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1 대한수혈학회지 : 제 27 권제 3 호, 2016 The Korean Journal of Blood Transfusion Vol. 27, No. 3, , December pissn eissn Review Article 줄기세포기반수혈용적혈구생산 김현옥 연세대학교의과대학진단검사의학교실 Production of Transfusable Red Blood Cells from Stem Cells Hyun Ok Kim Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea Blood transfusion is a well-established cell therapy. However, blood available for transfusion is a limited resource and is available only through donations by healthy volunteers. Moreover, the perpetual and widespread shortage of blood products, problems related to transfusion transmitted infections, and new emerging pathogens have elicited an increase in demand for artificial blood. Therefore, research for alternative RBC substitutes has begun in the 1960s. Hemoglobin-based oxygen carriers (HBOC) and perfluorocarbon-based oxygen carrier (PBOC) were two popular study subjects; however, research on these substitute candidates was halted due to unsatisfactory results and safety issues, including death, in the 1990s. Since then, worldwide efforts to produce RBC have shifted over to stem cell-derived RBC production using cord blood and G-CSF-mobilized peripheral blood stem cells, and some progress has been made. In terms of practical usefulness, however, large-scale production and cost effectiveness are still problematic. Recently, human embryonic stem cells (hesc) and human-induced pluripotent stem cells (hipsc) have shown the potential to produce RBCs as unlimited cell sources. These two methods using hescs and hipscs are also cost-effective since autologous and O, D negative blood RBCs will be used for alloimmunized patients with multiple alloantibodies or rare blood types (high incidence antigens) as well as universal blood production. We will review the current research on in vitro RBC production from hematopoietic stem cells and pluripotent stem cells and assess future directions in this field. (Korean J Blood Transfus 2016;27: ) Key words: Artificial blood, Stem cells, In vitro production 서론 인공지능이소개되고과학기술이눈부시게발 전하고있으나아직까지수혈용혈액은헌혈로만공급이가능하다. 전세계적으로약 112.5백만단위의헌혈이이루어지고있지만이중고소득국 Received on November 27, Revised on December 7, Accepted on December 7, 2016 Correspondence to: Hyun Ok Kim Department of Laboratory Medicine, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea Tel: , Fax: , hyunok1019@yuhs.ac This project was supported by a grant from the Korean Healthcare Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI10C1740). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright C 2016 The Korean Society of Blood Transfusion

2 Korean J Blood Transfus Vol. 27, No. 3, , Dec 가에서반을사용하고있으며, 이는전세계인구의 19% 에해당하는숫자이다. 1) 아프리카에서는 27개국에서연간 4백만단위의혈액공급이가능하여, 국가간의혈액사용량과혈액관리는극과극의차이를보이고있는것이현실이기도하다. 수혈로인한가장큰보건상의위협은감염전파이다. 세계는이미수혈감염으로에이즈, 광우병의경험을갖고있으며최근에는에볼라바이러스, 지카바이러스, 메르스전파를하는코로나바이러스등새로운바이러스감염이출현할때마다모든바이러스는혈액을통해감염될수있다는공포를갖고있다. 이는수혈의안전성에큰위협이되고있으며, 이를예방하기위해많은국가에서는필사적인노력과막대한검사비용을지출하고있다. 또한수혈의가장위험한요소중의하나는 ABO 혈액형부적합수혈이다. 이에대한교육과시스템지원으로실수의오류는많이감소되었으나아직까지도수혈사망사고의중요한원인이되고있다. 2) 국내에서도최근들어심한혈액부족상황이이어지고있다. 헌혈량감소는이미세계적인추세이다. 헌혈인구인젊은층의감소, 혈액사용대상자인노령층의증가, 개인주의에의한헌혈동기감소, 새로운감염질환출현등으로엄격한헌혈자선별검사의강화등이그이유이며, 계절적인요인인동절기마다이런현상은반복되고있다. Park 등 3) 은 2006년도에보고한논문에서 10 년내에혈액부족상황이발생할것을경고한바있으며 2015년도에 60세이상의노년층의혈액사용량이 60세이하의환자가사용하는혈액량보다많아질것이라는것을보여주는예측그래프를발표한바있다. 수혈용혈액은희귀한혈액등아주특수한경우를제외하고는국가간혈액교환이이루어지고있지않으며, 전적으로자국민의헌혈에의존해야하는자원이다. 따라서전세계 적으로인구의고령화, 새로운감염원의발생등으로수혈용혈액은장기적으로 30년내에는부족할것이라는예측과수혈을통한감염전파는아직도해결되지못한과제로서국가간안전한혈액을확보하기위한노력은치열하고인공혈액을생산하고자하는기술개발은세계적으로계속경고되는혈액부족현상에대해어쩌면당연한과학자들의열망이기도하였다. 인공혈액연구는 1960년대중반부터시작되었으며, 헌혈에의존하는혈액대체물질로 perfluorocarbon과같은 oxygen carrier 또는 hemoglobin solution과같은인공혈액을개발하였으나모두수혈후유리산소 (oxygen radical) 에의한장기독성사고로더이상의개발이중지된상태이다. 4) Erythropoietin은적혈구생산을증진시키는약물로만성빈혈환자에서사용이가능하나그기전이체내에서스스로적혈구생산을유도시키는것으로급성실혈에서는그효과를기대할수없으며악성혈액종양환자에서출혈과같은부작용을일으킬수있어그적용대상이한정적이다. 그러나 2000년대들어줄기세포가의학분야에서재생의학이라는새로운패러다임으로소개되었고이를기반으로하는많은연구과제중수혈의학에종사하는연구자를중심으로줄기세포로부터적혈구를생산하고자하는연구가시작되었다. 본원고에서는앞부분에서인공산소운반체의연구결과를간단히소개하고이연구가모두실패할수밖에없었던이유를살펴본후줄기세포기반적혈구생산기술개발의과정과줄기세포로부터수혈용혈액생산을위해해결해야할부분이무엇인지등을살펴보고앞으로의그발전방향에대해고찰하고자한다

