저산소상태에서배양한제대혈유래 간엽줄기세포의특성에대한연구 지도교수 이기종 이논문을석사학위논문으로제출함 2014 년 7 월 1 일 연세대학교보건환경대학원 의생명과학전공 양정윤

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1 저산소상태에서배양한제대혈유래 간엽줄기세포의특성에대한연구 연세대학교보건환경대학원 의생명과학전공 양정윤

2 저산소상태에서배양한제대혈유래 간엽줄기세포의특성에대한연구 지도교수 이기종 이논문을석사학위논문으로제출함 2014 년 7 월 1 일 연세대학교보건환경대학원 의생명과학전공 양정윤

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4 감사의글

5 차례 그림차례 i 표차례 ii 약기호표 iii 국문요약 iv 제1장서론 1 제2장재료및방법 제대혈채취 줄기세포분리및배양 줄기세포표면항원분석 줄기세포의골분화유도및확인 줄기세포노화확인 Real-time PCR 통계분석 7 제3장결과 Hypoxia에서의줄기세포증식율증가 Hypoxia에서의줄기세포표면항원 Hypoxia에서줄기세포의골세포분화능증가 Hypoxia에서줄기세포노화지연 Hypoxia에서의줄기세포능조절관련유전자발현량증가 21 제4장고찰 23 제5장결론 29 참고문헌 30 Abstract 37

6 그림차례 Figure 1. Photomicrographs of UCB-MSCs cultured under hypoxia and normoxia 11 Figure 2. Ex vivo expansion of UCB-MSCs cultured under hypoxia and normoxia 12 Figure 3. Characterization of surface markers in cultured UCB-MSCs 14 Figure 4. Photomicrographs of osteogenic differentiated UCB-MSCs cultured under hypoxia and normoxia 17 Figure 5. Osteogenic differentiation of UCB-MSCs cultured under hypoxia and normoxia 18 Figure 6. Cellular senescence of UCB-MSCs cultured under hypoxia and normoxia 20 Figure 7. Expression of OCT4, NANOG and SOX2 in UCB-MSC cultured under normoxia and hypoxia 22 - i -

7 표차례 Table 1. Obstetric characteristics of mothers and neonates 8 Table 2. Immunophenotypic characterization of UCB-MSCs cultured under hypoxia and normoxia 15 - ii -

8 약기호표 MSC : mesenchymal stem cell UCB : umbilical cord blood OCT4 : octamer-binding transcription factor 4 Rex I : reduced expression 1 CPDA : citrate phosphate dextrose adenine MNC : mononuclear cell MEM : minimum essential medium EDTA : ethylenediaminetetraacetic acid CPD : cumulative population doubling CD : cluster of differentiation FITC : fluorescein isothiocyanate HLA : human leukocyte antigen PE : phycoerythrin ALP : alkaline phosphatase PCR : polymerase chain reaction RNA : ribonucleic acid ISCT : International Society for Cellular Therapy HIF : hypoxia inducible factor - iii -

9 국문요약 간엽줄기세포는자가재생능력과다양한세포로의분화능력이있어서세포치료및조직공학분야에활용되고있다. 그러나간엽줄기세포는분리시에획득할수있는세포수가한정되어있어서, 임상적용을위해서는체외배양을통해충분한세포수를확보하는과정이요구된다. 이에본연구에서는인체내골수환경이저산소분압상태인것에착안하여, hypoxia와 normoxia에서배양한제대혈유래간엽줄기세포의증식과노화, 분화, 줄기세포능조절유전자발현변화등을비교분석하여 hypoxia 배양이간엽줄기세포의배양법으로타당한지를검증하고자하였다. 5 units의제대혈에서분리한간엽줄기세포를 hypoxia (3% 산소상태 ) 와 normoxia ( 일반적인대기압산소상태, 약 20% 산소상태 ) 에서각각계대배양한결과, hypoxia에서배양된간엽줄기세포는 normoxia에서배양된간엽줄기세포보다증식률이높았고, 노화가지연되었으며, 골세포로의분화능도더우수한것으로관찰되었다. 또한, 줄기세포능조절유전자인 OCT4, NANOG 및 SOX2의 mrna 발현이 hypoxia에서유의하게발현이증가하였고 hypoxia에의한간엽줄기세포의세포표면항원발현변화는관찰되지않았다. 본연구에서제대혈유래간엽줄기세포를 hypoxia 상태로배양을하면간엽줄기세포의증식능이향상되었고, 노화율이감소하였으며, 분화능이유지됨을확인하였다. 이러한결과는 hypoxia 배양조건이간엽줄기세포의줄기세포능유지에적합한배양법임을시사하는것으로간엽줄기세포를이용한세포치료및조직공학분야에 hypoxia 배양방법을적용한다면줄기세포능유지에따른치료효과의개선과배양기간단축에따른비용을크게줄일수있을것으로기대한다. - iv -

