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1 대한생식의학회지 : 제 37 권제 1 호 2010 미즈메디병원불임의학연구소 지희준 * 최순영 정다연 Differentiation of Human Embryonic Stem Cells into Germ Cell and Culture Condition for Single Embryonic Stem Cells Dissociated by Enzyme Hee Jun Chi *, Soon Young Choi, Da Yeon Chung Mizmedi Hospital ART Research Center Objective: The present study was carried out to induce differentiation of human embryonic stem cells (hescs) into germ cells and to establish a culture condition for single hescs dissociated by enzyme. Methods: Embryonic body (EB) was formed by hanging drop culture for 3 days from hescs colony. The EBs were cultured in the medium supplemented with retionic acid (RA) or/and bone morphogenetic protein-4 (BMP4) for 14 days to differentiate into germ cells. Germ cell specific markers, c-kit and VASA were used for immunohistochemistry of EB. Human ESCs colonies were dissociated into single cells by Collagenase, Tryple and Accutase, and then colony formation rate of the single cells was examined. Rho-associated kinase inhibitor (ROCK inhibitor, Y27632) was added into the culture medium of single cells to reduce the apoptotic damage during the dissociation. Results: Single cells dissociated with Tryple or Accutase showed higher colony formation rates compared to the cells dissociated with Collagenase. Seeding of cells/well (4 well dish) was efficient to obtain high colony formation rate compared to other concentrations of seeding cell. Addition of Y27632 significantly increased the colony formation rate of the single cells dissociated by Tryple. Immunohistochemistry of EB with c-kit and VASA markers showed a weak fluorescence signals compared to the signals from the testicular tissue. Conclusion: Dissociation with Tryple was useful to obtain healthy single cells and addition of Y27632 was beneficial for survival and colony formation of the single cells. Unlike other studies, we just observed a dim fluorescence staining of the germ cell markers, probably caused by the short-term culture for the differentiation of EB compared to other studies. [Korean. J. Reprod. Med. 2010; 37(1): ] Key Words: HESCs, Germ cells, Tryple, Retionic acid, Y27632 배아줄기세포 (ES cell) 는생식세포 (germ cell) 를포함한신체를구성하는모든형태의세포로분화되는잠재력을가진세포로알려져있다. 이러한특성을이용하여배아줄기세포로부터신경세포, 1 주관책임자 : 지희준, 우 ) 서울시강서구내발산동 701-4, 미즈메디병원불임의학연구소 Tel: (02) , Fax: (02) ivf129@mizmedi.net 심근세포, 2 조혈세포, 3 인슐린분비세포 4 등다양한체세포로의분화와관련한연구들이진행되었다. 또한배아줄기세포를체외에서장시간배양할경우이들이자발적으로생식세포특이적유전자의발현을나타내었으며 5,6 난포와유사한구조를갖는세포로분화됨으로써생식세포분화연구에대한촉매제가되었다

