344 Kook In Park, et al. Transplantation of Neural Stem Cells 서론 신경줄기세포 는미분화된상태로계속증 식하는자가갱신 을보이고신경계를이루는신경 원세포 희소돌기아교세포 및성상 세포 로분화하는분화의다능성을보이는세포로정 의한다신

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1 Review Article Neonatal Med 2013 Aug;20(3): pissn eissn Therapeutic Application of Neural Stem Cells for Neonatal Hypoxic-ischemic Brain Injury Kook In Park, M.D., PhD., Kyoyeon Goo, M.D., Ph.D., Kwangsoo Jung, B.A.*, Miri Kim, B.A.*, Il-Sun Kim, M.Sc., Seokhwan Yun, M.Sc.*, Il-Shin Lee, PhD., Jeong Eun Shin, M.D., Ha Yang Yu, M.D., Ho Seon Eun, M.D., Jung Eun Kim, M.D., Ran Namgung, M.D., PhD., and Chul Lee, M.D., PhD. Department of Pediatrics, BK21 Project for Medical Science*, Yonsei University College of Medicine, Seoul, Korea 신생아저산소성허혈성뇌손상에서신경줄기세포의치료적적용 연세대학교의과대학소아과학교실, BK21 의과학사업단 * 박국인 구교연 정광수 * 김미리 * 김일선 윤석환 * 이일신 신정은 유하양 은호선 김정은 남궁란 이철 ABSTRACT Neural stem cells (NSCs) are characterized by a capacity for self-renewal, differentiation into multiple neural cell lineages, and migration toward damaged sites in the central nervous system (CNS). NSCs expanded in culture could be implanted into the brain where they integrate into host neural circuitry and stably express foreign genes. It hence appears that transplantation of NSCs has been proposed as a promising therapeutic strategy in neurological disorders. During hypoxic-ischemic (HI) brain injury, factors are transiently elaborated to which NSCs respond by migrating to degenerating regions and differentiating towards replacement of dying neural cells. In addition, NSCs serve as vehicles for gene delivery and appear capable of simultaneous neural cell replacement and gene therapy (e.g. with factors that might enhance neuronal differentiation, neurites outgrowth, proper connectivity, neuroprotection, and/or immunomodulatory substances). When combined with certain synthetic biomaterials, NSCs may be even more effective in engineering the damaged CNS towards reconstitution. Human NSCs were isolated from the forebrain of an aborted fetus at 13 weeks of gestation and were grown as neuro spheres in cultures. After the characterization of human NSCs in preclinical testing and the approval of the IRB, a clinical trial of the transplantation of human NSCs into patients with severe perinatal HI brain injury has been performed. The existing data from these clinical trials have shown to be safe, well tolerated, and