10 중심단어 : 제대혈, 간엽줄기세포, hypoxia, 노화, 증식능, 분화능, 줄기세포능 - v -

11 제 1 장서론 간엽줄기세포 (mesenchymal stem cells, MSCs) 는자가재생 (self-renewal) 과다분화능 (multi-lineage potential) 을가지고있어조직공학에서매우유용한자원으로활용되고있다 (1,2). 이러한간엽줄기세포를얻을수있는성인골수나지방조직은임상의주요한자원이기는하나, 기증자의나이에따른영향으로인해노령인환자의조직에서분리한간엽줄기세포일수록치료에효과적이지못할수있으며, 채취과정이외과적인침습과정을거쳐야하는한계를가지 고있다 (3,4). 따라서대안이될수있는태반, 탯줄, 양막을포함한태아조 직들을새로운자원으로연구되고있으나, 이조직들에서세포를분리하는과정은쉽지않았다 (5-7). 이에비해제대혈 (umbilical cord blood, UCB) 은태아분만후폐기하는탯줄에서채취하므로윤리적인규제가적고, 채취방법이비침습적이기에비교적쉽게얻을수있다는장점을가지고있다. 이전까지간엽줄기세포에대한연구대부분이배아나골수, 지방과같은조직에서분리한줄기세포위주로진행되어온이유는이러한조직의간엽줄기세포분리율이높기때문이며, 제대혈은여러장점이있음에도불구하고다른조직에비해줄기세포분리율이낮아효율성면에서여전히논란의여지가남아있다. 그러나최근많은연구자들이다양한배양방법을이용하여 65% 까지제대혈에서의줄기세포분리율을높이고있어 (60-62), 제대혈의줄기세포에대한연구가증가할것으로예상할수있다. 또한, 기존 hypoxia 배양에대한골수와지방조직에서분리한간엽줄기세포의증식과노화, 분화, 자가재생능력을비교한연구결과가집중되어있는반면 (13-21), 제대혈에서분리한간엽줄기세포의 hypoxia 배양에관한연구결과는많지않았다. 제대혈은 1988년최초로판코니빈혈환자에게제대혈이식이이루어지면서, 골수와말초조혈모 - 1 -

12 세포를대체할조혈모세포공급원으로주목받기시작하였고, 제대혈내에존재하는비조혈세포성분중간엽성섬유세포양세포등의존재가보고되면서 (6 3) 제대혈간엽줄기세포에대한연구가다른조직에비해늦게시작되었지만, 제대혈에서분리한간엽줄기세포가성인간엽줄기세포보다더미성숙하며, 생체외에서쉽게증식할수도있다는 (10) 특징으로인해골수유래간엽줄기세포보다제대혈유래간엽줄기세포의임상적용을더용이하게한다 (11,12). 간엽줄기세포를치료목적으로임상적용을하는연구자들에게는세포의증식과노화는매우중요한관심사이다. 간엽줄기세포의임상적용을위해충분한세포수가필요하며이에순수간엽줄기세포들을체외에서계대배양하는과정을거치게된다. 그러나이체외증식배양과정동안세포는노화하기때문에 (2 0,21), 이러한세포의노화를배양방법개선으로지연할수있도록하는것이세포치료제분야의연구자들이해결해야할과제가되었다. 조직과세포의배양을했던연구자들은체외에서세포를배양하기위해사용하는영양성분, 성장인자, ph 완충액등의사용에있어서정교한주의가필요하며 (53), 세포배양배지의산소농도는세포성장을위해적절한대기 (ambient air) 조건이요구된다고하였다 (54). 그리고, 조직의산소농도를직접측정할수있게된이후부터배아조직에서의산소농도가매우낮음을증명하였으며 (55), 줄기세포들이있는조직들또한산소농도가낮아줄기세포들을유지시킬수있는환경 (niche) 을가지고있는것을확인하였다 (56-58). 인체내특히골수 niche에서는여러미세환경신호전달체계로간엽줄기세포의자가재생과분화능력에영향을주며, niche에서의산소분압또한간엽줄기세포기능에있어중요한역할을한다 (13-16). 이는최근 2% 산소분압상태 (hypoxia) 에서골수에서분리한줄기세포특유의유전자발현 (OCT-4, Rex-1) 과세포외기질단백질 (fibronectin) 의합성, 그리고인체지방조직에서분리한간엽줄기세포의분화능력도증가하는것으로확인하였다 (17-19). 또한, 골수유래간엽줄기 - 2 -

13 세포를 2% hypoxia에서배양하였을때, 세포의증식능력은증가하고, 세포내에서의 glucose와 lactate 대사과정은더효율적으로작용하며세포의노화속도가감소했다고하였고 (22), 태반에서분리한간엽줄기세포의증식과분화능도 hypoxia에서증가한다는최근연구결과도보고되었다 (59). 이에본저자는다른성체조직보다얻기쉽고미성숙하여유용성이높은제대혈에서분리한간엽줄기세포를임상에더효율적으로적용하기위하여배양방법을개선하는방안으로 hypoxia를적용하고자하였고, hypoxia에서배양한제대혈유래간엽줄기세포와통상적으로배양하는 normoxia 조건의줄기세포와의비교를통해더효율적인배양방법인지를확인해보고자하였으며, 간엽줄기세포의특징으로는세포의증식과노화, 표면항원형, 분화능력그리고줄기세포능조절관련유전자의발현량을비교하였다. 이를통해 hypoxia에노출된제대혈유래간엽줄기세포가일반 normoxia 방법으로배양한세포보다세포의증식과분화능, 줄기세포능조절관련유전자의발현량이뛰어나면서세포의노화는지연되고, 줄기세포의고유한표면항원은유지하여효율적인배양방법임을제시하고자하였다