2 대한생식의학회지 이처럼배아줄기세포의자발적인생식세포로의분화가능성이제시되었지만생식세포는체세포와는다른독특한분화과정과발달경로를가지고있어서특별한배양조건과분화방법이요구되었고 8 이와관련한다양한연구들이진행되었는데생쥐의배아줄기세포를생쥐의난소과립막세포 (ovarian granulosa cells) 와공동배양함으로써형태적으로난자와유사한세포로분화시켰으며 9 생쥐의 EB (embryonic body) 를 testicular cell conditioned medium 에서배양한경우에도, 난소유사구조 (ovarian-like structure) 로분화되었다. 10 또한체외에서원시생식세포 (primordial germ cell, PGC) 의증식과특성화에중요한역할을하는것으로알려진 retionic acid (RA) 11,12 와 bone morphogenetic protein-4 (BMP4) producing M15 cells을이용하여생식세포로의분화를유도하였다. 13 최근에는영장류인 cynomolgus monkey embryonic body (EB) 의일부세포괴에서초기생식세포인 gonocyte의특이발현유전자인 VASA 의발현을확인하였으며 15 인간배아줄기세포로부터형성된 EB를 BMP4, BMP7, BMP8b 등을이용하거나, 14 돼지난소섬유아세포 (mitotically inactivated porcine ovarian fibroblasts) 와공동배양하였을경우에도초기생식세포로의분화성공이보고되었다. 16 생식세포의분화는다양한특이유전자들의발현을통해확인하였는데이들분화된세포들은난자특이유전자인 Figalpha, GDF-9, and ZP1-3 뿐만아니라정소특이유전자인 SCP3를발현하였다. 9 이외에도다양한특이유전자인 VASA, c-kit, Mvh, Stella, Dazl, Piwil 2, Pdrd 1, Rex 14, Rnf 17, Bmp8b, Stra-8, Haprin, LH-R, Sycp1, Sycp3, EMA-1, FE-J1, Fragilis, Acrosin, acetylated alpha-tubulin) 등의발현을관찰한연구들이보고되었다. 5,6,11,12,14,15,17 최근에는이러한유전자발현을역으로이용하여배아줄기세포로부터분화된생식세포에서 VASA 의발현을시각적으로관찰할수있는형광표지유전자 (GFP, LacZ) 를생쥐배아줄기세포내에삽입한 knock-in ES cell line을구축하여 germ cell 분화과정을연구한연구결과가보고되었으며 13 Kee 등은최근에본연구와 같은목적으로인간배아줄기세포를대상으로외래유전자인 VASA-GFP를삽입한 knock-in ES cell을만들어분화유도후초기생식세포를분리한다음이들을유전자조절을통해다시반수체의정자세포로분화시키는데성공을하였다. 18 이처럼외래유전자를삽입또는특정세포만을선별하기위해서는배아줄기세포의효과적인단일세포로의분리방법이필요한데이를위해 Trypsin 19,20 외에 recombinant-trypsin (Tryple) 21 과혼합효소인 Accutase 등이사용되었으며 22 분리후배양액에 ROCK (Rho-associated kinase) inhibitor (Y-27632) 를첨가하여단일세포분리에의한 apoptosis를줄여생존성을향상시키는연구가진행되었다. 22,23 한편 Novak 등은생쥐배아줄기세포에서생식세포로분화를시켰을경우, 분화된유사생식세포들이반수체가되기위한감수분열이라는마지막장벽을넘어서지못한다는결과를보고하였으나 17 같은해 Nayernia 등은생쥐의배아줄기세포로부터 spermatogonial stem cells (SSCs) 을구축한뒤이들세포가체외에서 meiosis를통해반수체 (haploid) 생식세포로의분화를확인하였으며이들세포를생쥐난자에미세주입하여수정및산자생산에성공함으로써배아줄기세포로부터분화된생식세포가정상적인수정과발달능력을갖추었다는상반된결과를보고하였다. 24 이처럼배아줄기세포로부터생식세포로의분화연구를통하여많은지식과경험이축적되면서생쥐에서는인위적으로분화된생식세포를이용하여배반포형성 25 및산자의생산 24 까지성공을거두었고인간의배아줄기세포로부터는반수체생식세포로의분화에성공함으로써 18 수정능력이있는생식세포분화의가능성도열어놓았다고할수있다. 이에본연구는형광표지유전자 (GFP, LacZ) 와생식세포발현유전자 (VASA) 를결합한외래유전자를인간배아줄기세포내에삽입하여새로운기능을부여한 knock-in hecs cell line을구축하여생식세포로분화를유도함으로써생식세포발생에대한근본적인기전을규명하고이해하고자하는궁극적

3 제 37 권제 1 호, 2010 지희준 최순영 정다연 인목표를두고그기초단계연구로서배아줄기세포의생식세포로의분화유도조건과유전자삽입을위한단일세포배양조건의확립을위해본연구를수행하였다. 연구대상및방법본연구는본원의배아연구기관 IRB 심의를통과하여 2008년 6월부터 2009년 12월까지 1.5년간연구를수행하였던연구로서사용된재료및방법은다음과같다. 1. 인간배아줄기세포 (human embryonic stem cells) 배양및유지미분화상태의인간배아줄기세포주 (hescs) 인 Miz-4는세포성장억제물질인마이토마이신 C를처리한 mouse embryonic fibroblast (MEF) 세포위에서공배양을하였다. 