of neurologicallysome benefits. Therefore, long-term and large scale multicenter clinical study is required to determine its precise therapeutic effect and safety. Key Words: Neural stem cells, Neonatal hypoxic-ischemic brain injury, Cell therapy Received: 10 July 2013 Accepted: 13 August 2013 Correspondence to: Kook In Park, M.D., Ph.D. Division of Neonatology, Department of Pediatrics, Severance Children s Hospital, and Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, 50 Yonsei-ro, Seodae moon-gu, Seoul , Korea Tel: , Fax: , kipark@yuhs.ac Copyright(c) By Korean Society of Neonatology. All right reserved.

2 344 Kook In Park, et al. Transplantation of Neural Stem Cells 서론 신경줄기세포 는미분화된상태로계속증 식하는자가갱신 을보이고신경계를이루는신경 원세포 희소돌기아교세포 및성상 세포 로분화하는분화의다능성을보이는세포로정 의한다신경줄기세포는신경계발생초기에는측뇌실 을둘러싸고있는뇌실층 의신경상피 세포 이며발생중기부터측뇌실과피막 을연결하는긴세포돌기를보이는이극세포 인방사교세포 가신경줄기세포이다방 사교세포는뇌실층에서계속대칭비대칭증식 하며딸세포 는내뇌실하층 으로이동하여중간신경전구 세포 혹은중간변천증폭세포 가된다본세포는계속증식하면서전체대뇌를이 루는신경원세포로분화하고일부는신경교세포로도분화된다 또한방사교세포는뇌실층에서떨어져나가외뇌실하층 으로이동하여계속증식하여외뇌실하 층에서방사교세포중간신경전구세포혹은중간변천증폭세포가 되고본세포들은계속증식하여전체대뇌를이루는신경원세포 로분화하여대뇌형성에중요한작용을하며일부는신경교세포 로분화한다 방사교세포는성인에이르러서는성체신경줄기세 포인뇌실하층의성상세포로변이된다성체신경줄기세포인성상세포는형세포라고하며를발현하고비교적증식을많이하지않으며형세포는빠르게증식하는중간변천증폭세포혹은중간신경전구세포인형세포로바뀌어증식하면서신경전구세포의숫자를늘리고다시신경아세포인형세포로변화하여증식하면서을따라서전방으로이동하여후구에도달한후과립층에통합되어과립신경세포가되고사구체층에서는사구체주위신경세포로분화하여후각기능에관여한다또전사인자를발현하는일부뇌실하층형세포는희소돌기아교세포로분화하여뇌량선조체해마채궁등에분포한다성체신경줄기세포는뇌실하층뿐만아니라뇌해마의치상회 의과립하층에도두종류의세포로존재하는데형줄기세포는와를발현하며비교적증식을하지않는조용한세포이고긴신경돌기를내어치상회의과립층을가로질러해마의분자층에도달하는형태를띤다형줄기세포는는발현하나는발현하지않 으며빠르게증식하고긴세포돌기는없다해마의신경줄기세포계보는형세포에서형세포로형세포는중간신경전구세포가되고전구세포는신경아세포로분화하고이주하여과립세포층에서성숙한글루탐산과립신경세포가되며축삭돌기를해마의과지역으로보내어주위신경세포에접합되고신경회로에통합되어학습및기억능력에관여한다형및형줄기세포는성상세포로도분화하며중간신경전구세포는희소돌기아교세포로도분화한다또한신경줄기세포는상기에서언급한뇌의과립하층및뇌실하층뿐만아니라시상하부와편도체대뇌피질및척수등에서도천천히증식하면서신경세포를생성함이보고되었는데아직그존재여부가확증된상태는아니다따라서신경줄기세포는과립하층및뇌실하층외에성체중추신경계의보다광범위한지역에주로휴면상태로존재하면서소수의신경원세포및교세포를생성하거나일부극소수의세포가다능성신경줄기세포로전환되어새로운신경세포를생성할가능성도있다이상으로손상되면재생되지않는것으로알려져있는포유류의중추신경계에신경줄기세포가존재하고본세포는일생을통하여증식. 분화하여새로운신경원및교세포를생성한다또한생체외에서신경줄기세포를대량배양할수있고신경원및교세포로분화유도할수있으며중추신경계에이식한경우이식세포의생착이주분화및외부유전자의발현등을확인하게됨에따라난치성신경계질환에서신경줄기세포를이용한신경재생치료의가능성에관심이증가하고있다 중추신경계에존재하는신경줄기세포의기능적의의 중추신경계에존재하는신경줄기세포는신경재생기능에참여하는데신생아저산소성허혈성뇌손상인경우등의의영향으로뇌손상부위에서원거리에존재하는뇌실하층의줄기세포가뇌손상부위로특이적으로이주하여신경재생에참여함이확인되었고와같은성장인자들을측뇌실에주입하여뇌실하층줄기세포를뇌손상부위로이주케할수있으며뇌손상시혹은 등의성장인자를뇌에주입할경우대뇌피질선조체뇌중격시상시상하부등에서신경줄기세포로부터일부새로운신경세포가생성됨이보고되었다따라서향후신경손상시내인성신경줄기세포의신경재생기능을이용하여줄기세포의증식이주분화신경연결등을조절함으로