14 제 2 장재료및방법 2.1 제대혈채취메디포스트 ( 주 ) 의학연구윤리심의위원회 (Institutional Review Board, IRB) 의심의를통과한제대혈기증동의서에자발적으로동의및서명을한산모에게서 2005년이전출산시분만을담당한산부인과의사가태아만출직후태반이나오기전에제대를결찰하고항응고제 (Citrate Phosphate Dextrose Adinime, CPDA-1) 24.5ml을포함한채취백 ( 녹십자, 대한민국 ) 에채취절차에따라제대혈을채취하였다. 채취한제대혈 5 units은실온을유지하면서 40시간이내에운반, 세포분리를하였으며, 세포생존도 95% 이상임을확인하였다. 2.2 줄기세포분리및배양채취한제대혈은 Ficoll-Hypaque 용액 (d=1.077g/cm 3, Sigma, St. Louis, USA) 을이용하여원심분리하고, 단핵세포층 (mononuclear cells, MNCs) 을분리하였다. 분리한단핵세포층을 10% 우태혈청 (Fetal Bovine Serum, Gibco) 을포함한 α -MEM (α-minimum essential medium, Gibco) 에세포수 /cm 2 의밀도로배양하였다. 배양조건은 5% 이산화탄소배양기에 37, 습도 95% 이상을유지하고, 배양액은 1주일에 2번씩교환하였다. 분리한간엽줄기세포들은배양용기표면의 80% 이하로증식배양하여, 계대배양을실시하였다. 계대배양시 trypsin-edta(gibco) 로세포를수집하고, 세포수 /cm 2 로분주하였으며, 배양조건은일차배양과동일한조건에서시행하였다. P6(Passage 6, 6번째계대배양 ) 에서 hypoxia(3% O 2 ) 와 normoxia( 약 20% O 2 ) 로배양조건을분리하여각각의산소조건에서계대배양을하였다. 이배양단계에서세포의증식은 trypan blue dye exclusion 방법으로분석하였다. 산소분압이다른배양조건에 - 4 -

15 따라세포의증식에영향을주는지를확인하기위해각세포들의계대배양기 간동안 Cumulative Population Doubling(CPD) 을계산하였다. CPD 의계산식은 다음과같다. CPD = log(n/n O )/log2 (N : 배양후회수한세포의수, No : 초기세포의수 ) CPD 는배양조건을분리하는 P6 부터분석하였다. 2.3 줄기세포표현항원분석배양한세포들의표면항원을분석하기위해세포를 human CD14-FITC, CD45-FITC, HLA DR-FITC(Becton-Dickinson, San Jose, CA, USA), CD29-PE, CD73-PE, CD90-PE, CD166-PE(Pharmingen, Los Angeles, CA, USA), CD105-PE(Serotec, Kidlington, UK) 항체들과 15분동안실온에서반응시켰고, 대조염색은 mouse isotype 항체로하였다. 염색한세포는인산완충식염수 (phosphate buffered saline, PBS) 로세척하고, 1% paraformaldehyde로고정하였다. 간엽줄기세포의면역표현형은 FACSCalibur(BD Bioscience) 를사용하여유세포분석으로확인하였다. 2.4 줄기세포의골분화유도및확인 2 주동안세포를골분화유도배지에서배양하였고, 배지는 1 주일에 2 번씩 교체하였으며, 각세포의증식률에따라세포수를 /cm 2 밀도로배양하 였다. 골분화유도배지는 α-mem에 10% 우태혈청, 0.1 um examethasone(sigma), 10 mm β-glycerol phosphate(sigma), 50 um L-ascorbic acid 2-phosphate(Sigma) 를포함하여사용하였다. 2주간골분화를유도하고골세포로의분화는 von Kossa 염색과 ALP(alkaline phosphatase) 염색으로확인하였다. von Kossa 염색은먼저세포를 methanol 용액으로고정시킨후, silver nitrate - 5 -

16 용액으로자외선아래에서 1시간동안반응시키고, 5% sodium thiosulfate 용액에 2분, Mayer s haematoxylin 용액 (Sigma) 로대조염색을하였다. ALP 염색은먼저세포를 60% acetone으로고정한후, fast violet B salt를포함한 naphthanol AS-MX phosphate 용액 (Sigma) 에실온에서 30분동안반응한후, Mayer s haematoxylin 용액으로대조염색하였다. 2.5 줄기세포노화확인세포의노화를확인하기위해계대배양중 1주일동안배양한세포의수가 2배이상의증식을보이지않는시기의세포를 6 well plate에 /well의밀도로 1일배양하여 Senescence β-galactosidase Staining Kit R (Cell Signaling Technology) 로염색하였다. 염색후현미경으로전체세포수중염색된세포 ( 노화세포 ) 의수를비율 (%) 로확인하였다. 2.6 Real-time PCR O 2 분압에따른조건으로세포를배양하는중 P8 세포를이용하여 realtime PCR을시행하였다. 모든 RNA는각각의세포로부터 TRIzol R (Invitrogen Technology, Grand Island, New York, USA) 을이용하여추출하였다. cdna 합성을위하여 1ug의 RNA를동일하게사용하였으며, Superscript II Reverse Transcriptase Kit(Invitrogen Technology) 로하였다. Real-time PCR은 LightCycler TM 480 (Roche, Mannheim, Germany) 을사용하였고, mrna의양을확인하기위해다음과같은 SYBR primer를사용하였다. β-actin은 (forward) 5 -CCAACC GCGAGAAGATGA-3, (reverse) 5 -CCAGAGGCGTACAGGGATAG-3 을, OCT-4 는 (forward) 5 -CAATTTGCCAAGCTCCTGA-3, (reverse) 5 -CGTTTGGCTGAAT ACCTTCC-3 을, NANOG는 (forward) 5 -AGATGCCTCACACGGAGACT-3, (reverse) 5 -TTTGCGACACTCTTCTCTGC-3 을, SOX-2는 (forward) 5 -TGCTGCC - 6 -