이때 hescs는기초배양액인 DMEM/F12 (50:50, Gibco BRL, Gaithersberg, MD) 에 20% (v/v) serum replacement (Gibco), 1 mm L-glutamine (Gibco), 1% nonessential amino acids (Gibco), 100 mm β-mercaptoethanol (Gibco), 4 ng/ml bfgf (Sigma, St. Louis, MO) 를첨가하여조성한배양액을사용하였다. 배양액은매일갈아주며, 7일간격으로새로운지지세포에계대배양하였다. 2. hescs colony 의 single cell 분리, 배양및 colony formation 유도 hescs의특성을손상하지않고 single cell로분리한다음다시 colony 를재구성하는배양조건을확립하기위해서기존에널리사용되던 Collagenase (Sigma, Type IV, 1 mg/ml) 를비롯하여최근에상품화된 Tryple (Gibco, 1 ml/well) 또는 Accutase (Chemicon, Temecula, CA, 1 ml/well) 등의효소들을이용하여가장적합한효소를선택하고자하였다. 일정농도의효소를처리한뒤각효소의최적의반응시간을할애한후분리된세포들을회수하여세척한뒤 hescs의배양과동일한조건으로배양을하였다. 또한 hescs의특성상 single cell 배양이쉽지않기에일정농도의 single cell이함께배양되어야상호작용에의해 colony를형성하는데이로울것이라는가정하에 single cell seeding 농도를달리하여적절한 single cell seeding 농도를구하고자하였다. 한편배아줄기세포의특성상 single cell로분리하여배양하였을경우, 세포활착력이떨어져배양접시또는지지세포위에부착되지못한세포들이결국 apoptosis로빠져 colony formation 능력이현저하게감소하게되는데세포활착력향상효과가있다고알려진 Rho-associated kinase inhibitor (ROCK inhibitor, Y27632, Calbiochem, La Jolla, CA) 를 10 μm 농도로배양액에첨가하여 colony formation rate의향상효과를조사하였다. Colony formation은 hescs의대표적인전분화능 marker인 alkaline phosphatase (AP) 로염색을하여염색이된 colony의수를측정하였다. 3. hescs 의 germ cell로의분화유도 hescs의 germ cell로의분화유도를위해 hescs colony를 Collagenase (0.25 mg/ml) 를 20분간처리하여떼어낸뒤 hanging drop에서 3일간배양하여 embryonic body (EB) 를형성하였다. 이렇게형성된 EB를 Bone morphogenetic protein-4 (BMP4) 100 nm 또는 Retionic acid (RA, Sigma) 각각 100 nm 농도로첨가된배양액과함께 EB의 3차원적구조를지속적으로유지하도록 ultra-low attachment surface dish (Corning, Wilkes Barre, PA) 에서배양함으로써생식세포로의분화를유도하였다. 4. EB조직의 germ cell로의분화확인일정기간 EB를분화유도한뒤이를파라핀을이용한조직절편을한뒤초기 germ cell에서발현되는유전자인 c-kit (primordial germ cell marker) 과 VASA (gonocyte marker) 등을 germ cell specific marker 로이용하여면역형광염색방법으로 germ cell로의분화가이루어졌는지를확인하였다. 분화유도한 EB 를 4% paraformaldehyde 용액에서 1시간동안고정시킨후에파라핀블럭을만들었다. 파라핀으로고

4 대한생식의학회지 Table 1. Colony formation of single ES cells dissociated with enzymes Enzymes Exposure time Cell recovery Concentration of seeding cells No. of colonies (%) (7 day culture) Collagenase 60 min 2.2± /ml/well Failure (0%) a Tryple 5 min 5.4± /ml/well 4±2.5 (0.4%) b Accutase 5 min 3.0± /ml/well 5.8±3.0 (0.58%) b Values are mean ± S.D. a,b Numbers in columns with different superscripts are significantly different (p<0.05) Hee Jun Chi. Differentiation of Human Embryonic Stem Cells into Germ Cell and Culture Condition for Single Embryonic Stem Cells Dissociated by Enzyme. Korean J Reprod Med 정된샘플은 5 μm 두께로잘라서슬라이드에올린후에 5% normal goat serum과 1% BSA로 blocking을시킨다음일정농도로희석된 marker를 (chicken anti-human VASA 1/100, c-kit 1/100) 이용하여면역형광염색을실시하였다. 대조군으로사용된정소조직은폐쇄성무정자증으로예상되는환자의현미경적정소조직검사이후폐기처리되는남은조직파편일부를본연구에사용하였다. 5. 