3 Neonatal Med 2013 Aug;20(3): 써신경재생을유도할수있는방법도개발할수있다신경줄기세포는단순히뇌의특정부위에서일생을통하여증식하면서새로운신경세포를생성할뿐만아니라이러한신경세포생성은환경적및유전적요인들에영향을받아변화는가소성을보이며특정뇌기능에도영향을미친다특히운동좋은환경스트레스감소호르몬임신카레섭취등은해마치상회에서신경세포생성을촉진시키나노화는신경세포생성을감소시키고동물의종및주에따라신경세포생성정도도다르며뇌질환저산소성허혈성뇌손상뇌졸중간질우울증파킨슨병알츠하이머병헌팅톤병등도영향을미친다신경세포생성은신경세포의증가신경회로의구조적변경신경접합증가신경접합력의변화등을초래하여신경가소성을증가시키는데해마치상회에서새로생성된신경원세포는주위신경세포들에비해역치가낮고에의한억제효과에민감하지않아학습과기억능력을증가시킨다또한최근에는다양한약물들이신경줄기세포의증식과분화에영향을미친다고보고되었는데항우울제항정신약물항콜레스테롤제발기부전치료제등을들수있고따라서약물개발시화학약물이인지기능에미치는영향등을조사하여부작용발생을최소화하여야할것이다이분야의연구는현재시작단계에있으며향후노화와연관된기억능력감소치료제항우울제개발뇌인지및감각기능항진제개발등을기대할수있을것이다 가함유된무혈청의특정배지에서미성숙미분화된세포가증식하여형태로배양되고분화조건시에는신경원세포및교세포로분화한다본를단일세포로분리하여같은세포성장배지에서증식시킬경우단일세포차원에서미성숙상태로다시증식하여를형성할수있고역시분화조건하에서세포분화의다능성을보일경우신경줄기세포의존재를확인하는방법이다그러나모든줄기세포가단 일세포차원에서 를형성하는것은아니 며한개의줄기세포가반드시한개의 를형성하는 것도아니고 를형성하였다고하여반드시한개의 줄기세포가있다고할수없어만으로는정확히신뢰성있게줄기세포의존재와빈도를측정할수없다따라서향후신경줄기세포및전구세포줄기세포가아닌세포를분리할수있는새로운측정방법들이개발되어야할것이다신경줄기세포배양방법에있어서최근에는차원세포배양법이개발됨에따라세포와세포상호관계에대한연구를진행할수있고시험관에서신경계질환모델을확립하여신경계에대한약물개발및독성검사를시행하는연구가진행중에있으며세포성장을도와주는자가조립와그리고와같은생활성분자로세포배양용인공미세환경등의개발로신경줄기세포의성장과분화를시험관내에서조절하고생체내이식실험에적용중에있다 신경줄기세포의배양 신경세포의시험관배양법및배지개발의비약적발전으로신경계로부터신경전구세포를채취및분리하여다양한성장인자를사용하여대량으로배양할수있게되었는데쥐의태아전뇌에서단일세포단위의전구세포를배양함으로써포유류의중추신경계에도계속자가갱신하고분화의다능성을보이는신경줄기세포가존재함이확인되었고성체신경계에서도신경줄기세포를시험관내에서부유하는미성숙미분화된세포덩어리인 형태로배양할수있게되었다따라서이러한신경줄기세포의배양방법개발은생체내외에서신경줄기세포의존재여부를확인해줄뿐만아니라줄기세포의자가갱신및분화기전연구에도중요하며인간신경줄기세포도배양하게됨에따라중계연구와세포치료제개발에도크게기여할수있게되었다시험관내에서신경줄기세포의존재빈도및활동등을측정하는간편한방법으로를사용하여왔다세포증식인자 신경줄기세포로부터특이신경세포로의분화 신경계발달초기에신경줄기세포는신경계에분포하는위치에따라지역특이적인신경세포로적절히증식및분화한다즉신경줄기세포는지역특이성을보여신경계에위치하는지역정보를함유하고있으나신경계의다른위치로이식될경우이식된부위의환경적요인에의하여지역특이성이변화하는가소성을보인다또신경줄기세포는신경계발달단계에따른시간적특이성도보이는데발달단계초기줄기세포는다양한신경세포로분화될수있지만후기에추출된줄기세포는분화가능성이제한적이며각줄기세포에시간특이적발달프로그램이내재되어있어세포배양시발달신호의단계적발현이재현된다신경줄기세포는비대칭적증식을통해서신경원세포로먼저분화되고계속증식을통하여신경교세포를생성하기시작하는데이러한시간적분화형태의변화는세포내부에프로그램되어있거나외부환경적요인에의해서도일어난다따라서신경계를이루는다양한신경세포는신경계발달시공간적및시간적신호에의해

4 346 Kook In Park, et al. Transplantation of Neural Stem Cells 특성화되는신경줄기세포에의해생성되는데이러한공간적및시간적신호에의해신경계형태가특성화되는분자기전의규명이신경줄기세포생물학의주요한연구목표이며이러한지식은배아줄기세포와유도전분화능줄기세포로부터특이신경세포로의분화유도시에적용될수있다최근까지신경줄기세포의증식과분화등을조절하는많은환경인자유전자및화학물질등이보고되었고이러한인자들을이용하여비교적균질의분화신경세포를배양하여여러가지신약개발독성검사및세포치료에적용하는연구가진행되고있으며최근에는배아줄기세포및유도전분화능줄기세포를이용하여보다효율적이고균질한특정신경세포로의분화유도실험이활발히이루어지고있다그러나배아줄기세포및유도전분화능줄기세포는무한정증식하고다양한세포로의분화유도가능성을보이지만종양형성등의안전성문제가아직완전히해결되지않은상태이다따라서향후가까운미래에서는세포증식력과분화가능성이제한되어있지만비교적안전한신경줄기세포를대상으로배양조건의변화와분화세포의분리등전통적인방법을이용하여비교적균질한신경세포아형을획득하고세포치료임상시도에적용될가능성이높다고하겠다 신경줄기세포의치료적유용성 퇴행성신경계질환 은급 만성으 로국소성 다소성 혹은범발성 으로중 