-CCC GGA AGA TGG TGA TGG GAPDH: Glyceraldehydes-3-phosphate dehydrogenase GeneBank accession No NM_ EU DQ AMF

2-phosphate 50 µg/ml Indomethacin 100 µm β-glycerophosphate 100 mm TGF-β1 10 ng/ml Isobutyl methyl xanthine 500 µm Flow cytometer(facscalibur; BD Biosciences, USA) 를이용하여관찰하였다.

3 돼지지방조직및골수유래성체줄기세포의성상분석과다능성에관한연구 39 Table 2. Differentiation induction media Adipogenesis Osteogenesis Chondrogenesis Dexamethasone 100 nm Dexamethasone 10 nm Dexamethasone 100 nm Insulin 500 ng/ml Ascorbic acid 2-phosphate 200 µm Ascorbic acid 2-phosphate 50 µg/ml Indomethacin 100 µm β-glycerophosphate 100 mm TGF-β1 10 ng/ml Isobutyl methyl xanthine 500 µm Flow cytometer(facscalibur; BD Biosciences, USA) 를이용하여관찰하였다. 이들항체는형광물질이결합된 antimouse CD29(BD Biosciences), anti-rat CD44(BD Biosciences), anti-mouse CD45(Fitzgerald, USA), anti-mouse CD90(BD Biosciences), anti-mouse CD105(Abcam) 를사용하였다. 분화유도및염색분화를유도하기위해각각의분화조성에맞춘배지에 21 일간배양하였다 (Table 2). 이때, 3 일주기로분화배지를교체하였다. 결 돼지의지방조직, 골수에서분리한세포는배양일주일내에세포군락 (colony) 을형성하였고 5 일주기로계대배양 과 Fig. 1. (A) Adipose tissue-derived MSC (pad-msc), (B) Bone marrow-derived MSC (pbm-msc). Morphological characteristics of pmscs at passage 1 (p1). The adherent cells exhibited a typical fibroblast-like morphology. 40, Scale bars =100µm. 하며세포모양을관찰하였다. 1 차계대시지방, 골수유래세포는섬유아세포모양으로부착해서자라는것을확인하였고이러한모양은 2, 3 차계대까지지속되었다 (Fig. 1). 성체 Fig. 2. (A) Expression of pluripotent transcriptional marker, Oct4, Sox2 and Nanog in pad-msc and pbm-msc at passage 3 by RT-PCR. GAPDH was used as a housekeeping gene. (B) Specific immuno-reactivity for Oct4, Nanog (nucleus), and SSEA-4 (cytoplasm) was observed in pmscs. DAPI: 4,6-diamidino-2-phenylindole, FITC: fluorescein isothiocyanate, GAPDH: glyceraldehydes-3-phosphate dehydrogenase, RT-PCR: reverse transcription polymerase chain reaction. 600.

40 이아영 최경임 나진주 소병재 이경우 장기윤 송재영 차상호 Fig. 3. Expression of cluster of differentiation (CD) markers in pad-msc and pbm-msc at passage 3. Each sample was counted with 10,000 cells by a flow cytometer.