통계분석연구결과에대한통계분석은 Student t-test를이용하여실시하였으며 p<0.05인경우통계학적유의성이있는것으로판정하였다. 결과 1. 효율적인단일세포분리방법특정유전자를삽입하기위해서는 hescs의단일세포배양이필수적이기에 hescs의효율적인단일세포분리방법을구축하고자하였다. hescs colony 를 single cell로만들기위해서크기가유사한 150 개의 hescs colony (colony 1개당약 1~ 개의세포로구성 ) 를각각 50개씩 Collagenase, Tryple 또는 Accutase 등의효소로처리하여 single cell로분리한다음이들의 single cell 회수율을조사하였고분리된 single cell들을각 well당 개의농도로 seeding하여 7일간배양한후이들의 colony formation을비교하였다 (Table 1). hescs colony를 single cell로분리하는데 Collagenase를사용하였을경우, 60분의긴시간동안처리를하여도 single cell로분리되는효율성이상대적으로낮고회수율도낮았다. 또한분리된 single cell을배양하여 colony formation을유도하였지만단한차례도성공하지못하였다. 반면 Tryple 과 Accutase를이용하였을경우상대적으로짧은처리시간인 5분이내에쉽게 single cell로분리가용이하였으며이들을체외배양하였을경우 colony formation이관찰되었다. 이러한결과를통해 ES cell을 single cell로분리한뒤이를다시 colony로재구성하는데있어서는 Tryple 또는 Accutase를사용하는것이시간적인면에서유리할뿐만아니라처리과정중에세포에대해상대적으로손상을덜주는방법이라는것을확인하였다. 효소를이용하여 hescs을 single cell 로만든다음이들을배양하여 colony를형성하는과정을 Figure 1에제시하였다. Single cell에서처음으로 colony를형성할때는세포수가적고치밀하지못한엉성한 colony 형태를나타내지만 (Figure 1-E) 이러한 colony를계대배양하였을때이들 colony의모양이정상적인상태로다시치밀화된다는것을 (Figure 1-F) 확인하였고이들의 colony 수는 alkaline phosphatase (AP) staining을통해염색된 colony를계수하여측정하였다. 2. 분리된단일세포의적정한 seeding 농도 Tryple로분리된 single cell을농도를달리하여

5 제37권 제1호, 2010 지희준 최순영 정다연 A B C D E F Figure 1. Colony formation of single hescs dissociated by Tryple. (A) single hescs seeded on MEF feeder layer (magnification 200), (B) early colony formation on day 7 after seeding ( 200), (C) colony formation on day 14 after seeding ( 200), (D) alkaline phosphatase (AP) staining of early colony on day 7 ( 100), (E) AP staining of colony on day 14 ( 100), (F) colony formation after two passages of subculture ( 200). Hee Jun Chi. Differentiation of Human Embryonic Stem Cells into Germ Cell and Culture Condition for Single Embryonic Stem Cells Dissociated by Enzyme. Korean J Reprod Med Table 2. Colony formation rate according to the concentrations of seeding single cells Concentrations of seeding cells No. of colonies (%) (7 day culture) 1 103/ml/well 3.6±3.5 (0.36%)a 3 가 지속적인 성장을 보여주지는 못하였다. 반면 세포 수를 well 당 5 103개 seeding한 경우 colony formation의 유의한 증가를 나타냈으며 지속적인 계 대배양을 할 경우에도 건강한 colony를 유지한다는 것을 확인하였다 개의 세포를 seeding한 경 b 5 10 /ml/well 37.3±28.7 (0.74%) 우에는 건강한 colony를 얻을 수 있었지만 colony 1 104/ml/well 47.0±18.5 (0.47%)a,b formation rate는 5 103개를 seeding하는 것 보다 비 Values are mean ± S.D. a,b Numbers in columns with different superscripts are significantly different (p<0.05) 교적 낮은 효율성을 나타내었다. 