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1 종 설 대한생식의학회지 : 제 34 권제 4 호 2007 서울대학교의학연구원인구의학연구소 1, 의과대학산부인과학교실 2 김윤영 1 오선경 1,2 최영민 1,2 * Feeder Cells for Culture of Human Embryonic Stem Cells Yoon Young Kim 1, Sun Kyung Oh 1,2, Young Min Choi 1,2 * 1 Institute of Reproductive Medicine and Population, Medical Research Center, 2 Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul, Korea [Korean. J. Reprod. Med. 2007; 34(4): ] 서 인간배아줄기세포 (human embryonic stem cell; hesc) 는확립된이후, 1 난치병을치료하기위한세포치료 (cell-based therapy) 에있어훌륭한세포공급원이될수있을것으로기대를모아왔다. 이후지속적인연구를통하여배아줄기세포뿐아니라여러조직또는기관내에존재하는줄기세포들에관한사실들이속속밝혀지고있다. 한정된분화능을가진것으로알려졌던성체줄기세포 (adult stem cell) 는유래된세포와다른종류의세포로분화할수있는교차분화 (transdifferentiation) 능력이있음이밝혀졌으며, 2 이미분화된체세포 (somatic cell) 들도역분화 (dedifferentiation) 기작을통해다시전분화능을가진상태로돌아갈수있다는것이알려졌다. 3 최근에는양수 (amniotic fluid; AF) 에존재하는줄기세포의존재또한밝혀졌다. 4 이러한줄기세포들은줄기세포의특성을공통적으로나타내며, 그기원에따라각기다른특성을지니고있어, 배아줄기세포외에또다른세포공급원의역 론 주관책임자 : 최영민, 우 ) 서울특별시종로구연건동 28, 서울대학교의과대학산부인과학교실 Tel: (02) , Fax: (02) ymchoi@snu.ac.kr 할을할것으로기대되고있다. 인간배아줄기세포는배반포 (blastocyst) 의내세포괴 (inner cell mass) 로부터유래된세포로체외배양시미분화상태 (undifferentiated state) 를유지하며, 자가증식 (self-renewal) 능력및모든종류의세포로분화할수있는전분화능 (pluripotency) 을가진세포이다. 이러한특성에기인하여배아줄기세포는세포치료에있어적합한세포공급원의하나로간주되고있다. 최초로인간배아줄기세포가확립된이후, 1 여러인간배아줄기세포주의확립및유지가보고되었으며, 5,6 배아줄기세포를특정세포로분화시키기위한연구들이진행되어, 삼배엽성세포로의분화유도가성공적으로이루어지고있다. 7~9 또한배아줄기세포의미분화유지기작, 전분화능을조절하는신호전달체계 (cell signaling) 및전사인자 (transcription factor), 배양조건 (culture condition) 의개선등에관한연구가활발하게진행되고있다. 그러나, 배아줄기세포로부터특정계열의세포로분화유도된세포들을세포치료에이용하기위해서선결되어야할과제들이있는데, 미분화상태를유지하기위한이종유래의지지세포 (feeder cell) 를사용함으로써유발될수있는병원균의전달

2 대한생식의학회지 Table 1. Composition of media generally used for human embryonic stem cell culture Types of Media Media Composition MEF culture medium DMEM 85 / 90%, FBS or FCS 15 / 10% STO culture medium DMEM 85 / 90%, FBS or FCS 15 / 10% hesc culture medium KO DMEM or DMEM/F12 80 / 85%, KO-SR 20 / 15%, 0.4 or 4 ng/ml bfgf Conditioned medium KO DMEM or DMEM/F12 80 / 85%, KO-SR 20 / 15%, 8 ng/ml bfgf MEF; mouse embryonic fibroblast, FBS; fetal bovine serum, FCS; fetal calf serum, hesc; human embryonic stem cell, KO DMEM; knockout DMEM, KO-SR; knockout serum replacement, bfgf; basic fibroblast growth factor (transmission of unknown pathogen) 방지와, 환자에게이식했을경우발생할수있는기형종 (teratoma) 의형성및면역거부반응 (immune rejection) 의억제등을꼽을수있다. 