배시현 Clinical application of liver stem cells: Beyond research 범위간절제술후간부전상태의치료로줄기세포를이용한임상시험결과들이보고되고있다. 자가증식과특정장기로분화, 유지하는기능을보유한성체줄기세포로는골수, 제대혈, 말초혈액에존재

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1 KDDW KASL-KSG Joint Symposium 1 Clinical application of liver stem cells: Beyond research 간줄기세포의임상적용 배시현 가톨릭대학교의과대학내과학교실 Abstract Sustained liver injury may cause development of fibrosis with subsequent portal hypertension, liver failure and hepatocellular carcinoma (HCC) leading to a significant morbidity burden on health care worldwide. Liver transplantation is the most effective and curative therapy for patients with acute hepatic failure or end stage liver disease such as decompensated cirrhosis. However, the shortageof donor livers is a major obstacle in the clinical field, and OLT leads to the requirement of lifelong immunosuppression; additionally long-term side effects of OLT are of much concern. Therefore, stem cell transplantation has been studied as an alternative therapy for or at least a bridge to OLT. Among hematopoietic stem cell, mesenchymal stem cell(msc), umbilical cord blood cells, fetal liver progenitor cells, adult liver progenitor cells, and mature hepatocytes as stem cells, Bone marrow mesenchymal stromal cells (BM-MSCs) are known to have self-renewal and multi-potentiality properties and have been considered as alternative cell sources for liver or hepatocyte transplantation because of their high capability for self-renewal and differentiation without ethical or tumorigenic problems. Indeed, stem cell therapies have shown promising benefits for hepatic fibrosis in pre-clinical and clinical studies. In liver damage, MSC are able to differentiate into hepatocytes, stimulate the regeneration of endogenous parenchymal cells, migrate to damaged sites, and enhance fibrous matrix degradation. Furthermore, many clinical data on stem cells have been reported favorable effects for restoration of hepatic function and histological grading of fibrosis in patients with cirrhosis. We review the recent reports of human clinical trials on BM-derived MSC cells for restoration of hepatic functionand highlight the challenges facing clinicians to use the stem cells to save lives. Keywords: Bone marrow, Mesenchymal stem cell, Hepatic regeneration, Cirrhosis 서론 전세계적으로만성간질환으로인한사망률이급속히증가하고있지만, 현재까지급성간부전이나비대상성간경변증의치료로확립된방법은간이식뿐이다. 이처럼간부전환자의가장좋은치료법은간이식이지만, 공여자의절대적인부족, 높은의료비용, 수술의합병증, 지속적인면역억제제투여와만성신부전, 이식후심혈관합병증등의문제점을가지고있다. 최근, 간이식을대체할만한방법으로줄기세포가만성간질환환자치료에도움이될것이라는희망을주고있어, 급성간부전, 비대상성간경변증이나광 54 대한간학회

2 배시현 Clinical application of liver stem cells: Beyond research 범위간절제술후간부전상태의치료로줄기세포를이용한임상시험결과들이보고되고있다. 자가증식과특정장기로분화, 유지하는기능을보유한성체줄기세포로는골수, 제대혈, 말초혈액에존재하는조혈모세포 (hematopoietic stem cell) 와중간엽줄기세포 (mesenchymal stem cell, MSC) 등이있으며, 그중에서도중간엽줄기세포는특정상황에서간세포로분화하며, 그외내분비 (endocrine), 주변분비 (paracrine) 효과등을통해질병치료효과를보임으로써여러난치병질환의치료뿐아니라간질환치료에도성체줄기세포의이용이가시화되고있다. 1,2 본글에서는간질환치료로중간엽줄기세포의임상적용에대해고찰하고자한다. 본론 중간엽줄기세포는증식능력과분화능력이외에도, MHC class 1의발현이낮고 MHC class 2의발현이되지않아, 생체내면역반응을유발시키지않기 ( 면역반응억제능력 ) 때문에이론적으로이종이식 (xenograft) 에서조차면역억제제사용없이이식이가능하다. 3 또한중간엽줄기세포는 stromal cell-derived factor-1α (SDF-1α)/C-X-C chemokine receptor type 4 (CXCR4) 체계나면역반응유도성장인자및사이토카인 (TGF-β1, IL-1β,TNF-α, PDGF-AB) 등에반응하여손상된조직이나장기주변으로이동하는것으로보고되었다. 4 결국손상된부위에서다양한종류의영양인자 (HGF, FGFs, VEGF, EGF, GDNF, BDNF, PDGF 등 ) 를분비하여손상조직의재생과정에중요한주변분비역할을하며, 미세환경에서손상세포로직접분화 (trans-differentiation) 가가능한것으로알려져있다. 1 현재가장많이사용되고있는중간엽줄기세포의원천은골수이며, 자가골수유래중간엽줄기세포 (autologous bone marrow-derived mesenchymal stem cells) 를활용한간경변치료임상연구들이수행되고있다. 1. 간섬유화에대한중간엽줄기세포의작용중간엽줄기세포는특정배양조건에서뼈, 연골, 근육, 인대등의중간엽조직들로분화할수있는다능성세포이다. 일반배지조건에서분화없이세포배양용기의바닥에부착하여자라충분한양을쉽게확보할수있고증식력이우수한것이특징이다. 또한세포끼리자가분비 (autocrine) 와주변분비 (paracrine) 효과를가지며, 손상조직으로직접적인세포이동을하며, 5 낮은면역원성 (immunogenecity) 을갖는다는점에서최근조혈모세포보다활용도가더높다. 6 사람의골수와제대혈에서분리해낸중간엽줄기세포를실험실내에서확인할수있는표지자는 CD105+(SH-2+), CD73+(SH-3/SH4+), CD90+, CD44+, CD34-, CD45-, CD14- 등이있다. 7,8 현재중간엽줄기세포의조직재생에있어분자수준의기전은밝혀져있지않으나, 손상받은조직에서사이토카인들이분비되면이들은 niche에머물고있는중간엽줄기세포를활성화시켜동원한다. 활성화된중간엽줄기세포는상처입은조직으로부터추가적인 homing 신호를받고중간엽줄기세포가성숙된세포 55