추신경계에신경세포가사멸하거나기능부전을보이는난치성 질환으로광범위한범주의질환이다이러한퇴행성신경계질환 에서신경줄기세포의치료적유용성은 신경계질환모델의중 추신경계에신경줄기세포를이식하면이식세포는생착생존이 주및숙주신경회로에통합되어시간적공간적신경발달미세 환경신호혹은신경계질환특이적인신호에반응하여적절한신 경세포로분화되어기능부전을보이거나사멸한신경세포를대 체하고손상된신경회로를재건하여신경재생을유도하며 시 험관내에서미리신경원세포전구세포 희 소돌기아교세포전구세포 성상세포 로분화유도한후숙주신경계에이식하여손상된신경세포를 보다효율적이고특이적으로재생할수있고 이식세포는숙주 신경계에통합되어신경접합을형성하고적절한신경전달물질 을분비하여국소재신경지배 를형성한다 이식 세포는숙주신경계에서뇌손상부위로특이적으로이주하거나 전체신경축에걸쳐광범위하게이주하므로국소성다소성혹 은범발성신경계질환에서손상결함을보이는세포효소신경 영양인자신경전달물질수초 세포외기질 세포표면물질등을제공할수있다이식세포는숙주신경세포의생존이주분화재생등에영향을미치는다양한신경영양인자등들을분비하여신경보호및재생을유도한다신경줄기세포는다양한항염증싸이토카인혹은면역제어물질 α β 등을분비하여신경재생을촉진한다혈관생성촉진물질등을분비하여신경재생을유도하고신경줄기세포는비바이러스성에의하여생체외에서쉽게유전자형질이입이가능하며신경보호및재생을촉진시키는물질의발현을유도할수있다간편하고안전한방법으로중추신경계에이식되어생착된후외부에서이입한유전자산물을직접적지속적그리고조절되는양상으로분비할수있고생분해성합성고분자화합물과공동배양시신경줄기세포의성장증식및분화가촉진되므로본세포를조직공학적방법으로이용하여신경조직의재생을유도할수있으며최근에는세포성장을돕는자가조립와그리고와같은생활성분자로세포배양용인공미세환경등을개발하여신경줄기세포의성장과분화를시험관내에서조절하고생체내이식술에적용할수있다 신생아저산소성허혈성뇌손상동물모델에서줄기세포이식 저산소성허혈성뇌손상신생아의는신생아기에사망하며중등도뇌손상인경우생존아의에서중증뇌손상인경우이상에서중증신경발달학적후유증이동반된다지난수십년동안고위험신생아에대한주산기치료가획기적으로발전하였고새로운신경보호제개발을위한많은연구가진행되었음에도불구하고최근치료적저체온증적용시중등도이상의저산소성허혈성뇌손상에서제한된신경보호효과를보인다는결과외에신생아저산소성허혈성뇌손상에대한특별한치료법이없으며주로일반적대증요법이진행되고있다따라서줄기세포를이용한세포치료는특별한치료법이없는신생아저산소성허혈성뇌손상에대한새로운신경재생치료법으로연구되고있다신생아저산소성허혈성뇌손상에대한줄기세포치료연구는크게태아혹은성체중추신경계유래신경줄기세포와골수혹은제대혈등의비신경계조직유래줄기세포를이용한연구로나눌수있다생후일된미성숙생쥐에서국소성저산소성허혈성뇌

5 Neonatal Med 2013 Aug;20(3): 손상을유발후쥐설치류신경줄기세포를뇌손상부위에이식한 결과공여세포는일시적증식과뇌손상부위로특이적으로이주 함을보이고손상부위와주변대뇌피질접경부위에확고히생착 하며신경원세포희소돌기아교세포및성상세포로분화하여뇌 손상으로손실된신경세포를재생함을보였다 신경줄기세포는 뇌손상시발현되는다양한인자 등들에반응하여뇌 손상부위로특이적으로이주함을보이는데신생아저산소성허 혈성뇌손상동물모델에 발현신경줄기세포를뇌손상반 대편뇌에이식할경우이식세포는뇌량 및대뇌 반구간교련 등을통하여 α α 농도가증가된뇌손상부위로이 주함을보였다 이러한결과는뇌손상시발현되는염증반응물 질인 에대하여 를발현하는신경 줄기세포내신호전달반응에의하여세포의증식이주및분화 등의신경재생적반응이일어남을나타낸다뇌손상부위에이 식된신경줄기세포는약 정도에서신경원세포로분화하지만 신경원세포로의분화를촉진하는 를 과발현시킨신경줄기세포를뇌손상부위에부위에이식할경우 공여세포는뇌손상부위에서는뇌손상접경부위에서약이상에서신경원세포로분화하고발현신경원세포로분화함을보였다실제신생아저산소성허혈성뇌손상동물모델에인간신경줄기세포를이식한경우도줄기세포는뇌손상부위에생착하고광범위하게이주함을보이며신경원및교세포로분화하고다양한신경전달물질을발현하는신경세포로분화함을확인하였으며주위의숙주신경세포와신경접합도형성하였다바이러스벡터를이용하여다양한신경영양인자등를과발현하게한인간신경줄기세포를뇌손상부위에이식한경우도잘생착하고보다많은공여세포가신경원세포로분화하였으며발현된신경영양인자로인하여숙주신경세포에대한신경보호및영양효과도확인되었다신경행동검사에서도이식군이대조군에비해뇌경색증범위가감소하고신경기능및학습과기억능력도향상됨이확인되었다또한와같은신경계발달시신경원세포생성을촉진시키고대뇌발달에중요한역할을하는전사인자를발현케한인간신경줄기세포를저산소성허혈성뇌손상모델에이식한경우이식세포의생착신경세포로의분화신경돌기신전신경접합형성등이모두촉진되고손상된피질척수로재생이강화되어신경기능도복구도크게강화됨을보였다이러한결과는신경줄기세포에성장인자싸이토카인신경발달관련유전자등을발현케한후저산소 성허혈성뇌손상부위에이식할경우공여세포뿐만아니라숙 주신경세포에도작용하여신경세포의분화유도신경돌기성장 촉진손상된신경회로복구신경세포생존보호강화신경접합 촉진등을유도할수있음을보였고한번의신경줄기세포이식 으로손상된신경세포를대체하는세포치료와적절한신경영양 인자를동시에제공하는유전자치료가가능함을제시하였다 