이러한과제들을개선하기위해서는인간배아줄기세포의배양조건중생쥐유래지지세포의대체또는배제, 동물유래물질을배제하고성분이알려진배양액 (defined media) 의개발등이필수적이다. 현재가장광범위하게사용되고있는배아줄기세포배양방법은생쥐유래지지세포와동물유래성분의 knockout serum replacement (KO-SR, Invitrogen) 가포함된배양액을사용한다 (Table 1). 이러한이종유래물질들을포함하는조건에서배양된배아줄기세포로부터특정한세포로분화유도하여세포치료에이용할경우, 이종유래의레트로바이러스에대한노출 10 또는병원균의전달등의여러가지문제들을고려해야만한다. 따라서배아줄기세포의배양시생쥐지지세포를대체할수있는다양한종류의인간지지세포의개발, 성분이알려진배양액개발등과같은배아줄기세포배양조건개선을위한연구가활발히이루어지고있다. 본고찰에서는본연구실에서개발한인간유래지지세포를이용한인간배아줄기세포의배양에관한연구결과를포함하여, 최근까지수행된다양한인간지지세포를이용한배양, 지지세포를배제한조건배양액 (conditioned media; CM) 을이용한배양및성분이알려진배양액개발등인간배아줄기세포의배양조건의개선을위한연구들에관하 여살펴보고자한다. 지지세포의역할지지세포는배아줄기세포의미분화상태를유지하는데기여하는영양인자 (trophic factor) 들을분비하고, 세포접촉으로매개되는미분화유지기작과연관되어있는것으로알려져있다. 11 지지세포에서분비되는신호전달물질들은상호간의다양한네트워크를구성하여배아줄기세포의미분화유지또는분화의개시를조절하는데기여한다. 지지세포로부터분비되는물질들은신호전달물질인 Wnt, Bone Morphogenetic Proteins (BMPs), Transforming Growth Factor (TGF) β 등과세포외기질 (extracellular matrix) 성분이있다. 12 Wnt 단백질은배아발생및분화에다양한역할을하며, 13 배아줄기세포의자가증식에도기여하는것으로알려져있다. 14 BMPs는초기배아의분화, 성장및발달에다양한역할을하며, 15 TGF β 군에속하는단백질중의하나인 Activin A는지지세포가없는경우에도배아줄기세포의전분화능유지에기여를한다는사실이보고되었다. 16 인간배아줄기세포의확립에앞서확립 연구된생쥐배아줄기세포 (mouse embryonic stem cell) 의배양시에생쥐섬유아세포 (mouse embryonic fibroblast; MEF) 를지지세포로사용한배양법이널리이용되어왔다. 17 그러나생쥐배아줄기세포는 leukemia inhibitory factor (LIF) 를첨가하여배양할경우지지세포없이도미분화상태의유지가가능하다

3 제 34 권제 4 호, 2007 김윤영 오선경 최영민 는사실이알려져있으나, 18 인간배아줄기세포에서는 LIF가미분화유지에기여를하지않는다는것이보고되었다. 19 최초로인간배아줄기세포를확립한 Thomson 등의연구에서는생쥐섬유아세포를생쥐배아줄기세포의배양에서와같이인간배아줄기세포의미분화상태유지에이용하였다. 1 이후보고된다른연구에서도인간배아줄기세포주의확립및배양에생쥐섬유아세포가널리사용되고있다. 본연구실에서는생쥐유래 STO (mouse embryonic fibroblast, CRL-1503, ATCC) 세포를지지세포로이용하여인간배아줄기세포주의확립및배양을보고한바있다. 6 현재인간배아줄기세포의배양에가장널리쓰이는지지세포는생쥐섬유아세포인 MEF 또는 STO 세포이다. 적정한밀도로자란지지세포들을감마선조사 (γ-irradiation, 30 Gy) 또는 mitomycin C (0.01 µm, 2시간이상 ) 처리방법을이용하여세포분열을억제시켜다시배양접시에깐후, 인간배아줄기세포의콜로니를배양하는데사용한다. 생쥐로부터유래된섬유아세포를지지세포로이용하는배양법은이종세포와의직접적접촉이이루어지고있어, 세포치료에사용될배아줄기세포의배양법으로는적절하지않다. Martin 등은이러한배양법으로배양된인간배아줄기세포는세포막에통해전달되는이종유래의면역반응을유발할수있는 sialic acid, Neu5Gc를포함하고있음을확인하였다. 20 위에서언급한문제점들을극복하기위하여인간지지세포를이용한배양또는성분이알려진배양액의개발등에관한연구는필수적이라고할수있다. 