김현옥 : 줄기세포기반수혈용적혈구생산 본론 1. 산소운반체 (Artificial Oxygen Carriers) 의개발 안전한혈액대체물질을개발하고자하는노력은 1960년대초부터시작되었으며, 1970년대이후에는혈액부족문제로인하여큰의료회사들이중심이되어적극적으로개발에나서게되었다.

1) Perfluorocarbon-based oxygen carrier (PBOC) Perfluorocarbon (PFC) 은 carbon과 fluorine의혼합물로서인체에무해하고가스용해도가높은합성유기물이다. PBOC 중가장대표적인상품은 1978년개발된 Fluosol-DA (Green Cross Co.

, Costa Mesa, CA, USA) 는혈색소를변형해서만든제품들에비해재료공급에제한이없으며가열로소독할수있는장점으로수술중수혈량을줄이려는목적으로 1992년에개발되었으나뇌졸중유발로임상시험이중단되어, 현재 PBOC 제품은모든임상시험이중단된상태이다.

이는혈색소분자가 nitric oxide (NO) 분자와결합하여혈관확장및혈소판활성억제기능을저해하기때문인것으로보고있다. 이후 nanomedical technology 기술이접목되면서혈색소내글로빈을여러개연결한혈색소중합체의형태로 Hemopure R (Biopure Inc.