4 40 이아영 최경임 나진주 소병재 이경우 장기윤 송재영 차상호 Fig. 3. Expression of cluster of differentiation (CD) markers in pad-msc and pbm-msc at passage 3. Each sample was counted with 10,000 cells by a flow cytometer. Two kinds of cells were stained with each specific CD marker (filled histograms) and isotype matched controls (open green line). Expression level of markers represents the mean percentage of all assessed cells stained by the antibodies, respectively. Fig. 4. pad-msc and pbm-msc were seeded at per well, counted and passaged every 5 days. Proliferative capacities of pmscs were expressed as cumulative population doubling level (CPDL) using the formula CPDL = ln(nf/ Ni)ln2, where Ni and Nf are initial and final cell numbers, respectively, and ln is the natural log. 줄기세포는 미분화 상태에서 자가재생을 통해 증식할 수 있 는 능력을 가진 세포로서 이를 위해서는 다양한 특정 유전 자가 역할을 한다 [1, 9, 15, 21]. 그 중, 대표적인 초기전사 인자인 Oct4, Sox2, Nanog의 발현을 확인하기 위해 3계대 세포의 RNA를 추출하여 RT-PCR을 통해 두 가지 세포 모 두 이들 유전자의 발현을 확인하였다(Fig. 2A). 또한, 면역염 색법을 통해 핵 안에 위치한 Oct4, Nanog와 세포질에 위치 한 SSEA-4의 발현을 관찰하였다(Fig. 2B). 표면특이마커의 발현을 확인하기 위해 유세포 분석기를 통해 CD29, CD44, CD90, CD105 그리고 CD45 발현 유무를 관찰하였다. 성체 Fig. 5. Differentiation potential of pmscs. After induction for 21 days, adipogenesis (A and B), chondrogenesis (C and D) and osteogenesis (E and F) were demonstrated by Oil red O, Alcian blue staining and Alizarin red staining, respectively. 200, Scale bars = 100 µm. 줄기세포 마커인 CD29, CD44, CD90, CD105는 발현되었 지만, 조혈줄기세포 마커인 CD45는 발현되지 않았다(Fig. 3). 세포 증식률 확인을 위해 5일 주기로 3계대부터 10계대까 지 배양하여 지방, 골수 유래 성체줄기세포의 증식률을 조사 하였다(Fig. 4). 전체적으로 지방 유래 줄기세포가 골수 유래