3. ROCK inhibitor (Y27632)의 첨가가 단일세 Hee Jun Chi. Differentiation of Human Embryonic Stem Cells into Germ Cell and Culture Condition for Single Embryonic Stem Cells Dissociated by Enzyme. Korean J Reprod Med 포 colony formation에 미치는 영향 Tryple과 Accutase를 사용하여 단일세포로 분리한 feeder cell 위에 seeding할 때 세포의 농도에 따른 후 이들을 well 당 5 103개의 농도로 seeding하여 colony formation rate를 비교하였다 (Table 2). 분리된 colony formation을 유도할 때 체외배양액에 첨가한 single cell을 feeder cell이 깔린 4 well-dish의 각 well Y27632의 효과를 조사하여 Table 3에 나타내었다 당 1 10, 5 10, 1 10 개/1 ml로 농도를 달리하여 hescs colony를 single cell로 분리하는데 Tryple을 3 seeding한 결과, 1 10 개의 적은 수의 single cell을 사용한 군에서 보다 높은 colony formation rate를 seeding하여도 colony가 형성되지만 형성된 colony 나타내었고 Y27632를 첨가하였을 경우 양쪽 군 모

6 대한생식의학회지 Table 3. Effect of ROCK inhibitor (Y27632) on colony formation of single hescs Enzymes ± Y27632 Concentration of seeding cells No. of colonies (%) (7 day culture) Tryple /ml/well 5.67±1.15 (0.11%) a,b Tryple + Y /ml/well ±5.03 (2.12%) c Accutase /ml/well 2.67±0.58 (0.05%) a Accutase + Y /ml/well 17.67±2.08 (0.34%) b Values are mean ± S.D. a,b,c Numbers in columns with different superscripts are significantly different (p<0.05) Hee Jun Chi. Differentiation of Human Embryonic Stem Cells into Germ Cell and Culture Condition for Single Embryonic Stem Cells Dissociated by Enzyme. Korean J Reprod Med 두에서 colony formation이증가하였으나 Tryple군에서보다유의하게높은 colony formation rate를나타내었다. 따라서 Y27632를배양액에첨가하는것이분리된세포의지지세포위에활착력을향상시키고세포를분리하는과정에서발생하는 apoptotic damage를감소시켜 colony formation rate를증가시킨다는것을확인하였다. 4. hescs 의생식세포로의분화유도 hescs를생식세포로분화유도하는물질로알려진 RA와 BMP4를이용하여생식세포로분화시키고자하였다. 분화유도를하기위해서계대배양직전의 hescs colony 중그크기가크면서분화가이루어지지않은건강한 colony만을선별하여 Collagenase에 20분간노출시켰다가피펫팅으로 colony를물리적인손상없이배양접시바닥에서떼어낸후세척을하여분화용배양액이담긴배양접시로옮겨 EB의형성을유도하였다 (Figure 2). RA와 BMP-4를이용한분화유도는 low attachment dish를이용하여가능한한바닥에부착되지않는 3차원적구조를유지한상태로배양을하였다. 이러한배양조건에서 EB가 cyst 형태로변화하며성장이이루어지는현상을나타내었으나 BMP-4를첨가한조건에서배양된 EB는상대적으로 cyst 형성이떨어지고이미형성된 EB도부서지면서작아지는현상을나타내었다 (Figure 2-E). 그리고두가지물질을함께첨가하여분화유도를하였을경우 (Figure 2-F), RA 단독첨가한실험군과 (Figure 2-D) 차이를나타내지않아두분화유도제의공동상승작용 (synergy) 은발견할수없었다. 이러한결과를관찰한이후, 분화유도에 RA를주로활용하였다. 5. 생식세포로분화유도후생식세포특이유전자의발현형성된 EB를 RA가첨가된배양조건하에서 14일간배양하여생식세포로의분화를유도한후이들을조직절편하여슬라이드에도포한뒤생식세포로분화하였는지여부를초기생식세포에서발현하는유전자인 c-kit과 VASA 유전자에대한형광표지인자를이용하여생식세포로의분화여부를조사하였다 (Figure 3). 대조군으로사용된정소조직은생식세포특이유전자인 c-kit과 VASA 의발현에대한명확한표지인자의형광염색을나타낸반면분화유도된 EB 절편으로부터는이들유전자의표지인자에대한매우희미한형광염색을나타냄으로써생식세포로의분화에성공하였다고확신을할수는없었다. 고찰일반조직이나세포괴에서 single cell을분리하는데많이사용되는 Collagenase를 hescs colony를 single cell로분리하는데사용하였을경우, 다른체세포와는달리 60분동안처리를하여도 single cell 로분리되는효율성이낮아처리후 single cell의회수율도낮았다. 또한분리된 single cell에서 colony

differentiated in the presence of BMP4, (F) EB differentiated in the presence of RA and BMP4. Hee Jun Chi.