17 TCTTTAAGACTAGGAC-3, (reverse) 5 -CCTGGGGCTCAAACTTCTCT-3 으로하였다. 각 primer들은 Universal Probe Library Assay Design Center (Roche) 에서구성한것으로하였다. mrna의상대적인비교는 comparative threshold cycle (2 - Ct ) 방법과 β-actin을대조로하여계산하였으며, normoxia에서의세포 mrna 발현양을 1.0으로정의하여비교하였다. 2.7 통계분석비교는 paired Student s t-test와 one-way ANOVA로하였으며, 결과는 3회이상각각실험한결과값으로평균 ± 표준오차로하였다. SIGMAPLOT 프로그램을사용하였으며, p-value<0.05인경우통계적으로유의한차이가있는것으로하였다

18 Table 1. Obstetric characteristics of mothers and neonates - 8 -

19 제 3 장결과 3.1 Hypoxia에서의줄기세포증식율증가제대혈에서분리한간엽줄기세포를 hypoxia와 normoxia의조건에서각각배양하였을때, 산소분압에따른세포증식의차이를비교분석하였다. 줄기세포를계대배양하여세포의모양과배양용기표면의 confluency를육안으로확인하고, 증식한세포의수를 CPD와 cumulative cell number로계산하였다. 제대혈에서분리한저밀도단핵세포들을 10% 우태혈청을포함한배지에서배양했을때, 섬유아세포와모양이유사한부착세포집단을이루었다. 배양 1주일후부터제대혈유래간엽줄기세포는섬유아세포처럼생긴부착세포가군집 (colony) 을이루기시작하였다. 군집을이루지못하거나부착하지못한원형의세포들이첫배양에서는보이지만, 이후계대배양을거치면서없어졌다. 세포들은플라스틱배양용기에서부착하여자라며, 세포는방추사모양을가진섬유아세포 (spindle-shaped fibroblast-like) 형태를보였다. 이세포들은 hypoxia와 normoxia에따라나눈두그룹에서계대배양을거치면서후기 (old passage) 로갈수록세포의형태가방추사모양에서세포질이넓고세포가커지는형태로변하였다 (Fig. 1). 이러한관찰은두그룹에서모두나타났다. 세포의 CPD는세포를분리한제대혈간의차이가있었으며, 결과는다음과같았다. UCB MSC-1의 CPD는 hypoxia/normoxia에서 1.84/1.78(P6), 4.03/2.78(P7), 6.15/3.43(P8), 7.67/3.62(P9), 9.11/2.62(P10), 9.25/3.17(P11) 을나타냈고, UCB MSC-4도비슷한결과를보였다. UCB MSC-2는 4.89/3.89(P6), 10.51/6.33(P7), 15.07/9.53(P8), 19.22/12.04(P9), 23.32/14.83(P10), 26.24/18.48(P11), 28.16/21.03(P12), 27.71/21.12(P13), 28.78/21.71(P14) 을보였다. UCB MSC-3은 - 9 -

20 0.51/1.1(P6), 1.79/1.46(P7), 3.85/0.98(P8), 5.18/0.48(P9), 5.45/0.15(P10) 으로, UCB MSC-5는 5.02/4.84(P6), 9.91/9.34(P7), 14.49/13.7(P8), 18.51/17.57(P9), 22.24/21.1(P10), 25.34/24.05(P11), 27.03/24.7(P12), 27.94/25.29(P13) 으로나타냈다. 또한 P10에서 UCB MSC-1의 cumulative cell number는 hypoxia/normoxia에서 / , UCB MSC-2는 / , UCB MSC-3는 / , UCB MSC-4는 / , UCB MSC-5는 / 로확인하였다 (Fig. 2)

21 Figure 1. Photomicrographs of UCB-MSCs cultured under hypoxia and normoxia. At passage 6 and 9, UCB-MSCs mainly consist of bipolar fibroblast-like cells. (P, passage. Scale bar = 500 μm)

22 Figure 2. Ex vivo expansion of UCB-MSCs cultured under hypoxia and normoxia. (A) Cumulative population doubling(cpd) of UCB-MSC cultured under hypoxia and normoxia. (n=5) (B) Cumulative cell number of UCB-MSC cultured under hypoxia and normoxia. (passage 10, n=5)

23 3.2 Hypoxia 에서의줄기세포표면항원 제대혈에서분리한간엽줄기세포 (P3) 와이세포들을 hypoxia 와 normoxia 에서계대배양 (P9) 후줄기세포의특이적인표면항원들이발현되는지를 확인하였다. 조혈관련항원 CD45 와 CD14, 조직적합항원 HLA-DR, integrin 수용체항원 CD29, matrix 수용체항원 CD106, 간엽줄기 / 전구세포항원 CD73와 CD105, 기타항원 CD90와 CD166로 ISCT(International Society for Cellular Therapy) 의규정에따라표면항원을분석하였다. 분석한결과, 제대혈에서분리한간엽줄기세포에서간엽줄기 / 전구세포항원 CD73과 CD105에대해양성이었으며, 조혈관련항원 CD45와 CD14 음성, 조직적합항원 HLA-DR 음성, integrin 수용체관련항원중 CD29 양성, matrix 수용체항원 CD166 음성이었다. 기타항원 CD90과 CD166은양성이었다. 표면항원의발현양성결과는 95% 이상, 음성결과는 5% 이하로균질한양상을나타냈다 (Fig. 3). 또한, hypoxia와 normoxia에서계대배양한세포의면역표현형도동일한결과를나타내었으며, 두조건간의차이는없었다 (Table 2). 이는제대혈에서분리한세포가간엽줄기세포의특이적인표면항원을나타내고있으며, 계대배양중줄기세포가산소농도에영향을받지않고표면항원의특징을그대로유지하고있음을보여주었다