신경줄기세포의높은신경재생능력에도불구하고중증저산소 성허혈성뇌손상인경우급격한조직괴사로인하여뇌실질에큰 와동 을형성한다따라서중증뇌손상부위에이식된줄기 세포는신경조직및성형 의부족으로인하여신경재생 능력이제한되며뇌손상부위에혈액공급도원활하지않아생존 도감소한다따라서이식세포의조직화와성장이주및분화를 유도하는성형인생분해성합성고분자화합물비계 를이용하여신경줄기세포와공동배 양한후신경줄기세포비계복합체를저산소성허혈성뇌손상 부위에이식하였는데이식세포의확고한생착뇌손상부위감 소혈관생성촉진다양한신경세포로의분화신경원세포로의 분화및신경돌기신전촉진염증반응의감소및뇌량을통한양 측대뇌반구사이신경연결형성등이유도됨을보였다 이러한 결과는향후고분자화합물비계와신경줄기세포를이용하여중 증저산소성허혈성뇌손상시신경조직공학적치료가가능함을제시하였다한편중추신경계에직접이식된신경줄기세포는신경손상질환부위에이식세포가특이적으로이주하고생착하여손상된신경세포로분화함을보이나성숙한성체중추신경계에이식된공여세포는일반적으로생체내이주범위가제한적이며신경계손상부위에직접이식할경우도일부세포는주변신경조직부위로광범위하게이주함을보인다한편손상된신경계에직접줄기세포를이식할경우는수술적처치를하여야하는데이러한침습적방법을피하기위하여줄기세포를혈액내주사하는경우중추신경계에이주생착하는공여세포는극히제한적이다따라서생체내로이식된신경줄기세포가신경손상혹은질환부위로특이적이며효과적으로이주생착및이탈을방지하는기술이요구된다이러한문제를해결하기위하여먼저줄기세포내로초 자성들을이입하여사용할수있는데생체내로나노입자가이입된줄기세포를이식한후에자석을이용하여자장을걸어주면이식세포의생체내이동과생착을외부에서제어조절이가능하다예비연구에서나노입자를인간신경줄기세포에효과적으로이입할수있었고자성을이용하여줄기세포를신경손상혹은질환부위에특이적으로이주생착및분화시킬수있었다따라서나노공학과줄기세포의융합연구를통하여신경계에새로운비침투적줄기세포이식술을개발할수있고신경계에이식된줄기세포의이 주생착및분화를외부에서제어조절가능하며나노입자를통

6 348 Kook In Park, et al. Transplantation of Neural Stem Cells 하여줄기세포에다양한신경재생촉진보호물질분화유도물질등을제공하며생체내이식된줄기세포를효과적으로추적할수있는영상기술을개발할수있어앞으로첨단융합연구가기대된다또한신경줄기세포는저산소성허혈성뇌손상부위에이식후사멸된신경세포를재생할뿐만아니라항염증작용면역제어조절작용등의 에의한치료적기능도보이는데뇌졸중모델에서정맥주사된신경줄기세포는뇌손상부위에생착된후대부분미분화된세포로존재하고이러한미분화세포는항염증작용신경상처조직형성감소숙주신경세포보호등의작용을하는것으로보고되었고뇌출혈모델에서정맥주사된신경줄기세포는비장에축적되어면역제어조절작용에의하여신경보호효과를보였다이러한연구결과들은모두신생아저산소성허혈성뇌손상에서신경줄기세포의치료적유용성을보여주지만신경줄기세포의신경보호재생기전이식세포와숙주신경세포와의적절한신경회로형성여부이식후장기간에걸쳐공여세포의생체내에서의기능급성및만성뇌손상에서신경줄기세포의치료효과비교등에대한연구가더필요한실정이다비신경계조직유래줄기세포로먼저인간제대혈줄기세포를저산소성허혈성뇌손상모델에서복강정맥경동맥등에주사한결과뇌손상부위에생착된공여세포는별로없었고신경세포로의분화는드물었으나이식군에서신경기능개선효과를보였고인간제대혈유래중간엽기저세포를뇌손상부위에이식한경우일부공여세포는성상세포로분화하고역시신경기능개선효과를보고하였다이러한결과는이식된제대혈줄기세포가손상된신경세포를재생하기보다는신경영양인자싸이토카인항염증인자면역제어조절인자등을발현하여신경세포보호효과를보이는것으로생각된다이러한결과를바탕으로현재저산소성허혈성뇌손상을보이는만삭신생아를대상으로자가제대혈줄기세포투여의안전성및적용가능성을평가하는임상상시험이미국대학에서시행되고있으며세포투여후신경학적검사와뇌검사등이진행되고있다성체뇌졸중모델에서골수유래단핵구세포및골수유래중간엽기저세포이식이신경보호와재생효과가있음이보고되었고최근에는환자를대상으로본세포를이식하여긍정적인결과를보고하였다신생아저산소성허혈성뇌손상모델에서골수유래중간엽기저세포를심장에이식한경우공여세포는양측대뇌반구에생착함을보이고신경기능개선을보였으나 대부분의세포는성상세포및소신경교세포로분화하였으며뇌손상정도를개선시키지못하였고중간엽기저세포를뇌손상부위에직접이식한경우공여세포는극히일부분만신경세포로분화하고주로항염증효과에의하여내인성신경줄기 세포에의한신경세포생성이촉진되고신경기능이개선됨등을 보고하였다 그러나향후신생아저산소성허혈성뇌손상모델 에서골수유래기저세포이식이뇌졸중모델과유사한신경기능 개선효과를보이는지확인하고아급성혹은만성신생아저산소 성허혈성뇌손상에서도이식효과를평가하기위한더많은연구 가필요하다 신생아저산소성허혈성뇌손상환자에서줄기세포이용세포 치료확립을위해서는동물실험등에서세포치료의안전성문제 가일단확보되면실제임상상연구에서세포이식방법이식세 포수이식세포종류및세포이식시기등을평가하는연구가함 께고려되어야한다현재까지가장적절한세포이식방법은아직 결정되지않았는데저산소성허혈성뇌손상모델에서대부분의 세포이식은뇌손상실질부위에바로세포를이식하였으나너무 침투적이어서뇌손상을줄수있고측뇌실내에이식할경우뇌 실질내로이식세포가광범위하게확산되나측뇌실에서멀리떨 