7 제37권 제1호, 2010 지희준 최순영 정다연 A B C D E F Figure 2. Formation of EB and differentiation of EB to germ cells (magnification 200). (A) detached hescs colony, (B) EB formation after 3 day culture, (C) EB differentiated in the basic medium DMEM F-12 for 14 days, (D) EB differentiated in the presence of retionic acid (RA), (E) EB differentiated in the presence of BMP4, (F) EB differentiated in the presence of RA and BMP4. Hee Jun Chi. Differentiation of Human Embryonic Stem Cells into Germ Cell and Culture Condition for Single Embryonic Stem Cells Dissociated by Enzyme. Korean J Reprod Med A B C D Figure 3. Immunohistochemistry of EB with germ cell specific markers (magnification 200). (A) testicular tissue stained with DAPI and c-kit marker, (B) testicular tissue stained with DAPI and VASA marker, (C) EB stained with c-kit marker, (D) EB stained with VASA marker. Hee Jun Chi. Differentiation of Human Embryonic Stem Cells into Germ Cell and Culture Condition for Single Embryonic Stem Cells Dissociated by Enzyme. Korean J Reprod Med

8 대한생식의학회지 formation이이루어지지않은것은 Collagenase의처리과정에의해 hescs의세포표면또는기능적인부분에손상을받아지지세포위에활착이나증식이이루어지지않은것으로생각된다. Tryple이나 Accutase 등은 Collagenase보다세포손상력이강한 Trypsin 을포함하고있지만처리시간이 5분이라는매우짧은시간때문에오히려세포에손상을주지않은것으로사료된다. Tryple 과 Accutase를이용하여분리한군들간에 colony formation rate에서차이를나타내지는않았다. 그러나단일세포분리에따른 apoptosis를감소시키는 Y27632를첨가하여배양하였을때 Tryple 과 Accutase를사용한두그룹모두에서 Y27632 를첨가하지않은그룹들에비해유의한 colony formation의증가가나타났으며이러한 Y27632의분리된 single cell의생존성에대한긍정적인효과는다른연구자들의연구결과에서도보고되었다. 22,23 Li 등은이러한 Y27632의효과가분리된 cell 의활착력을높여주어다른세포에비해 anokis (detachment induced apoptosis) 에민감한 hescs의생존성을높여주었다고설명하고있다. 22 본연구에서도 Y27632의유익한효과를 apoptosis와연결시켜확인하려고하였으나 FACS 장비를활용하는데필요한충분한세포를확보하는현실적인어려움이있어서이와관련한실험은다음연구에수행할계획이다. Y27632의긍정적인효과는특히 Tryple 을이용한군에서두드러지게나타나 Accutase를이용한군에비해유의하게높은 colony formation rate 를나타내었다. 이러한결과는같은조건하에서는 Tryple 이 Accutase 보다는 single cell로분리하는데보다세포에손상을덜준다는것을의미하는것이며 Tryple이 single cell로분리하는데 3~5분정도소요되는데비해 21 Accutase의경우이보다 2배에가까운 8분을필요로하였다는이전의연구결과들이 22 이를뒷받침한다고할수있다. 분리된 single cell 농도를달리하여 feeder cell 위에 seeding한결과, 농도에따라 colony formation rate는차이를나타내지않았다. 그러나계대배양을 위해장기간형성된 colony를배양한결과낮은농도보다는일정수이상의높은농도의세포를 (well 당 개 ) 뿌리는것이보다건강한 colony 를형성하는데도움이된다는것을확인하였다. 이러한결과는배아줄기세포특성상단일세포로생존및증식이어렵다는점을확인시켜주었으며일정농도의세포들을함께배양하는것이 single cell간의상호작용뿐만아니라 paracrine signals 등이이들 single cell의생존과 colony formation에도움을준다는주장과 26 일치한다고할수있다. Colony formation rate (%) 는 seeding한세포수와형성된 colony 수의비율로나타내었으며 colony의형태를쉽게구별하고이들이전분화능을유지하고있는지여부를확인하기위하여가장널리사용되는 stem cell marker인 alkaline phosphatase (AP) 로염색하여 colony 수를측정하였다. 본연구에서는우선적으로효소적으로분리된단일세포의 colony formation에중점을두었기에몇차례의계대배양후의전분화능유지여부에대한실험은시행하지못하였다. 이와관련한실험은보다안정된단일세포분리및배양방법이구축된후에다양한전분화능표지인자를사용하여확인할예정이다. 그리고효소를이용하여분리된 single cell로부터형성된 colony는물리적으로작은세포괴로나뉘어새로형성된 colony 에비해얇고엉성한형태를나타내었다가몇차례의계대배양을통해정상적인형태의 colony의형태를갖추게되는데이러한현상은이들세포가새로운변화에대해적응하는시간이필요한것으로생각되며이러한형태적인특성은 Ellerstrom 등의연구 21 에서도관찰되었다. Retionic acid를이용하여 14일동안분화유도후생식세포특이유전자에대한표지인자 (c-kit, VASA) 를이용하여면역조직학적검사를수행한결과대조군인정소조직은유전자의발현이선명하게확인되었으나 EB 절편으로부터는매우희미한 germ cell marker의형광염색만이확인가능하였기에생식세포로의분화에성공하였다고확신을할수는없었다. 그러나 Richards 등도 RA를이용하여 15일