3 제 21 차대한간학회추계학술대회 로증식및분화되도록촉진하고, 새롭게형성된성숙세포는상처받은조직으로이동하여손상을입은조직을재생시키는데기여함으로서급 만성간질환의합병증을감소시킬수있다. 8,9 2. 간성상세포와중간엽줄기세포와의상호작용간성상세포 (Hepatic Stellate Cells, HSCs) 는정상적상태에서는휴지기에있다가간손상에의해활성화되면세포내의지방과레티노이드성분을잃고 α-sma (alpha-smooth Muscle Actin) 를발현하는증식성의근섬유모세포 (myofibroblast) 로바뀌게된다. 이렇게전이분화된근섬유모세포는세포외기질 (extracellular matrix) 을분비하여 Disse강주위에침착되면서간섬유증이생기고결국간경변으로발전한다. 이러한섬유화과정에중간엽줄기세포는 TNF-alpha, interlukin-10 (IL-10), Hepatocyte Growth Factor (HGF) 등의사이토카인을통해간성상세포의활동을억제시키고, TRAIL, Fas 신호를통해간성상세포의세포자멸사 (apoptosis) 를유발한다 (Fig. 1) 또한골수유래중간엽줄기세포는내생간세포 (endogenous hepatocytes) 의증식을자극할뿐만아니라간세포를새롭게생산시켜손상받은조직이정상조직으로회복하는데영향을미친다고보고되었다. 10,11 이기전은 IL-10, TNF-α, HGF 를통해골수유래중간엽줄기세포의주변분비작용 (paracrine effect) 으로설명할수있다. 12 간경변환자의간섬유증이회복될때중간엽줄기세포는간성상세포의사멸유도, Tissue Inhibitors of MMP (TIMMP) 억제, collagenase 활성도를증가시킨다. 3. 임상연구 2006 년 Terai 등 13 이 9 명의비대상성간경변환자를대상으로자가골수유래중간엽줄기세포를말초정맥으 로주입하는임상연구를진행하였다. 24 주후혈액화학검사에서알부민이증가하였으며간경변의중증도 Figure 1. Changes in clinical data. The levels of serum INR(A), total bilirubin (B), albumin (C) and ALT (D) were serially tested duringfollow-up. 56 대한간학회