인간유래지지세포를이용한배아줄기세포의배양 생쥐섬유아세포를대체하기위한다양한인간유래지지세포들이개발되어인간배아줄기세포의확립및유지에이용되고있다 (Table 2). 본연구 Table 2. Mouse- and human-derived feeder cells used for culture of human embryonic stem cells Origin Type of feeder cell Reference Mouse Human Mouse embryonic fibroblast (MEF) 1, 5, 26, 46~58 STO 6 Adult fallopian tube epithelial cells 25 Adult muscle 25 Adult skin 25 Adult marrow stroma 28 Amniotic fluid fibroblast 21 Fetal muscle 24 Fetal skin 25 Foreskin 10, 26, 27 hesc-derived fibroblast 31~33 Placenta 29, 30 hesc; human embryonic stem cell 실에서는인간유래지지세포들중양수세포를지지세포로사용하여인간배아줄기세포를성공적으로배양할수있음을보고하였다. 21 양수세포는태아의이상을진단하는산전유전진단에가장많이이용되어온세포지만, 그기원과특성에대해서는정확하게알려지지않았다. 양수에서유래한세포는태아조직과양막 (amnion) 으로부터유래된세포들이섞여있으며, 22 줄기세포의대표적미분화표식인자인 Oct4를발현한다는사실이알려짐으로써줄기세포와유사한가능성을가진세포로새로이대두되고있다. 23 최근에양수세포내에존재하는줄기세포가삼배엽성세포로성공적으로분화될수있음이확인되어세포치료의새로운공급원이될수있는가능성이확인되었다. 4 이러한양수세포의특성에착안하여김등은양수세포에서분리한섬유아세포를인간배아줄기세포의배양에지지세포로이용하여, 배아줄기세포의미분화상태및전분화능이성공적으로유지될수있음을보고하였다. 21 이들은 SNUhES2 세포주를

non-treated with mitomycin C, (D) SNUhES2 P71-5 cell colony at day 7 on hafc non-treated with mitomycin C; B, Morphology of colony of SNUhES2 cell on day 7 grown on feeder-free condition.

4 대한생식의학회지 A B Figure 1. haf fibroblast as feeder cells for hesc (SNUhES2) culture. 21 A, Morphology of SNUhES2 cell line grown on haf feeder layer. (A) SNUhES2 P71-5 cell colony at day 2 on hafc treated with mitomycin C, (B) SNUhES2 P71-5 cell colony at day 7 on hafc treated with mitomycin C, (C) SNUhES2 P71-5 cell colony at day 2 on hafc non-treated with mitomycin C, (D) SNUhES2 P71-5 cell colony at day 7 on hafc non-treated with mitomycin C; B, Morphology of colony of SNUhES2 cell on day 7 grown on feeder-free condition. haf; human amniotic fluid, hesc; human embryonic stem cell, hafc; human amniotic fluid cell 이용하여 59계대동안미분화특성을유지할수있었고, 성공적으로배아체 (embryoid body; EB) 를형성하였으며, 이배아체에서분화관련유전자들의높은발현을확인하였다. 따라서양수세포로부터유래된섬유아세포가인간배아줄기세포의배양에있어서지지세포의역할을충분히수행할수있음을증명하였다 (Figure 1). 생쥐섬유아세포를대체하여인간세포를지지세포로이용한연구는 Richards 등에의해최초로연구되었다. 24 이들은태아의근육 (fetal muscle; FM) 과피부 (fetal skin; FS) 및성체나팔관상피 (adult fallopian tube; AFT) 세포등과인간혈청 (human serum) 을포함한배양액을이용하여배아줄기세포의미분화상태를성공적으로유지하였다. 