어진부위는이식세포가이주생착하기힘들며폐쇄성뇌수종을 일으킬수도있다최근신경계질환모델에서줄기세포의정맥내 주사는간편비침투적안전한방법이며주로세포사멸을억제 하여신경기능을개선시킨다고보고되었으나 단지소수의이 식세포만뇌손상부위에생착하고폐콩팥간및비장등에공 여세포가축적되므로임상적용전에세포이식에따른다른장기 에서의안전성및독성등에대한평가가이루어져야할것이다 신생아저산소성허혈성뇌손상에서뇌의병리적환경이뇌손상 후시간에따라급격히변화하므로세포이식시기결정에중요한 영향을미치는데뇌손상후 일사이에가장대사적생화학 적및분자적활성도가높기때문에이시기가세포이식에가장적 합하다고보고하였고뇌손상일후세포를이식하면세포 증식및내인성신경재생기전이가장활발하다고하였다 그러 나만성저산소성허혈성뇌손상모델에서줄기세포이식의유효 성을보고한바는별로없어만성뇌손상에서줄기세포이식의임 상적적용은한계점이있다 신생아저산소성허혈성뇌손상및뇌졸중을포함한신경계질 환에서세포치료를위하여가장적합한세포는아직결정된바없 지만신경줄기세포이식이세포치료의표준이라고간주된다그 러나일부연구에서공여세포의낮은생존율제한된세포이주 미분화혹은신경교세포로의분화등으로신경재생에제한을보 인다고보고하였고자가신경조직혹은사망한태아조직획득과 충분한양의신경줄기세포배양이어려운점동종세포이식에따 른면역거부반응및종양형성가능성신경줄기세포유래특이 신경세포로분화유도후이식할경우뇌손상급성기에즉각적인사용이어렵고면역반응및배양액에의한오염등의가능성이제기될수있다골수및제대혈유래중간엽기저세포사용시에도적절한신경세포로의분화유도가되지않는점외에상기신경

7 Neonatal Med 2013 Aug;20(3): 줄기세포와유사한안전성유효성및세포배양에소요되는시간에의하여즉각적인세포이식이어렵다는점을들수있다한편제대혈및골수유래단핵구세포는자가세포를즉각적으로간편하고편리하게주산기시혹은동결보관후필요할때사용할수있는장점이있는반면에아직세포특성이확립되어있지않고세포이식의안전성및유효성도평가되어야한다 신경줄기세포임상적용을위한선행연구 신경줄기세포의증식과분화등의행동을조절하는많은환경인자유전자및화학물질등이보고되었고이러한인자들을이용하여비교적균질의분화신경세포를배양하여여러가지신약개발독성검사및세포치료에적용하는연구가진행되고있으며최근에는배아줄기세포와유도전분화능줄기세포를이용하여보다효율적이고특정신경세포로의분화유도실험이활발히이루어지고있다그러나배아줄기세포및세포는다양한신경세포로의분화유도가능성을보이지만종양형성등의안전성문제가아직완전히해결되지않은상태이며최근리프로그램줄기세포는유전적및후생유전적변형염색체이상단일유전자돌연변이유전자발현형태의변화등도보고되고있어향후더많은연구가필요한실정이다따라서가까운장래에는세포증식능과분화가능성이제한되어있지만비교적안전한신경줄기세포를대상으로배양조건의변화와분화세포의분리등전통적인방법을이용하여세포치료임상시도에적용할가능성이높다고하겠다실제미국는라이소좀축적질환중하나인유전성희귀신경질환인환아를대상으로미국허가하에인간신경줄기세포를뇌이식하는임상상시험을진행하여세포치료에따른안전성에특별한문제점이없다고보고하였으나희귀질환이어서환자참여가줄어계속적인임상연구는중단된상태이다또한최근에는또다른희귀유전성신경질환이며백질장애질환인환아를대상으로인간신경줄기세포이식임상상시험결과가보고되었는데역시안전성에특별한문제점은없었으며뇌검사상일부이식세포에의한뇌백질에수초화가관찰됨이보고되었다그러나실제전세계에서공식적으로아직퇴행성신경계질환환자를대상으로신경줄기세포를포함한다양한줄기세포이용세포치료의효능이증명되었거나허가된경우는없는실정이다이러한사실에도불구하고우리나라를포함한세계적으로일부난치성질환환자를대상으로합리적인이유없이 혹은과학적및임상적기초자료가불충분함에도불구하고실제 임상에서다양한줄기세포이용세포치료가일부시행되고있는 데이러한행위는항상효과를과대평가하고부작용발생은과소 평가하는경향을보인다 따라서난치성퇴행성신경계질환에 서줄기세포이용세포치료가성공하기위해서는임상시험을위 한명확한계획서가필요하고여기에는줄기세포임상적용에필 요한단계적인기초연구및임상연구이정표설정그리고임상시 험에따른윤리적사회적경제적법적행정적규범들의확립도 필요하다 인간신경줄기세포의임상적적용을위해서도아직많은연구 가필요한데 신경계발달과정에서신경줄기세포의다양한전 구세포혹은특이신경세포로의분화기전규명과줄기전구세포 특이표지인자가발굴 신경줄기세포의증식성장분화기전 연구를통하여세포이식에충분한수의신경세포를안전하게대 량배양증식하고적절한신경세포로의분화유도기술확립 이식된신경줄기세포가신경계내에생착이주분화및숙주신 경계에통합되는분자생물학적기전규명 내인성신경줄기세 포에의한신경계재생기전규명및신경치료에이용방법개발 신경줄기세포에치료적유용성을보이는유전자혹은단백질의 안전하고효율적전달방법개발 퇴행성신경계질환의병태생 리기전규명을통하여각각의질환치료에줄기세포이용세포치 료의적절성및경제성을먼저평가하고이식세포종류확인 각각의난치성신경계질환의병태생리에따른기능적이식술개 발 질환모델동물에서세포이식의확실한신경기능개선효과 확인과장기간에걸쳐부작용발생에대한평가 종류의성체줄기세포배아줄기세포 다양한다른 세포혹은리프로그래 밍세포유래신경세포이식과태아혹은성인중추신경계유래신 경줄기세포이식간에세포치료제로서의안전성및유효성비교 평가 신경줄기세포의항염증작용면역제어조절작용및성 장인자발현등으로인한신경보호작용규명 신경줄기세포의 치료적유용성기전규명및신경계질환의병태생리상호작용연 구를통하여가장적절한줄기세포이식방법이식시기등에관 한연구 다양한원인에의한복합퇴행성신경계질환에서줄 기세포이용세포치료와더불어다양하고다원적인융복합치료 법을함께적용하는전략개발등에대한연구들이진행되어야할 것이다 중증신생아저산소성허혈성뇌손상환아에서인간신경줄기세포이식임상시험 본연구팀은임신주에유산된태아의사체조직을보건복지부생명윤리법안을따르고연세의대세브란스병원기관생명윤