9 제 37 권제 1 호, 2010 지희준 최순영 정다연 동안 hescs를 germ cell로분화유도하였을때본연구결과처럼매우희미한유전자 (VASA) 발현의형광염색을관찰하였으나 30일분화유도시에는강한유전자발현을관찰하였다. 따라서본연구에서도분화유도배양기간을길게하였다면좀더확실한유전자발현을관찰할수있었으리라생각된다. 생쥐의 EB로부터분화유도를한경우에는 7 일후면생식세포의특이유전자를발현하는것이관찰되었지만 11 인간의 EB는이보다더긴 30일정도의분화유도기간을필요로하는것으로종간에차이가있는것으로생각된다. 16 한편자료를제시하지는않았지만본연구에서도 Lacham-Kaplan 등의연구방법과유사하게갓태어난생쥐의난소와정소조직을배양한 conditioned medium을이용하여 hescs의생식세포분화를유도하였지만 EB가 conditioned medium 내에서는 3 차원적구조를유지하지못하고 culture dish 바닥에부착되어 single layer와유사한형태로바뀌기에파라핀절편을이용한 immunohistochemistry를수행할수가없어서이들배양액에대한분화효과를제대로확인할수가없었다. 이에대한연구는향후실험디자인을다시세워수행할예정이다. 이상의연구결과로 hescs colony를 Tryple로단일세포로분리하여 Y27632를첨가한배양액에서배양하고 well당약 개의단일세포를 seeding 하는것이보다건강하고많은 colony를얻을수있다는것을확인하였다. 한편생식세포분화유도제로 RA를이용하였을때희미하지만생식세포특이유전자의발현을확인하였으며분화유도기간을연장할경우좀더뚜렷한생식세포특이유전자의발현을관찰할수있으리라생각된다. 아직은초기단계의연구라서더많은지속적인연구가필요하다. 참고문헌 1. Ben-Hur T, Idelson M, Khaner H, Pera M, Reinhartz E, Itzik A, et al. Transplantation of human embryonic stem cellderived neural progenitors improves behavioral deficit in Parkinsonian rats. Stem Cells 2004; 22: Mummery C, Ward D, van den Brink CE, Bird SD, Doevendans PA, Opthof T, et al. Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 2003; 107: Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thomson JA. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 2001; 98: Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M. Insulin production by human embryonic stem cells. Diabetes 2001; 50: Chen HF, Kuo HC, Chien CL, Shun CT, Yao YL, Ip PL, et al. Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation. Hum Reprod 2007; 22(2): Clark AT, Bodnar MS, Fox M, Rodriquez RT, Abeyta MJ, Firpo MT, et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Hum Mol Genet. 2004; 13(7): Hubner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, et al. Derivation of oocytes from mouse embryonic stem cells. Science 2003; 300(5623): Kiatpongsan S. From embryonic stem cells to functioning germ cells: science, clinical and ethical perspectives. J Med Assoc Thai 2007; 90(10): Qing T, Shi Y, Qin H, Ye X, Wei W, Liu H, et al. Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells. Differentiation 2007; 75(10): Lacham-Kaplan O, Chy H, Trounson A. Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes. Stem Cells 2006; 24: Kerkis A, Fonseca SA, Serafim RC, Lavagnolli TM, Abdelmassih S, Abdelmassih R, et al. In vitro differentiation of male mouse embryonic stem cells into both presumptive sperm cells and oocytes. Cloning Stem Cells 2007; 9(4): Silva C, Wood JR, Salvador L, Zhang Z, Kostetskii I, Williams CJ, et al. Expression profile of male germ cell-associated

10 대한생식의학회지 genes in mouse embryonic stem cell cultures treated with all-trans retinoic acid and testosterone. Mol Reprod Dev 2009; 76(1): Toyooka Y, Tsunekawa N, Akasu R, Noce T. Embryonic stem cells can form germ cells in vitro. Proc Natl Acad Sci U S A 2003; 100(20): Kee K, Gonsalves JM, Clark AT, Reijo Pera RA. Bone morphogenetic proteins induce germ cell differentiation from human embryonic stem cells. Stem Cells Dev 2006; 15(6): Teramura T, Takehara T, Kawata N, Fujinami N, Mitani T, Takenoshita M, et al. Primate embryonic stem cells proceed to early