24 이후 Richards 등은태아근육, 피부및성체나팔관상피, 근육 (adult muscle; AM), 피부 (adult skin; AS) 세포등을지지세포로이용하여, 인간배아줄기세포주를 30계대이상성공적으로배양하였다. 25 또, 인간유래지지세포를배양하는데인간혈청을이용하여배양액에서이종물질의함량을감소시키고, 여러종류의인간세포를지지세포로사용하여, 지 지세포의종류에따라배아줄기세포의미분화상태를유지하는능력에차이가있다는사실을확인하였다. 25 이후여러종류의인간유래지지세포를이용한인간배아줄기세포의확립및유지가보고되었다. Hovatta 등은상업적으로판매되는인간포피섬유아세포 (human foreskin fibroblast, CRL-2429, ATCC) 를지지세포로이용하여배아줄기세포를배양하였으며, 26 Amit 등은인간포피세포들을분리하여지지세포로사용함과더불어인간혈청을포함한배양액을이용하여, 인간배아줄기세포를성공적으로배양하였다. 27 Cheng 등은성인골수 (bone marrow) 세포를지지세포로이용하였으며, 28 이외에도태반에서유래된섬유아세포를지지세포로이용한사례도보고되었다. 29,30 인간유래의세포를지지세포로사용한연구외에도, 인간배아줄기세포로부터섬유아세포 (hescderived fibroblast) 로의분화를유도하여지지세포로이용한연구또한보고되었다. Xu 등은바이러스감염방법을통해 htert 유전자를삽입한섬유아세포세포주 (HEF1-hTERT) 를만든후, 31 이세포

5 제 34 권제 4 호, 2007 김윤영 오선경 최영민 로부터조건배양액을획득하여배아줄기세포의배양에이용하였다. Stojkovic 등은배아줄기세포로부터분화된섬유아세포를이용하여배아줄기세포의미분화상태유지하였고, 32 Wang 등또한배아줄기세포로부터얻은섬유아세포를이용하여인간배아줄기세포주의확립및유지에성공하였다. 33 그러나인간지지세포를이용한배아줄기세포의배양역시지지세포의안정적인확보및배양, 수명, 획득시기에따른변이등의요인에의해배아줄기세포를다량으로배양하는데이용하기어렵다는문제점을지니고있다. 지지세포의사용을배제한배양 (Feeder-free culture) 인간지지세포와함께조건배양액과세포외기질을이용한배양또한활발히연구되었다. 이방법은지지세포와의직접적인접촉없이, 지지세포와의 conditioning ( 세포분열이억제된생쥐또는인간지지세포를인간배아줄기세포배양액에서, 12시간이상배양한후수거하여사용 ) 을통해얻은조건배양액과세포외기질을배양에이용한다. 34 지지세포를배제한배양에서사용된세포외기질로는생쥐육종으로부터유래된 laminin과 16,29,34,35 기저층 (basal lamina) 의구성성분인 fibronectin 등이있다. 27,36 지지세포를배제한배양에서가장널리사용되는또다른세포외기질인 Matrigel (BD Sciences) 은역시생쥐육종으로부터유래된것이지만 laminin, collagen IV를비롯한다양한성장인자들이포함되어있다. 34 지지세포를배제한배양은 2001년처음으로보고되었는데, Xu 등은생쥐섬유아세포로부터얻어진조건배양액과 laminin, collagen IV, fibronectin 및 matrigel 등을이용하여인간배아줄기세포의성공적인배양에성공하였다. 34 이들은조건배양액을얻는지지세포의종류에따라배아줄기세포의미분화상태를유지시키는능력에있어차이가있다는사실또한확인하였다. Carpenter 등은생쥐섬유아 세포로부터얻은조건배양액과세포외기질을이용하여 4개의배아줄기세포주를배양하고각각의특성을분석하여, 지지세포를사용하지않고배양된배아줄기세포도그특성을오랜기간동안유지할수있음을확인하였다. 35 Wang 등은지지세포에서유래한조건배양액을사용하지않고, 배아줄기세포의미분화유지기작에기여한다고알려져있는 BMP 신호전달체계의길항제 (antagonist) 인 noggin 과고농도의 basic fibroblast growth factor (bfgf) 를이용하여지지세포를배제한상태에서의배아줄기세포의미분화상태유지에성공하였다. 37 지지세포와함께배양하여얻어지는조건배양액은지지세포에서배출된다양한종류의성장인자및미분화유지를위한알려지지않은수용성인자 (soluble factor) 등을포함하지만, 배양액을얻은시기및지지세포의종류에따른미분화유지정도차이가존재한다. 또한, 지지세포를배제한배양법은기존의배양법과비교하여, 생쥐지지세포와의직접적인접촉이없다는장점이있지만, 조건배양액과세포외기질의사용에따른동물유래물질들을여전히포함하고있다는점에서세포치료에사용될인간배아줄기세포의배양법으로는적합하지않다는문제점이있다. 또한미분화상태를유지하는기간과정도가기존의생쥐지지세포를이용한방법보다길지않고자연적분화가일어나는정도가높은것으로알려져있다. 34 따라서기존의생쥐지지세포를대신하여인간배아줄기세포의확립과유지에사용되기보다는, 주로유전자전달등의특정한실험에서생쥐유래세포와의섞임을방지하기위해단기간동안사용되고있다. 성분이알려진배양액 (Defined media) 배아줄기세포의배양시널리사용되고있는지지세포와배아줄기세포배양액의조성은동물유래의물질을포함한다 (Table 1). 동물유래물질을포함한배양액에서배양된배아줄기세포를이용할경우이종물질에의한면역거부반응의유발및