8 350 Kook In Park, et al. Transplantation of Neural Stem Cells 리위원회의허가와보호자의동의서를받고획득한다음태아중 추신경계의종뇌 부위를해부하여동물혈청은포함 되지않고 이포함된 에인간 신경줄기세포를신경구 형태로배양하였다본신 경구는시험관내에서 세포수가배증식되는데 소요시간이약일정도로빠르게증식하였고 년이상계속계 대배양이가능하였으며염색체검사상정상소견을보였고증 식조건에서약 이상의세포가 와같은미성숙신경줄기세포의표지자를발현하여신경줄기전 구세포형태를보였다 분화조건에서인간신경줄기세포는 β 를발현하는초기신경원세포 를발현하는희소 돌기아교세포전구세포 를발현하는성상세포로분화하였 고 등의신경전달물질을주로발현하였으며 전뇌 특이적인 와같은지역특 이유전자를발현하였다 본세포를 시설인세 브란스병원세포치료센터에서장기간계대배양하면서세포배양 마다시험관내에서증식분화형태를평가하였고주기 적인염색체검사혹은유전자의 검사를통하여유전자가정상세포임을확인하였다또 한마이코플라스마세균곰팡이바이러스 등병 원체에오염되지않고내독성검사도음성임을확인하였고동결보관후해동시에도세포특성변화가없음을확인하였다동물실험으로생후일된생쥐에서국소성저산소성허혈성뇌손상을유발하고주일후뇌손상부위에인간신경줄기세포를이식한결과공여세포는뇌손상부위및접경부위에광범위하게생착이주함을보였고신경원세포희소돌기아교세포성상세포로분화하고일부는미분화상태로있었으며이식된신경세포는등의신경전달물질을발현하고숙주신경세포와신경접합형성함을확인하였다숙주동물뇌조직학적검사상대조군에비해이식군에서신경염증반응이증가하지않았고뇌경색증범위는대조군에비해유의하게감소하지는않았으나신경학적검사상유의하게신경기능호전을보였고비정상적인신경행동소견이나뇌종양형성등은보이지않았다본연구팀은상기연구결과를바탕으로 중증주산기저산소성허혈성뇌손상환자에서동종인간신경줄기세포이식에의한세포치료법확립 이란연구자주도임상시험계획서를연세대학교의과대학세브란스병원기관생명윤리위원회에제출하여승인받았고년식약청에서임상시험시행을허가받았다본연구는비무작위전향적공개적임상연구이며출생시병력아프가점수심폐소생술실시여부동 맥혈가스검사뇌파검사이학적신경학적및방사선과적검사 상중증저산소성허혈성뇌손상을받은신생아를대상으로하였 고선천성기형선천성감염증유전성대사성염색체이상질 환출혈성뇌질환부당경량아자궁내성장지연소두증등이동 반되었거나저체온증치료및기타세포치료를포함한다른신경 보호치료를받은환자는제외하였다중증저산소성허혈성뇌손 상으로세브란스병원소아과에입원한환자를대상으로상기임 상시험기준에적합하며보호자가줄기세포이식치료에동의 한경우에한하여세브란스병원세포치료센터에서배양된인간 신경줄기세포를전신마취하에수술방에서신경외과의사가뇌 에이식하였다세포이식전과이식후정기적으로혈액검사이 학적및신경학적검사뇌파검사뇌 실시하였고세포이식위치는수술하루전뇌 이식당일수술방에서 는데손상된뇌실질혹은뇌실부위등에 검사검사를 를검사하고 로다시위치를확인하였 의미분화인간신경줄기세포를양측뇌에나누어서각각 직접주사하였다 농도씩 현재까지뇌손상후개월에서년이상경과한아급성및만 성저산소성허혈성뇌손상환자 명을대상으로인간신경줄기 세포를이식하였고동종세포이식후면역억제제는투여하지않았으며세포이식후년이상추적검사를실시하고있다줄기세포이식과직접관련된뇌출혈뇌막염뇌염뇌종양뇌수종경련통증강직면역쇼크신경학적기능악화비정상적인신경학적신경행동학적이상소견은관찰되지않았으며세포이식후개월년간격으로찍은뇌검사에서도이상소견이관찰되지않아인간신경줄기세포이식은안전하고적용가능한세포치료법임을보여주고있다세포치료의유효성여부는본임상연구가이중맹검무작위대조시험이아니고뇌검사상환자마다저산소성허혈성뇌손상의해부학적위치정도범위및신경병리소견이다양하며세포이식시환자의연령도다르며세포이식시기도아급성에서만성까지범위가넓고신생아의뇌는발달단계에있어연령마다발달기준이달라져세포이식후장기간추적검사가필요하므로현재로서는명확한평가를할수없다그러나일부환자는세포이식후뇌경색증의범위가유의하게감소하고신경학적기능과운동및인지기능의호전을보이고있다향후전체대상환자에서세포이식후적어도년이상추적검사가끝날경우임상자료를정리분석하여일차결과를발표하고자하며그후계속하여장기간에걸쳐세포치료의안전성및유효성결과를추적할예정이다