24 Figure 3. Characterization of surface markers in cultured UCB-MSCs. UCB-MSCs were cultured under hypoxia and surfaced stained with antibodies. Staining intensity was examined by flow cytometry. Data are shown as histograms. Green, isotype matched control Purple, antibody positive cells

25 Table 2. Immunophenotypic characterization of the UCB-MSCs cultured under hypoxia and normoxia. Data are presented as the mean±standard deviation (n=4)

26 3.3 Hypoxia에서줄기세포의골세포분화능증가간엽줄기세포의특징인다분화능이 hypoxia 영향을받는지를확인하고자 hypoxia와 normoxia에서배양한세포들의골분화능을비교하였다. 제대혈유래간엽줄기세포들을골분화유도를하지않은대조군과 hypoxia와 normoxia에서계대배양세포들을각각골분화유도배지로교체하여 2주간배양후현미경으로확인하였다. 육안확인결과, 대조군의세포가섬유아세포형태를보이는것에비해실험군 (hypoxia와 normoxia) 의세포모양이변형된것을확인할수있었으며 (Fig. 4), 실험군에서는골분화로인해칼슘이침착하여배지의색상이불투명한상태를보였다. 육안으로세포의상태를확인한후, ALP와 von Kossa 염색을한결과, 두조건에서배양한세포모두염색이된것으로나타났다 (Fig. 5A). 이두염색은줄기세포가골세포로유도, 분화되어칼슘이침착되는특성을이용하는염색방법으로 ALP염색에서는붉은색으로, von Kossa염색에서는검은색으로침착된칼슘이염색된다. Hypoxia와 normoxia간의염색의차이를확인하고자 hematoxylin으로대조염색을하였다. 비교결과, hypoxia 세포가 normoxia 보다 ALP와 von Kossa 염색액에더강하게반응함을확인하였다 (Fig. 5B). 위와같은결과로, 산소분압이낮은 hypoxia에서의간엽줄기세포가 normoxia의세포보다골분화능력이더우수하다고평가할수있다

27 Figure 4. Photomicrographs the osteogenic differentiated UCB-MSCs cultured under hypoxia and normoxia. UCB-MSC were cultured under hypoxia and normoxia for 14 days. Thereafter, cells were treated with osteogenic media for 14 days for osteogenic differentiation. Control: normoxic condition without osteogenic differentiation. Scale bar = 50 μm

28 Figure 5. Osteogenic differentiation of UCB-MSCs cultured under hypoxia and normoxia. UCB-MSCs were cultured under hypoxia or normoxia for two weeks in osteogenic induction media. Osteogenic cells were detected by alkaline phosphatase staining. Calcium matrix mineralization was detected by von Kossa staining. (A) high magnification image of cells. (B) low magnification of cell culture wells. Scale bar = 500 μm

29 3.4 Hypoxia에서줄기세포노화지연줄기세포는증식을거치면서세포노화가진행되며, 세포가사멸하는과정을거치게된다. 제대혈에서분리한간엽줄기세포가배양중의산소농도에따라노화진행에영향을받는지를확인하기위해세포노화염색을시행하였다. 계대배양과정중계대간세포수증가가 2배이하인세포들을대상으로 senescence-associated β-galactosidase(sa-βgal) 염색으로세포노화를확인하였고, 현미경으로염색된세포를확인하였다. Senescence β-galactosidase staining kit의 β-galactosidase는노화가진행하는세포에서만발색기질 X-gal에의해분해되어파란색으로염색되게한다. 각세포의노화염색을시행한계대배양단계는 UCB MSC-1은 P11, UCB MSC-2는 P13, UCB MSC-3은 P8, UCB MSC-4는 P11, UCB MSC-5는 P11이었고, hypoxia에서배양한세포가 normoxia 조건의세포보다세포질에염색이약하게되었음을현미경으로확인하였다 (Fig. 6A). Hypoxia의각세포들의염색이약하게되었을뿐아니라반응양성비율은 12.64±2.61%(normoxia 21.13±4. 1%), 6.55±1.79%(12.29±1.76%), 5.65±2.14%(22.99±5.79%), 19.38±2.89%(43.83±2.5 9%), 14.32±2.82%(25.34±4.0%) 로 normoxia 세포들의수치가상대적으로높았다 (Fig. 6B). 노화염색결과제대혈에서분리한간엽줄기세포는 hypoxia에서배양하였을때세포의노화가일반배양상태보다지연된다는것을확인하였다

30 Figure 6. Cellular senescence of UCB-MSCs cultured under hypoxia and normoxia. (A) β-galactosidase staining of UCB-MSC cultured under hypoxia and normoxia. Cells undergoing senescence are stained blue. Scale bar = 500 μm. (B) Quantification of β-gal positive cells. Cells were stained for β-gal, enumerated and data rendered by SIGMAPLOT. Error bars represent the means±sd (***; p=0.0001, ****; p<0.0001)