3 김현옥 : 줄기세포기반수혈용적혈구생산 본론 1. 산소운반체 (Artificial Oxygen Carriers) 의개발 안전한혈액대체물질을개발하고자하는노력은 1960년대초부터시작되었으며, 1970년대이후에는혈액부족문제로인하여큰의료회사들이중심이되어적극적으로개발에나서게되었다. 산소를운반하는적혈구기능의혈액대체물질은합성유기물인 perfluorocarbon (PFC) 을이용한 perfluorocarbon-based oxygen carrier (PBOC) 와적혈구내의혈색소를이용한 hemoglobin-based oxygen carrier (HBOC) 두종류가대표적이다 (Fig. 1). 1) Perfluorocarbon-based oxygen carrier (PBOC) Perfluorocarbon (PFC) 은 carbon과 fluorine의혼합물로서인체에무해하고가스용해도가높은합성유기물이다. PBOC 중가장대표적인상품은 1978년개발된 Fluosol-DA (Green Cross Co., Osaka, Japan) 라는제품으로임상실험이진행되었지만혈소판감소증, 감기유사증세, 뇌졸증등의부작용과투여시고압의산소가필요한점등사용상의불편함으로인하여 1994년생산이중단되었다. 그후같은제형으로 PFC를 lecithin으로둘러 싸서좀더안정적인제 3 세대 PBOC 로개발된 Oxycyte (Oxygen Biotherapeutics, Inc., Costa Mesa, CA, USA) 는혈색소를변형해서만든제품들에비해재료공급에제한이없으며가열로소독할수있는장점으로수술중수혈량을줄이려는목적으로 1992년에개발되었으나뇌졸중유발로임상시험이중단되어, 현재 PBOC 제품은모든임상시험이중단된상태이다. 5) 2) Hemoglobin-based oxygen carrier (HBOC) 최초의 HBOC는적혈구를깨서만든유리혈색소 (free hemoglobin) 였는데, 반감기가짧고, 자유혈색소로인한혈관수축작용이심하고, 산소라디칼의생성으로신독성과고혈압을유발하였다. 이는혈색소분자가 nitric oxide (NO) 분자와결합하여혈관확장및혈소판활성억제기능을저해하기때문인것으로보고있다. 이후 nanomedical technology 기술이접목되면서혈색소내글로빈을여러개연결한혈색소중합체의형태로 Hemopure R (Biopure Inc., Cambridge, MA, USA) 와 polyheme R (Northfield Laboratories Inc., Evanston, IL, USA) 등이개발소개되었으며이를좀더안정화시키기위해 liposome 형태로발전시키는연구를진행하였으나너무크기가작아체내에머무르는시간이짧고, 계속적인유리산소 Fig. 1. Comparative size of RBC, perfluorocarbon, and hemoglobin-based oxygen carriers. (A) Emulsion of perfluorocarbon. (B) PolyHb can maintain the patient for 12 h after reaching the hospital. (C) The 3rd generation modified liposome-encapsulated hemoglobin

4 Korean J Blood Transfus Vol. 27, No. 3, , Dec 의장기독성등으로다양한부작용을유발하고특히심장마비등가장치명적인사망사고등이발생하여현재로서는거의사용되지못하고있다. 4) 그러나혈액대체물질개발은기업들이경험한실패에도불구하고생산가가저렴하고, 실온이나냉장온도에서 1년이상보관이용이하고, 혈액형에상관없이사용될수있다는점등의장점으로이분야의연구는계속진행될전망이다. 2. 줄기세포로부터의적혈구생산 줄기세포기반인공혈액생산 기술개발은인공혈액개발의시급성을알고있는수혈의학전문가이면서줄기세포연구를하고있는연구자가진행할수있는연구로서각나라에서소수연구자에의해진행되고있다. 현재대표적인국외연구는미국국방과학연구소 (DARPA), 일본의리켄연구소와일본적십자혈액원, 영국은웰컴트러스트사와에딘버러대학의공동연구, 프랑스의혈액수혈연구소등에서진행되고있다. 최근중국에서도줄기세포기반적혈구생산관련한연구를간헐적으로보고하고있으나아직까지는뚜렷한연구성과는없다. 2000년부터시작된줄기세포기반의인공혈액생산기술은세계각국이원천기술선점을위한초기단계연구는완료되었다. 그러나시험관내소량생산의성공이며, 수혈의혈액량확보를위해반드시필요한대량생산을위한증식및배양기술은현재극복하지못한상태이며, 대량의초기세포원을확보하기위해최근에는역분화줄기세포를사용하여적혈구를생산하고자하는연구로이동하고있다. 1) 골수, 제대혈 CD34+ 세포로부터의적혈구생산 2002년프랑스연구팀인 Neildez-Nguyen 등 6) 은제대혈 CD34+ 세포로부터적혈구를만들어보 고하였으며이는줄기세포기반적혈구생산연구로최초의보고이다. 이때 CD34+ 세포로부터체외배양된적혈구는정상적혈구와비슷한혈색소농도, 모양, 수명을지녔지만, 탈핵이되지않아마지막탈핵과정은 NOD/SCID mouse 생체에서시키는과정을거쳐야만했다. 그후 2005년에같은연구팀에서체외에서쥐의중간엽줄기세포와공배양을통해체외배양에서만탈핵을시키는성과를보고함으로써 7) 이배양방식을기반으로적혈구생산연구가시작되었다. 이때세포원은제대혈, G-CSF로가동화된말초조혈모세포농축액등으로부터 CD34+ 세포를분리하여사용하였다. 전세계분쟁지역에군인을파병하고있는미국은미국국방과학연구소에서 Blood Pharming Project 를 2007년에시작하였고, 줄기세포로부터인공혈액개발을 Arteriocytes사 (Cleveland, OH, USA) 에위탁하여공동연구를진행하였으며, 8) 적혈구시제품을 2013년에출시할예정이라고보고한바있으나아직그시제품은출시되지않고있다. 한편일본에서도줄기세포로부터적혈구생산하는연구를 2006년도에시작하였으며, 9) Fujimi 등 10) 이골수에서성숙된적혈구아세포가대식세포주변에많이모여있다는사실에착안하여 in vitro 조성에서대식세포와의공배양을통해탈핵율을높인연구보고도많은관심을받았다. 그러나최근일본에서는역분화줄기세포로부터조혈모세포주를개발하면서이를기본으로하여적혈구분화기술의기반연구에중점을두고있다. 국내에서는 Baek 등 11,12) 의연구팀이이분야의유일한연구팀이며, 제대혈로부터적혈구생산기술을완성하여발표한바있다. 13) 프랑스연구팀은 2011년자가백혈구성분채집으로부터얻은 CD34+ 세포로부터 GMP 시설에서적혈구를생산하여 HbA 88%, HbF 10.7% 인배양혈액 2 ml 를인체내에성공적으로수혈한사례를처음보