4 배시현 Clinical application of liver stem cells: Beyond research 가개선될뿐만아니라간생검시증식세포핵항원 (proliferative cell nuclear antigen) 의표현율이낮아지는것을확인하였다. 또한 2007년 M. Mohamadnejad 등 14 이간경변환자 2명을대상으로한연구에서자가골수유래중간엽줄기세포를정맥주입했을때 ALT level 과 MELD score 가낮아지는등간기능이향상됨을확인하였다. P. Kharazaziha 등 15 이진행한연구에서는간경변환자 8명을상대로골수유래중간엽줄기세포치료한결과, MELD score, serum albumin, serum creatinine, bilirubin 수치의호전을보였다. 2010년국내에서김등은 10명의비대상성간경변증환자에자가골수내단핵구세포를정맥주사한후 6개월간관찰기간동안간기능개선, 간크기와간섬유화호전을확인하였다. 2 이와같이중간엽줄기세포의안전성과효과에관한연구에서, 간기능관련지표변화등의관찰을통해간기능개선에효과가있음이보고되었다. 15,16 주로자가골수를원천으로간동맥을통해중간엽줄기세포를주입하고있으며, 연구를통하여 serum albumin 수치증가, serum bilirubin, MELD score 의저하, 간의조직학적회복, 복수의완화등을유효평가지표로하고있다. 최근백등은 11명의알코올성간경변환자에서골수유래중간엽줄기세포의항섬유화효과를관찰하기위한비교연구에서 11명중 6명에서 Laennec fibrosis system에따라조직학적회복을보였고, 10명에서 Child-Pugh score, TGF-β1, α-sma이호전됨을관찰하였다. 임상연구기간동안특별한부작용은나타나지않았다. 17 현재우리나라에서이와관련한다기관무작위임상 2상연구진행에서대조군에비해치료군에서 Child-Pugh score 호전, 조직검사에서교원분포및섬유화호전을증명하였다. 저자의연구팀은급 만성간염에의한간부전환자 5명에게환자의골수에서분리한중간엽줄기세포를주입하는임상연구를수행하였다. 중간엽줄기세포의채취는국소마취상태에서환자의엉덩뼈능성으로부터골수를 330 ml 정도채취한후, 조혈모세포처리실에서 T-Cell Separation Kit (Sepax) 를이용하여단핵구만을분리후, CliniMACS 를이용하여 CD34+ 세포를고갈시켜중간엽줄기세포를포함하는단핵구세포들을획득하였다. 그중일부 (20 ml) 를얻어 RBC lysis 법을통해단핵구만을분리하여중간엽줄기세포의표지자인 CD90, CD73 을이용하여골수내중간엽줄기세포의 FACS 를통해분석한다. 채취한골수혈액에 CliniMACS를시행하여 CD34- MSC 를 310 ml 얻었고 310 ml 내에 40 ml 내에 CD34+ MSC 는냉동보관해두었다. 환자들의평균연령은 47세였으며남성이 2명, 여성이 3명이었다. B형간염환자가 2명, C형간염 독성간염 윌슨씨병환자가각각 1명씩이었다. 대부분간성혼수, 복수가심하고황달을동반한환자로간이식이시급한환자들이대상이었다. 결과에서간의합성기능의회복을보여주는주요수치인 알부민 수치향상 (Fig. 1), 간섬유증검사에서간탄력도는 kpa에서 kpa로섬유화현상이호전되었다 (Table 1). 특히희귀만성간질환인윌슨병환자의복수와간성혼수등증상이호전됐으며, 간크기는 ml에서 ml로 20.4% 증가했다 (Fig. 2). 18 본연구는단핵구내 CD34+ 와 CD34- 단핵구를별도분리하여중간엽줄기세포치료로안정성과효과를입증하였으며특히윌슨씨병과같은희귀난치성만성간질환에도임상적용을하였다. 57

결론 아직까지는급성간부전이나진행성간경변증에대한최선의치료법으로간이식이인식되고있다. 그러나 2012년질병관리본부장기이식관리센터가발표한통계에따르면기증자의부족으로인해간이식이필요한 6,000여명중 1,200여명만이식수술을받은것으로나타났다.

5 제 21 차대한간학회추계학술대회 Table 1. Transient liver elastography data (reference range fornon-cirrhotic liver < 5.3 kpa). Figure 2. CT scans of patient 4 at 1, 6 and 9 months after transplantation show a notably smoother surface and improved coarseness overtime. 결론 아직까지는급성간부전이나진행성간경변증에대한최선의치료법으로간이식이인식되고있다. 그러나 2012년질병관리본부장기이식관리센터가발표한통계에따르면기증자의부족으로인해간이식이필요한 6,000여명중 1,200여명만이식수술을받은것으로나타났다. 간이식의대안으로자가골수줄기세포를이용한치료법은기증자를찾지못해생명이위독한중증간질환자에게가교적인치료 (Bridge Therapy) 로활용될수있다. 성체줄기세포, 특히중간엽줄기세포는간세포로의분화능력과자가분비 (autocrine) 및주변분비 (paracrine) 능력을통해간섬유화를억제하는효과가있는 58 대한간학회