6 대한생식의학회지 병원균의전달가능성으로부터자유로울수없다. 따라서이러한위험성을감소시키고표준화된배양환경을조성하기위해최근에는지지세포를사용하지않고세포외기질과함께조성이알려진배양액을이용한연구가활발히연구되고있다. Ellerstrom 등은이종유래혈청에의한위험성을극복하기위해성인인간혈청이포함된배양액을이용한인간배아줄기세포의확립및배양이시도되어성공적으로인간배아줄기세포를유지하였다. 38 그러나인간혈청은동물혈청과마찬가지로획득된시기에따른차이, 알려지지않은구성성분, 인간배아줄기세포배양에있어동물혈청사용시보다낮은효율 ( 예 : 자연분화의증가, 낮은부착율 ) 등의문제점이있다. 39 배아줄기세포배양액에포함된혈청성분은현재 KO-SR로대체되어사용되고있다. 동물혈청을포함한배양액에서확립된배아줄기세포주들도 KO-SR 이포함된배양액을이용하여성공적으로미분화상태를유지하였으며, 40 KO-SR을포함한배양액에서배양된인간배아줄기세포는동물혈청을포함한배양액에서배양된인간배아줄기세포보다증가된성장율을나타내었다. 41 그러나동물혈청을대체한 KO-SR 또한소의혈청성분으로부터추출된알부민을포함하고있어완전히동물유래물질이배제되었다고는볼수없다. 27 최근에는구성성분이알려진배양액의개발에관한연구가활발히진행되고있다. Genbacev 등은태반에서분리된섬유아세포를지지세포로사용하여인간배아줄기세포주를확립하고, X-VIVO 10 (Cambrex Biosciences; 약제등급의인간 albumin, insulin, transferrin 등이포함되어있음 ) 배양액과고농도의 bfgf (80 ng/ml) 가첨가된성분이알려진배양액을이용하여배아줄기세포를배양하였다. 29 Li 등도 X-VIVO 10 배양액과고농도의 bfgf (8, 40, 80 ng/ml) 및 stem cell factor (15 ng/ml), flt3 ligand (15, 75 ng/ml) 등을첨가하여성분이알려진배양액에서배아줄기세포를배양하였다. 42 이밖에도 Vallier 등은인간배아줄기세포의전분화능이 Activin/Nodal 신호전달계를통해유지된다는사실을확인하고, 이신호전달물질을 bfgf와함께첨가하여지지세포없이, 성분이알려진배양액에서배아줄기세포를성공적으로배양한결과도보고하였다. 43 Ludwig 등은배양액에고농도의 bfgf, lithium chloride, GABA, pipecolic acid 및 TGF β를첨가하여성분이알려진배양액 (TeSR1) 을만들고인간유래세포외기질을사용하여인간배아줄기세포를확립하고, 미분화상태를유지하는데성공하였다. 44 그러나, TeSR1 배양액은배아줄기세포를다량으로배양하는데사용하기에는경제적인부담이커, 배양액의성분중일부를동물유래물질로대체한 mtesr1 배양액을개발함으로써, 이성분이알려진배양액의상용화를통하여성분이알려진배양액의적용범위를넓혔다. 45 결론이상에서와같이인간배아줄기세포의확립과배양에있어사용된다양한지지세포및배양액에관해살펴보았다. 또한, 본연구실에서연구된양수세포를지지세포로이용한인간배아줄기세포배양결과에관해살펴보고, 그밖의다양한인간유래지지세포, 지지세포를배제한배양 (feeder-free culture) 및성분이알려진배양액 (defined media) 에관하여살펴보았다. 현재활발히연구되고있는인간유래지지세포, 지지세포를배제한배양법, 성분이알려진배양액의개발등의연구들은궁극적으로세포치료에적합한인간배아줄기세포들의확립및유지에기여할것으로기대된다. 그러나, 상기의방법들은인간지지세포의확보및안정적인배양등이먼저이루어져야하기때문에인간배아줄기세포를다량으로배양하는데적합하지않다는한계를아직지니고있다. 이러한방법들을광범위하게적용하기에앞서지지세포종류에따른미분화유지의정도및기간, 전분화능의유지및정상핵형의유지등에미치는영향을비교분석하는등의후속연구들이좀더구체적으로이루어질필요

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