5 김현옥 : 줄기세포기반수혈용적혈구생산 고함으로써줄기세포기반으로생산된적혈구가수혈용혈액으로적합함을보여주었다. 14) 줄기세포기반초기연구진행과정을 Table 1에요약하였다. 15,16) CD34+ 세포로부터적혈구분화기술은연구자마다조금다른배양분화방법을소개하고있으나첫번째단계는 CD34+ 세포를조혈모세포증폭기본배지에 stem cell factor, IL-3, erythropoietin의사이토카인을넣어다양한혈액세포로분화가가능한적아구세포로증폭시키는단계 (7일) 이고, 2단계는적혈구로계속분화시키는과정 (7일) 이며, 3단계에서는이를가속화시키면서핵을탈핵시키는과정이된다. CD34+ 세포로부터시작하여성숙적혈구까지분화시키는데는약 21일정도소요된다. 본저자의연구실에서사용하는 RBC 생산 protocol은 Fig. 2에요약하였다. 13) 그러 나제대혈이나말초혈액으로부터수혈용적혈구를생산하기에는성체줄기세포로의증폭의한계와생산비용에대한지적이시작되었다. 즉, 적혈구농축액한단위에는 개의적혈구가들어있다. 골수 1 ml에는약 개의유핵세포가들어있으며, 이중 CD34+ 세포는 1.5% 이하이기때문에 500 ml의골수를채집하면이안에 CD34+ 세포는 개가있다. 말초혈액에는이보다조혈모세포가훨씬적고, 제대혈인경우라하더라도제대혈한단위용량이보통 ml이므로유핵세포가약 개포함되어있고, CD34+ 세포가약 2% 정도라고가정하면 개정도의조혈모세포를얻을수있게된다. 즉제대혈한단위로부터적혈구농축액한단위에들어있는적혈구의개수를만들기위해서는수백만배까지의증폭이 Table 1. Chronological history of in vitro RBC generation from CD34+ cells Year Reference Cell source Significant outcome 2002 Neildez-Nguyen et al. 6) CB CD34+ First trial of RBC production from cord blood CD34+ cells. Final RBC maturation in NOD/SCID mouse 2005 Giarratana et al. 7) CB CD34+ Adult CD34+ Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells 2006 Miharada et al. 9) CB CD34+ Development a method to produce enucleated RBCs efficiently in the absence of feeder cells Fujimi et al. 10) CB CD34+ Generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages 2008 Baek et al. 11) CB CD34+ The first report on generating clinical-grade RBCs by in vitro culture with human MSCs 2009 Boehm et al. 15) Human PB CD34+ RBC production using peripheral blood 2010 Baek et al. 12) CB CD34+ Enhanced enucleation of red blood cells in suspension by electrostatic interactions with culture plates 2011 Timmins et al. 16) CB CD34+ Large-scale approaches in RBC production using agitated bioreactor systems 2011 Giarratana et al. 14) Leukapheresis CD34+ First clinical trial in transfusion using cultured RBC generated from stem cells under good manufacturing practice conditions Abbreviations: CB, cord blood; PB, peripheral blood