6 배시현 Clinical application of liver stem cells: Beyond research 것으로알려져있다. 더욱이, 세포배양을통해충분한양을얻을수있고낮은면역원성 (immunogenecity) 을갖는다는점에서임상연구적용이용이하다는장점이있다. 여러기초및임상연구에서급성간부전및간경변치료에효과가보고되고있으나, 중간엽줄기세포이식에최적의세포타입, 세포수와치료경로 (route) 등에관해더욱많은연구가필요하다. 향후줄기세포치료의임상적효과와안정성을확인하고, 새로운치료법으로정착하기위해서는대규모무작위임상시험이필요하다. References 1. Bae SH. Clinical application of stem cells in liver diseases, The Korean Journal of Hepatology 2008;14: Kim JK, Park YN, Kim JS, Park MS, Paik YH, Seok JY,et al. Autologous bone marrow infusion activates the progenitor cell compartment in patients with advanced liver cirrhosis. Cell Transplantation 2010;19: Le Blanc K, Rasmusson I, Sundberg B,Gotherstrom C, Hassan M, Uzunel M, et al. Treatment of severe acutegraft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004;363: Hatch HM, Zheng D, Jorgensen ML, Petersen BE. SDF-1alpha/CXCR4: a mechanism for hepatic oval cell activationand bone marrow stem cell recruitment to the injured liver of rats. Cloning Stem Cells 2002;4: Lee KD, Kuo TK, Whang-Peng J, Chung YF, Lin CT, Chou SH, et al. In vitro hepatic differenciation of human mesenchymal stem cells. Hepatology 2004;40: NajimiM, Khuu DN, Lysy PA, Jazouli N, Abarca J, SempouxC, et al. Adult-derived human liver mesenchymal-likecells as a potential progenitor reservoir of hepatocytes. Cell Transplant 2007;16: Hong SH, Gang EJ, Jeong JA, Ahn C, Hwang SH, Yang IH, et al. In vitro differenciation of human umbilical cord blood-derived mesenchymal stem cells into hepatocyte-like cells.biochembiophys Res Commun 2005;330: Karp JM, LengTeo GS. Mesenchymal stem cell homing: the devil is in the details. Cell Stem Cell 2009;4: Tuan RS, Boland G, Tuli R. Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther2003;5: Abdel Aziz MT, Atta HM, Mahfouz S, Fouad HH, Roshdy NK, Ahmed HH,et al. Therapeutic potential of bone marrow-derived mesenchymal stem cells on experimental liver fibrosis. Clinical Biochem. 2007;40: Aurich I, Mueller LP, Aurich H, Luetzkendorf J, Tisljar K, Dollinger MM, et al. Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers. Gut. 2007;56: Parekkadan B, van Poll D, Megeed Z, Kobayashi N, Tilles AW, Berthiaume F, Yarmush ML, et al. Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells. BiochemBiophys Res Commun 2007;363: Terai S, Ishikawa T, Omori K, Aoyama K, Marumoto Y, Urata Y, et al. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Stem Cells 2006;24: Mohamadnejad M, Namiri M, Bagheri M, Hashemi S.M., Ghanaati H, ZareMehrjardi N, et al. Phase 1 human trial of autologous bone marrow-hematopoietic stem cell transplantation in patients with decompensated cirrhosis. World J Gastroenterol 2007;13: Kharaziha P, Hellstrom PM, Noorinayer B, Farzaneh F, Aghajani K, Jafari F, et al. Improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem cell injection: a phase I-II clinical trial. EurJ GastroenterolHepatol 2009;21: Mohamadnejad M, Alimoghaddam K, Mohyeddin-Bonab M, Bagheri M, Bashtar M, Ghanaati H, et al. Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis. Arch Iran Med 2007;10: Jang YO, Kim YJ, Baik SK, Kim MY, Eom YW, Cho MY, et al. Histological improvement following administration of autologous bone marrow-derived mesenchymal stem cells for alcoholic cirrhosis: pilot study. Liver International 2013;34(1): Park CH, Bae SH, Kim HY, Kim JK, Jung ES, Chun HJ, et al.a pilot study of autologous CD34-depleted bone marrow mononuclear celltransplantation via the hepatic artery in five patients with liver failure.cytotherapy. 2013;15(12):

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ

Pharmacotherapeutics Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee Univ Application of New Pathogenesis on the Drug Treatment of Diabetes Young Seol Kim, M.D. Department of Endocrinology Kyung Hee University College of Medicine & Hospital E mail : ycell2@yahoo.co.kr Abstract