31 3.5 Hypoxia에서의줄기세포능조절관련유전자발현량증가제대혈에서분리한간엽줄기세포를배양한산소조건에따라고유의자가재생능력의차이가있는지를확인하고자줄기세포능조절관련유전자로알려진 OCT4, NANOG, SOX2를이용하여 mrna의발현량을비교하였다. 분석은 normoxia 조건에서의세포 mrna 발현량을 1.0으로정의하여 hypoxia 의세포 mrna 발현량을상대적으로나타냈으며, 세포별로각각 4회씩반복하여 real-time PCR을시행하였다. Hypoxia에서배양한제대혈유래간엽줄기세포의 mrna 발현량은 normoxia 세포에비해 OCT4 1.34배 (±0.19), NANOG 2.26배 (±0.43), SOX2 1.88배 (±0.36) 로 3개유전자에서모두증가하였다 (Fig. 7). OCT4, NANOG, SOX2 유전자는간엽줄기세포의자가재생능력에영향을주는것으로알려져있으며, 이 3개의유전자가 hypoxia에서배양한간엽줄기세포에서더발현량이높은것은 hypoxia가간엽줄기세포의자가재생능력을향상시키는배양조건이라는결론을실험을통해서추측할수있었다

32 Figure 7. Expression of OCT4, NANOG and SOX2 in UCB-MSCs cultured under normoxia and hypoxia. UCB-MSC were cultured under normoxia or hypoxia for 18 days at passage 8. Expression of OCT4, NANOG and SOX2 were assessed by real time PCR. Data from UCB-MSC under normoxic conditions were normalized to 1 as the standard. Error bars represent the means±sd, n=3 ; * p<0.05, **p<

33 제 4 장고찰 자가재생과다분화능이라는매우유용한능력을가진간엽줄기세포는성인골수나지방조직에서얻을수있는유용한자원이나기증자의연령에따른영향으로인해세포치료제로서효과적이지못할수있을뿐아니라, 채취과정이외과적인침습과정을거쳐야하는한계를가지고있다 (3,4). 이에비해제대혈은윤리적인규제가적고동종이식시면역억제능력도있으며, 채취방법이용이하여간엽줄기세포를얻을수있는대안이되는자원이되었다 (8,9). 그러나, 제대혈의간엽줄기세포는다른조직에비해늦게연구가시작되었을뿐아니라, 분리율이낮아효율성면에서골수와지방조직에비해떨어지는편이다 (63). 이러한제대혈의간엽줄기세포의낮은분리율은많은연구자들에의해배양재료및방법들을개선하여 65% 까지분리율을향상시키고있다 (60-62). 게다가세포와조직배양방법에있어 ph와적절한영양성분, 성장인자들과더불어배양배지의산소농도에대한중요성을강조하여세포배양에일반적으로이용하는대기압조건 ( 약 20% 산소 ) 보다낮은 (<1-9%) 산소분압상태에서성체조직이잘유지하고있다는평가가증가하고있다 (23). 조혈모세포, 간엽줄기세포, 신경줄기세포들을포함한다양한줄기세포들이인체내저산소미세환경또는 niche 라는장소에있는것으로알려져있으며, 생체내의미세환경조건을모방한 hypoxia(<5% O 2 ) 에서분화하지않은줄기세포들의증식과노화, 사멸에영향을미치는것으로확인하였다 (24). 또한 2% 산소농도상태에서골수유래간엽줄기세포의증식능력과분화능력, 대사과정을 normoxia와비교하여평가한결과분화능력은차이가없었으며, hypoxia에서명확하게높은증식능력수치와 glucose와 lactate의효율적인에너지대사능력을보였다 (22). 반면

34 지방에서분리한간엽줄기세포의증식과대사과정뿐아니라분화능에서도영향을준다고도하였다 (19). Chow 등이줄기세포는 1-6% 의산소농도조건이골수에서의저산소미세환경과유사하여세포증식에있어적절한농도임을확인한적이있어 (25), 본연구에서는 3% 산소농도상태에서제대혈유래간엽줄기세포를배양하여증식과분화, 노화, 유전자발현의정도를일반산소분압조건과비교해보았다. 제대혈에서분리한간엽줄기세포들은배양기간내내 hypoxia에서누적증식률 (CPD) 이확연히증가하였다. 세포의증식률은세포개체간의차이가있기에 normoxia에서계대배양을진행할수록증식률이거의증가하지않고감소하는세포도있었고, 두조건간의차이가많이나지않는세포도있었으나, 제대혈 5 units에서분리한간엽줄기세포모두 normoxia보다 hypoxia에서증식률이높다는것이공통적인결과였으며, 이는이전다른조직에서분리한간엽줄기세포들과의연구와도같은결과를나타냈다 (17-19,22). 세포의노화확인은 senescence-associated β-galactosidase 염색으로하였으며, 이 senescence-associated β-galactosidase는노화세포에서만 monosaccharide로 β-galactoside의가수분해를촉진한다. β-galactosidase는노화가진행되는세포에서만발색기질인 X-gal에의해분해되어파란색의염료가나타나게한다 (51). 본세포노화염색결과에서도 hypoxia에서배양한세포의노화가천천히진행하고있음을확인하였으며, 이결과또한세포개체간의차이는존재하였으나 5종류세포모두 normoxia에서노화세포가상대적으로더많았음을확인하였다. 간엽줄기세포의분화능에대한비교실험에있어서도 hypoxia에서배양한세포가 normoxia 세포보다골분화가잘되는것을 ALP와 von Kossa 염색으로확인하였다. 이는골수에서분리한간엽줄기세포를두산소조건에서배양하여골분화능을확인하였을때차이가없었다고보고한결과 (22) 와는상반되게관찰되었다. 면역학적표현형을초기