6 Korean J Blood Transfus Vol. 27, No. 3, , Dec Fig. 2. Protocol of RBC production from cord blood-derived CD34+ cells. Abbreviations: HC, hydrocortisone; SCF, stem cell factor; EPO, erythropoietin; F68, poloxamer (reference 13 with permission). 필요한데이것이체외배양으로가능하지않았을뿐아니라사용해야하는배지량이계산상으로 1,000 L가필요하므로헌혈로얻는혈액과비교할때생산비용에경쟁력이없다는결론에도달하게되었다 ) 물론 bioreactor를통한대량생산도시도하여보았으나이역시좋은결과를얻지못하였다. 16) 따라서연구자들이새로운세포원을생각하게되었으며배아줄기세포또는역분화줄기세포를새로운시작세포원으로하여적혈구를생산하는기술로연구방향의전환점을맞게되었다. 2) 배아줄기세포와역분화줄기세포로부터적혈구분화생산기술개발줄기세포기반적혈구생산이경제성을얻기위해서는세포원을배아줄기세포나역분화줄기세포로부터시작해야한다는것이각국가연구자의공통된견해로 2010년경부터줄기세포원이제대혈이나말초혈액으로부터만능줄기세포로 전환되었다. 2008년일본 Hiroyama 등 20) 이 mouse 배아줄기세포로부터성숙적혈구를생산한연구를처음보고하였으며그이후에는인간배아줄기세포와인간역분화줄기세포로부터적혈구를생산하는연구가전세계적으로동시다발적으로보고되기시작하였다 (Table 2). 19) 또한이와동시에모든혈액을줄기세포로부터만들어진적혈구로대체하기보다는일부특수한상황에서의혈액시장을대체할수있는최종목표를갖게되었다. 즉만능공혈혈액인 O형 D 음성헌혈자로부터얻은혈액으로부터역분화줄기세포를얻어이를만능공혈혈액으로분화시키고, 희귀혈액형및복합항체보유자로쉽게적합한혈액을찾을수없는혈액수급제한환자를위해자가역분화줄기세포로부터적혈구를생산하고자하는기술에연구가집중되었다. 미국 NIH에등록된배아줄기세포주에는만능공혈자인 O형 D 음성혈액형인세포주가포함되

7 김현옥 : 줄기세포기반수혈용적혈구생산 Table 2. Chronological history of in vitro RBC generation from human ESCs and human ipscs Year Reference Cell source Significant outcome 2008 Lu et al. hesc First trial of differentiation mature hescs into functional oxygen-carrying erythrocytes on a large scale Lapillonne et al. hesc, hipscs First time the complete differentiation of human induced pluripotent stem cells into definitive erythrocytes capable of maturation up to enucleated red blood cells 2011 Chang et al. hipscs Production of fetal like RBCs(HbF) using hipscs produced from three somatic cell types 2011 Dias et al. hesc. hipscs Demonstrate the feasibility of large-scale production of erythroid cells from fibroblast-derived hipscs 2012 Kobari et al. hipscs Complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation from hipscs Abbreviations: hesc, human embryonic stem cell; hipsc, human induced pluripotent stem cell. 어있지않다고지적된바있다. 21) 즉제대혈이나배아줄기세포는미리혈액형을알고공여받는것이아니고무작위로받고특히배아줄기세포주를제조하기까지는많은윤리적인지적이있기때문에이를얻기에는어려움이있음을뒷받침할수있는자료이기도하다. 특히복합항체에의한동종면역이나희귀혈액을가진사람에대한적합한적혈구를생산해내기위한세포원을구하는것은거의불가능하다. 이런면에서역분화줄기세포인경우공여자가자신일수도있으며, 쉽게만능공혈자로부터체세포 ( 섬유모세포, 혈액, 소변등 ) 를얻을수있는장점이있다. 연구초기에역분화줄기세포를제작할바이러스벡터를사용하는경우바이러스의문제점과그수율의문제점이많이지적되었으나최근에는배양선택메디아의발전과바이러스를사용하지않고도 episomal plasmid를이용하거나 mrna 전달을통한역분화줄기세포를제작하는방법이소개되어보다쉽게역분화줄기세포를제작하는단계까지발전하였다. 17) 한편적혈구분화기술은줄기세포단계에서발 생하는유전성혈액질환에도적용할수있어 2015년, 미국존스홉킨스연구진은겸상적혈구질환자의혈액으로부터얻은단핵세포로부터유도한역분화줄기세포에서돌연변이가있는 -hemoglobin 유전자를정상으로치환한후, 이세포로부터정상적인적혈구가생성됨으로확인하여보고하였다. 22) 그러나이환자에서유전자조작으로치환된정상적혈구로분화시킨적혈구사진은아직도핵이있는정염성정적아구 (orthochromic normoblast) 가있어이를직접수혈할수있는성숙한적혈구까지완벽하게분화에성공하지못한기술이지만난치성유전성혈액질환에서병인규명과함께새로운치료기술을제안할수있는큰성과로평가받고있다. 줄기세포기반적혈구생산에관한연구로가장최근에보고된참고문헌으로 Dowey 등 17) 이발표한 plasmid vector를사용하여말초혈액으로부터 CD34+ 세포의분리과정없이역분화줄기세포를만드는연구논문과혈액에서배아줄기세포나역분화줄기세포를제작할때공배양세포없이, GMP grade로적혈구로분화시키는과정에서무혈청배양배지를