9 Neonatal Med 2013 Aug;20(3): 결론 신경줄기세포는미분화된상태로계속증식하는자가갱신을보이고한개의줄기세포로부터다양한신경원세포및교세포로분화하는분화의다능성을보이는세포를의미한다이러한신경줄기세포는태아기에서는신경계전반에걸쳐다양한해부학적부위에존재하여신경계를형성하며한번손상되면재생되지않는것으로알려져있는인간을포함한포유류의성체중추신경계에도존재하고일생을통하여증식분화하여새로운신경원및교세포를생성한다신경줄기세포는시험관내에서장기배양할수있고생체내이식시공여세포는생착이주및분화하여숙주신경계에통합되며외부유전자를발현하고이식전미리신경원및교세포로분화유도할수있고항염증인자면역조절인자신경영양인자및신경가소성촉진인자등을발현함이확인되어퇴행성신경계질환에서본세포를이용한신경재생보호치료가능성에관심이증가하고있다신경줄기세포를신생아저산소성허혈성뇌손상동물모델에이식한결과이식세포는뇌손상부위로특이적으로이주함을보이고손상부위에확고히생착하며다양한신경세포로분화하여신경세포를재생함을보였다신경줄기세포에성장인자싸이토카인신경발달관련유전자등을발현케한후저산소성허혈성뇌손상모델에이식할경우이식세포및숙주신경세포에으로작용하여신경세포의분화유도신경돌기성장촉진손상된신경회로복구신경세포생존보호강화신경접합촉진등을유도할수있음을보였고신경줄기세포이식으로손상된신경세포를대체하는세포치료와적절한신경영양인자항염증인자성장인자등을동시에제공하는유전자치료가가능함을제시하였다또한고분자화합물비계와신경줄기세포를이용하여중증저산소성허혈성뇌손상시신경조직공학적치료가가능함을제시하였고나노입자및줄기세포의융합연구를통하여신경계에새로운비침투적줄기세포이식술을개발할수있으며이식세포의이주생착및분화를외부에서제어조절가능하며이식세포를생체내에서효과적으로추적할수있는영상기술개발가능성을확인하였다본연구팀은임신주에유산되어사망한태아의종뇌부위에서인간신경줄기세포를배양확립하여생체내외에서특성을분석한후기관생명윤리위원회와식약청허가하에주산기중증저산소성허혈성뇌손상환아를대상으로인간신경줄기세포를이식하는연구자임상시험을시행하였다현재까지줄기세포이식에따른특별한부작용을관찰할수없었고일부환자에서신경기능의호전을관찰할수있어인간신경줄기세포이식은안전하고적용가능한세포치료법임을제시하였다따라서향후신경줄기세포이식경로이식시간및적절한대상환아선택치료적유용성이높은기능성신경줄기세포의개발조직공학및나노공학적융복 합연구를통한줄기세포기반복합기능성치료법개발많은환자를대상으로이중맹검무작위대조다기관임상시험시행및장기간에걸친줄기세포치료의안전성유효성을평가를통한세포치료법개발이필요하다 ACKNOWLEDGEMENT REFERENCES 1) Lui JH, Hansen DV, Kriegstein AR. Development and evolution of the human neocortex. Cell 2011;146: ) Merkle FT, Tramontin AD, García-Verdugo JM, Alvarez-Buylla A. Radial glia give rise to adult neural stem cells in the subventricular zone. Proc Natl Acad Sci USA 2004;101: ) Alvarez-Buylla A, Lim DA. For the long run: maintaining germinal niches in the adult brain. Neuron 2004;41: ) Menn B, Garcia-Verdugo JM, Yaschine C, Gonzalez-Perez O, Rowitch D, Alvalez-Buylla A. Origin of oligodendrocytes in the subventricular zone of the adult brain. J Neurosci 2006;26: ) Zhao C, Deng W, Gage FH. Mechanisms and functional implications of adult neurogenesis. Cell 2008;132: ) Ming GL, Song H. Adult neurogenesis in the mammalian brain : significant answers and significant questions. Neuron 2011; 70: ) Fowler CD, Liu Y, Wang Z. Estrogen and adult neurogenesis in the amygdala and hypothalamus. Brain Res Rev 2008;57: ) Sohur US, Emsley JG, Mitchell BD, Macklis JD. Adult neurogenesis and cellular brain repair with neural progenitors, precursors and stem cells. Philos Trans R Soc Lond B Biol Sci 2006; 361: ) Rakic P. Neurogenesis in adult primate neocortex: an evaluation of the evidence. Nat Rev Neurosci 2002;3: ) Imitola J, Raddassi K, Park KI, Mueller FJ, Nieto M, Teng YD, et al. Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine re-

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