35 간엽줄기세포와산소분압에따라계대배양을한세포군에서확인한결과, 초기간엽줄기세포의표현형은각조건에서계대배양을해도변하지않고유지한다는것을확인하였다. 이는간엽줄기세포의면역학적특징을그대로가지고있음을의미한다. 배아줄기세포의줄기세포능유지에는 OCT4, SOX2, NANOG등의유전자가작용한다 (29-31). Hypoxia가되면모든세포는 HIF(hypoxia-inducible factors) 유전자를발현시키며, 산소를조절하는 HIF-α와상보적인 subunit HIF-β가 HIF를구성하고있다 (26,27). 1992년 Semenza와 Wang이적혈구생성촉진유전자 (erythropoietin) 를유도하는 hypoxia 반응인자인 HIF-1을발견하였다 (41). HIF-1α에있는 proline은 prolyl hydroxylase로인해 hydroxylation 할수있으며, von Hippel-Lindau(VHL) 종양억제유전자와결합하고, 이것은 HIF-1α의 ubiquitinylation을유도하여, proteasome에의해 degradation이일어난다. prolyl hydroxylase의촉진은 O 2, Fe 2+, α-ketoglutarate, ascorbate에많이의존한다. 따라서 hypoxia에서는 prolyl hydroxylase를촉진할수없으므로, HIF-1은 증가하여세포핵으로이동하고핵에서 HIF-1β 와결합하며, 이 HIF-1 은 CBP/p300과의결합을촉진하게한다 (42). HIF-1은뇌, 심장, 신장, 폐, 간, 췌장, 태반, 골격근과사람의모든조직에서확인할수있으며, hypoxia가되면 HIF-1은암세포뿐아니라모든정상조직이생존하는데도움이되는전사인자 (transcriptional factor) 가된다. 또한 HIF-1의영향을받는 40개이상의 target 유전자가있으며, 이는산소운반, 당-에너지대사작용, 세포증식및생존에관여하는주요 3개그룹으로구분할수있다 (43). HIF-1 발현은 HIF-1이암세포의혈관생성에결정적인역할을하기때문에혈관질종양과연관이있다고도보고하였다 (44). 다능성줄기세포 (pluripotent stem cell) 는자가재생을통해수적증가와다양한조직세포로분화할수있는능력을가지고있는줄기세포로서, 세포치료를

36 위한다양한세포의공급원으로활용할수있을것으로기대하여, 많은연구자들이줄기세포자가재생에관련된기전에대해연구하고있다 (45,46). OCT4, NANOG, SOX2 전사인자역시줄기세포능조절기전과관련이있어이에대한연구도진행하고있다. OCT4 전사인자는초기에는산모의인자로난자에서활동하지만, 착상기간동안태아의상태를유지시키는역할을한다 (49). SOX2는 SRY (sex determining region Y)-box 2라는전사인자이며, 미분화한배아줄기세포의자가재생또는전분화능을유지하는필수적인역할을하고있다 (50). 또한, OCT4와 SOX2 단백질은 NANOG promotor에영향을준다고알려져있으며, NANOG 단백질또한전사인자로서작용기전에대한연구는아직완전하지는않으나, OCT4, SOX2, Rex-1등과함께서로상승적, 순환적으로작용하여줄기세포능에기여하는것으로보인다고하였다 (47-48). Hypoxia가되면배아줄기세포의전분화능유전자 OCT4를 HIF-2α가직접 upstream에서조절하고 (28), SOX2와 NANOG 또한 HIF-2α로인해발현이조절된다 (32). 미분화간엽줄기세포의줄기세포능을확인하고자한유전자 (OCT4, NANOG, SOX2) 발현실험에서 hypoxia 세포들이유전자발현이 normoxia에비해높은결과를보였다. 이는 hypoxia에서배양한줄기세포는자가재생능력의유지뿐아니라증가하는것으로나타났다. 본연구결과, 제대혈에서분리한간엽줄기세포가 hypoxia에서배양했을때세포의증식, 노화, 분화와자가재생능력의특성이다른조직 ( 골수, 지방, 태반등 ) 에서분리한간엽줄기세포와차이가없이 normoxia 배양방법보다우수하다는것을확인하였다. 간엽줄기세포는다양한조직환경의영향으로분화를할수있는줄기세포능을가지고있다. trophic mediator 로서의간엽줄기세포는면역억제또는재생촉진역할을함으로써생물적인작용 (bioactive) 을하는인자를분비하게하는중요한역할을한다 (33-35). 간엽줄기세포는주로골수에서

37 분리하였으나, 제대혈, 지방조직, 태반과같은다른조직에서도얻을수있다 (3 6-38). 이중제대혈은다른조직보다윤리적인제약이없고, 태아분만후버리는탯줄내혈액에서채취하므로침습적인수술을하는성체조직에비해쉽게얻을수있어간엽줄기세포의자원으로상당히유용하다. 또한, 제대혈에서분리한간엽줄기세포는다른성체간엽줄기세포보다훨씬미성숙하고생체외에서쉽게증식하고 (36), 골수에서분리한간엽줄기세포보다 유용성, 접근성, 다분화능이좋고, 암세포로의이동가능성이적다는등의 많은장점이있다고보고하였다 (11,39). 게다가골수나지방에서분리한간엽줄기세포보다제대혈에서분리한간엽줄기세포가장기간체외배양하였을때항염효과도높으며, 세포의노화는지연되는특징이있다고하였다 (40). 이러한장점들로인해제대혈에서분리한간엽줄기세포는골수유래간엽줄기세포보다임상에적용하기가더용이한것으로생각되어지고있다 (11, 12). 줄기세포치료제는인체의조직이나장기에질병이생겼을때줄기세포로부터만들어진새로운세포가병든세포를대체하는재생의학에서의능동적치료방법이다. 줄기세포의역사는오래되었으나, 현대적의미의줄기세포연구가주목받기시작한것은 1998년 Thomson등이인간배아줄기세포를처음만들기시작하면서부터였다 (46). 또한 Yamanaka 등이유도만능줄기세포를만드는역분화기술을개발하면서 (52) 줄기세포분야의발전과그활용폭이넓어졌다. 전세계적으로줄기세포의연구는정부및민간단체의적극적인재정지원을받는미국이줄기세포치료제개발임상연구및논문발표현황이가장활발하며, 일본이국가차원의지원으로역분화줄기세포발견을계기로발전하고있다. 이밖에도중국, 러시아, 영국, 이탈리아, 스페인, 독일, 호주, 싱가포르에서배아, 역분화및성체줄기 ( 골수, 지방, 제대혈등 ) 세포에대한연구및임상을진행하고있다. 국내에서도동종제대혈유래중간엽줄기세포 ( 무릎연골결손 ), 자가골수유래