5 돼지지방조직및골수유래성체줄기세포의성상분석과다능성에관한연구 41 줄기세포보다증식이빠르다는것을확인할수있었고골수유래줄기세포는 6 계대이후부터증식률이떨어지는반면, 지방유래줄기세포는 10 계대까지증식이지속되는것으로보아골수유래줄기세포보다지방유래줄기세포의증식률이월등히우수하다는것을확인할수있었다 (Fig. 4). 분화는지방, 골수및연골조직으로유도하였다. 분화배지에서 21 일간배양후, 각각의분화염색시약으로분화여부를확인하였다. 분화를유도한지방, 골수유래성체줄기세포를 Oil red O 를이용하여지방세포가붉게염색되는것을관찰하였고 (Figs. 5A and B) 연골조직으로유도한실험은 Alcian blue 로염색하여분화양성을확인하였다 (Figs. 5C and D). 또한, 골조직으로분화한실험은 Alizarin red 염색을통해정상적으로분화하는것을확인하였다 (Figs. 5E and F). 고 줄기세포란자가재생능력과증식력, 광범위한분화능을가지는상대적으로분화되지않은세포이다 [5]. 줄기세포는분화능에따라크게배아줄기세포, 성체줄기세포로나눌수있다. 배아줄기세포는배아포배 (blastocysts) 의 inner cell mass 를추출한세포로배아뿐만아니라모든세포로분화가가능하고성체줄기세포는이보다제한적으로분화될수있으나다양한조직에서추출가능하기때문에실용적인면에서많은연구가이루어지고있다. 초기전사인자로알려진 Oct4, Sox2, Nanog 는배아줄기세포에서강하게발현되는유전자로써자가재생과여러유전자의전사를조절한다. 또한, 줄기세포의중요한특성인다능성에관여한다고알려져있기때문에이들유전자를다분화능표지인자라고도일컫는다 [3, 11]. 본연구에서는추출, 분리, 배양한세포의 Oct4, Sox2, Nanog 발현을모두확인할수있었고지방유래줄기세포는계대 10, 골수유래줄기세포는계대 6 까지증식능을보유한결과를종합해보면, 이는본연구에서분리한세포가다능성과자가재생능을가진줄기세포임과동시에조직간차이를보임을시사하고있다. 성체줄기세포는일반적으로조혈모세포의표식자인 CD45, CD34 는발현하지않고반대로 CD29, CD44, CD90, CD105 는발현한다 [8]. 이러한표식자발현은종간의차이, 조직유래세포간의차이가있고또한세포배양시계대가후반대로진행될수록그발현이현저히감소한다 [18]. 최근다양하고새로운표면특이마커에의해줄기세포의특성분석을규명하는연구가활발히진행되고있다. 성체줄기세포는대표적으로지방세포, 연골세포, 골세포로분화가가능하며분화를유도하기위한분화배지의조성은일반적으로알려져있다. 분화시약의조성은다양한조직에서유래한세포에따라차이가있을수있다. 지방세포로의분화는 lipid vacuole 을 Oil red O 염색법을이용하여확인하거나 adipocyte protein 2(aP2), peroxisome proliferator activated receptor gamma(pparγ), lipoprotein lipase(lpl) 찰 등관련유전자의발현을측정하여확인할수있다 [17]. 골세포의분화는 calcium 축적과골기질을 Alizarin red 염색을통해확인하며 osteocalcin 과 osteonectin 의발현을관찰할수있다 [19]. 연골세포는 Alcian blue 염색을통해 glycosaminoglycan 의축적됨을알수있고 Sox9, Aggrecan, Collagen type II 의유전자발현유무로연골세포로분화됨을확인할수있다 [13]. 이밖에성체줄기세포는신경세포, 췌장내분비세포, 간세포, 혈관내피세포및심근세포로의분화또한보고되었다 [10]. 또한, 분리된성체줄기세포는배지조건에따라세포의증식능및분화능에차이를보인다고알려져있다 [12]. 본연구에서는돼지의지방조직, 골수에서분리한세포가초기전사인자, 표면특이마커의발현, 초기계대한세포의특정배양조건하에서지방세포, 연골세포, 골세포로분화함을확인함으로써다능성의특징을지닌세포임을확인하였다. 성체줄기세포를세포치료제, 재생의학등다양한생명의학연구에적용될경우그목적에따라원하는세포로정확히분화시켜야하기때문에이에대한배양및분화조건에대한추가적인연구가필요할것이다. 이번연구를바탕으로미래의세포기반재생의학적치료에도움이될것으로사료된다. 감사의글 본연구는농림수산검역검사본부수의과학기술개발연구사업 (P-AD ) 에의해수행되었습니다. 참고문헌 1. Babaie Y, Herwig R, Greber B, Brink TC, Wruck W, Groth D, Lehrach H, Burdon T, Adjaye J. Analysis of OCT4-dependent transcriptional networks regulating selfrenewal and pluripotency in human embryonic stem cells. Stem Cells 2007, 25, Bosch P, Pratt SL, Stice SL. Isolation, characterization, gene modification, and nuclear reprogramming of porcine mesenchymal stem cells. Biol Reprod 2006, 74, Carlin R, Davis D, Weiss M, Schultz B, Troyer D. Expression of early transcription factors Oct-4, Sox-2 and Nanog by porcine umbilical cord (PUC) matrix cells. Reprod Biol Endocrinol 2006, 4, Choi MJ, Byun JH, Kang EJ, Rho GJ, Kim UK, Kim JR, Park BW. Isolation of porcine multipotential skin-derived precursor cells and its multilineage differentiation. J Korean Assoc Oral Maxillofac Surg 2008, 34, Czyz J, Wiese C, Rolletschek A, Blyszczuk P, Cross M, Wobus AM. Potential of embryonic and adult stem cells in vitro. Biol Chem 2003, 384, da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 2006, 119 (Pt 11), Friedenstein AJ, Chailakhjan RK, Lalykina KS. The development of fibroblast colonies in monolayer cultures of

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( )Jkoa138.hwp J Korean Orthop Assoc 2006; 41: 911-919 인간제대혈유래간엽줄기세포군에서얻어진단일클론세포군간의세포특성차이 성지혜 백금옥 성기선 하철원 Difference in Cell Characteristics among the Monoclonal Cell Populations Obtained from the Human Umbilical Cord