gametogenesis in vitro. Cloning Stem Cells 2007; 9(2): Richards M, Fong CY, Bongso A. Comparative evaluation of different in vitro systems that stimulate germ cell differentiation in human embryonic stem cells. Fertil Steril 2010; 93(3): Novak I, Lightfoot DA, Wang H, Eriksson A, Mahdy E, Hoog C. Mouse embryonic stem cells form follicle-like ovarian structures but do not progress through meiosis. Stem Cells 2006; 24(8): Kee K, Angeles VT, Flores M, Nguyen HN, Reijo Pera RA. Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature 2009; 462; Hasegawa K, Fujioka T, Nakamura Y, Nakatsuji N, Suemori H. A method for the selection of human embryonic stem cell sublines with high replating efficiency after single-cell dissociation. Stem Cells 2006; 24: Nicholas CR, Gaur M, Wang S, Reijo Pera RA, Leavitt AD. A method for single-cell sorting and expansion of generally modified human embryonic stem cells. Stem Cells Dev 2007; 16: Ellerstrom C, Strehl R, Noaksson K, Hyllner J, Semb H. Facilitated expansion of human embryonic stem cells by single-cell enzymatic dissociation. Stem Cells 2007; 25: Li X, Krawetz R, Liu S, Meng G, Rancourt DE. ROCK inhibitor improves survival of cryopreserved serum/feederfree single human embryonic stem cells. Hum Reprod 2009; 24(3): Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology 2007; 25(6): Nayernia K, Nolte J, Michelmann HW, Lee JH, Rathsack K, Drusenheimer N, et al. In vitro-differentiated embryonic stem cells give rise to male gametes that can generate offspring mice. Dev Cell 2006; 11(1): Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 2004; 427(6970): Pyle AD, Lock LF, Donovan PJ. Neurotropins mediate human embryonic stem cell survival. Nat Biotechnol 2006; 24:

11 제 37 권제 1 호, 2010 지희준 최순영 정다연 = 국문초록 = 목적 : 본연구는인간배아줄기세포를생식세포로의분화를유도하고효소에의해분리된단일배아줄기세포의배양조건을확립하기위해수행하였다. 연구방법 : Embryonic body (EB) 는배아줄기세포 (hescs) colony를떼어내어 3일간 hanging drop culture 방법으로작성하였고, 이러한 EB를 retionic acid (RA) 와 bone morphogenetic protein-4 (BMP4) 를단독또는함께배양액에첨가하여 14일간배양함으로써생식세포로의분화를유도하였다. 분화를유도한 EB는생식세포발현유전자인 c-kit과 VASA의표지인자를이용한면역조직형광법으로분화여부를조사하였다. 줄기세포는 Collagenase, Tryple 그리고 Accutase 등의효소로각각분리하였고분리된단일세포들의 colony formation rate를조사하였다. 한편 Rho-associated kinase inhibitor (Y-27632) 를단일세포배양액에첨가하여단일세포분리과정중에발생하는 apoptotic damage 를감소시키고자하였다. 결과 : Tryple 또는 Accutase를이용하여분리한단일세포가 Collagenase에의해분리된세포에비해높은 colony formation rate를나타내었다. 단일세포를 cells/well (4 well dish) 농도로지지세포위에 seeding하였을때다른농도의세포를 seeding한것에비해높은 colony formation rate를확보하는데효과적이었다. Y27632의첨가는단일세포의 colony formation rate를유의하게향상시켰으며특히 Tryple로분리한단일세포에보다효과적이었다. EB의분화유도후 c-kit과 VASA의표지인자를이용한면역조직형광염색은대조군인정소조직에비해약한형광염색을나타내었다. 결론 : Tryple을이용한단일세포분리가건강한단일세포를얻는데가장효율적이었으며 Y27632 의첨가는단일세포의생존및 colony formation에유익하다는것을확인하였다. 다른연구와는달리본연구에서는단지생식세포표지인자의희미한형광염색만을관찰하였는데이러한결과는본연구의분화유도기간이상대적으로짧았던것이원인이었을것으로생각된다. 중심단어 : 인간배아줄기세포, 생식세포, Tryple, Retionic acid, Y

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歯 PDF * * The Effects of Oocyte Oocytes Intracytoplasmic ICSI) Kee TaekHoo Hai * SikChun Department of Obstetrics and Gynecology, School of Medicine, Kyungpook Taegu, *Department of Animal Science, Taegu Kyungbuk,