38 중간엽줄기세포 ( 급성심근경색 ), 자가지방유래줄기세포 ( 크론성누공 ) 으로줄기세포치료제의상용을허가하였고, 이외에도뇌질환 ( 뇌경색, 알츠하이머 ), 척수손상, 버거씨병, 비혈연조혈모세포이식보조, 이식편대숙주질환, 치루, 미숙아기관지폐이형성증, 근위축성측삭경화증, 스타가르트병, 하지허혈증등을대상으로연구및임상적용을시도하고있다. 간엽줄기세포를임상에적용하기위한필요세포수를대량으로얻으려면일반적으로최소 3주이상의체외증식기간이필요하다. 이 3주이상의배양기간은세포치료제로임상에적용하기에는짧은시간이아니기때문에많은세포치료연구기관들이배양기간을단축하기위해다양한시도를하고있다. 대부분의연구목적은배양시간단축뿐아니라세포의증식을위한일련의과정들이배양한세포의수율을최대로얻고자하는방안을찾고자한다. 효율적인방법으로배양조건을개선하게되면배양과관련된장비, 시설, 인력등의제반비용을최소화시킬수있을뿐만아니라, 궁극적으로노화의진행을지연한미성숙세포를임상에적용하여치료효과를극대화시키는것을목표로하고있다. 세포치료제로서다양한장점을가지고있는간엽줄기세포의배양에 hypoxia를적용함으로써, 단위시간당세포수를더많이얻을수있고, 배양기간을단축할수있으며, 일정기간배양후에도증식과분화능이유지되고있는줄기세포를확보할수있으며, 또한노화되지않은줄기세포를얻을수있기때문에간엽줄기세포를이용한임상적용에있어서줄기세포치료효과를크게높일수있을것으로생각된다

39 제 5 장결론 본연구를통해제대혈에서분리한간엽줄기세포를 hypoxia에서배양하였을때, 일반적인배양산소분압조건에서보다간엽줄기세포의증식과분화능, 줄기세포특유의줄기세포능이증가된것을확인하였다. 그리고, 간엽줄기세포의노화과정은 hypoxia에노출된세포가 normoxia 세포보다지연되고있음을확인하였으며, 면역표현항원에는영향을주지않았다. 또한, 골수나지방조직의간엽줄기세포의 hypoxia 연구결과와제대혈의간엽줄기세포의결과가동일하다는것을확인할수있었다. 다른조직에비해 간엽줄기세포분리율이낮은제대혈을단핵구분리 배양단계 (primary culture) 부터 hypoxia 를적용하여단점을극복하기위한추가연구를지속해야 할필요가있다고생각된다

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47 Abstract Characterization of umbilical cord blood derived mesenchymal stem cells cultured under hypoxic conditions Yang, Jungyun Department of Biomedical Life Science The Graduate School of Health and Environment Yonsei University Mesenchymal stem cells(mscs) have the ability to self-renew and differentiate into multi-lineage cells, thus highlighting the feasibility of using umbilical cord blood-derived MSCs(UCB-MSCs) for cell-therapy and tissue-engineering. However, the low numbers of UCB-MSC derived from clinical samples requires that an ex vivo expansion step be implemented. As most stem cells reside in low oxygen tension environments (i.e., hypoxia) such as in the bone marrow, I examined the effect of hypoxic cell cultures of UCB-MSCs. The following parameters were examined: proliferation, senescence, differentiation and gene expression. UCB-MSCs were cultured under 3% O 2 (hypoxia) and normal atmospheric conditions (~20% O 2, normoxia). I found that UCB-MSCs cultured under hypoxic conditions expanded to significantly higher levels compared to UCB-MSCs cultured under normoxic conditions. The senescence of UCB-MSCs was decreased during hypoxic culture conditions compared to normoxic culture conditions as determined by beta-gal assay. In regards to differentiation potential, cells cultured under

48 hypoxic and normoxic conditions both underwent similar levels of osteogenesis as determined by ALP and von Kossa assay. Furthermore, UCB-MSCs cultured under hypoxic conditions exhibited higher expression of OCT4, NANOG and SOX2 - genes indicative of stemness. Moreover, cells expanded under hypoxia maintained a stem cell immnunophenotype as determined by flow cytometry These results demonstrate that the expansion of human UCB-MSCs under a low oxygen tension microenvironment significantly improved cell proliferation and differentiation. Hypoxic culture can be rapidly and easily implemented into the clinical-scale expansion process in order to maximize UCB-MSCs yield for application in clinical settings and at the same time reduce culture time while maintaining cell product quality. Key words: umbilical cord blood, mesenchymal stem cell, hypoxia