8 Korean J Blood Transfus Vol. 27, No. 3, , Dec 사용하여효율높은적혈구를생산을보고한 Olivier 등 23) 의논문을추천한다. 3. 적혈구생산을위한역분화줄기세포주은행구축 2006년 8월에일본교토대학의신야야마나카박사는생쥐의피부세포를 Oct4, Sox2, Klf4, c-myc 유전자들로역분화시켜만능분화능을가진역분화줄기세포를만드는데성공했고그다음해에는인간역분화줄기세포도제작하여 2012년에노벨생리의학상을수상하였다. 24) 역분화줄기세포는만능줄기세포로서배아줄기세포와는달리생명에대한윤리적인논란을불러일으키지않기때문에각종질병의병인연구, 신약발굴, 줄기세포기반치료제개발등차세대세포치료제로서가장각광받는세포가되었다. 현재역분화줄기세포유래세포모델은심장질환에서정신분열증까지다양한질병연구에사용되고있음이이를뒷받침하고있다. 줄기세포기반인공혈액을생산하려는연구도위에서언급한이유로조혈모세포보다는역분화줄기세포를세포원으로사용하여연 구가활발히진행되고있다 (Fig. 3). 18,19) 이러는가운데프랑스의 Douay 연구팀은동종면역환자나희귀혈액형을가진사람을위한역분화줄기세포주은행을만드는것을제안하였다. 25) 프랑스에서 10년간 16,486명의동종면역환자를대상으로분석한결과단 3명의역분화줄기세포주를확보하면이런환자의 99% 까지적합한혈액공급이가능하다는자료와함께프랑스의희귀혈액형보유환자를대상으로할때 15명의 O형희귀혈액형을갖는정상인으로부터역분화줄기세포를제작하는경우거의 100% 까지적합한혈액을공급할수있음을계산상으로보여주었다. 물론지금까지의기술이역분화줄기세포로부터완전한수혈용적혈구를생산하기위해서는대량생산, 탈핵문제, 안전성과생산비문제등많은문제점이남아있다. 그러나최종산물인적혈구는 HLA 문제가없어다른질환에서의역분화줄기세포적용보다줄기세포기반인공혈액생산은가장빠르고안전하게사업화및임상적용이가능한세포치료제로개발될수있다. 따라서 O형 D 음성인공혈액생산을위해국내에서도역분화줄기세포 Fig. 3. Diagram representing feeder-free and serum-free erythroid differentiation of human pluripotent stem cells. Abbreviations: Meso, mesoderm; HA, hemangioblast; CMP, common myeloid progenitor; MEP, megakaryocyteerythroid progenitor; BFU-E, burst forming unit-erythroid; CFU-E, colony forming unit-erythroid; ProE, proerythroblast; BasoE, basophilic erythroblast; PolyE, polychromatophilic erythroblast; OrthoE, orthochromatic erythroblast; Retic, reticulocyte

9 김현옥 : 줄기세포기반수혈용적혈구생산 주은행설립에대해외국의연구사례를벤치마킹할필요가있다. 결론국민의건강과직결되는안전한혈액을안정적으로공급하고자하는노력은국가전략사업의하나이며, 혈액자원은시간이갈수록점차부족해지고있어인공혈액의시장성은매우큰미래의신사업으로받아들여지고있다. 이에 2000 년중반부터가속화되기시작한줄기세포기반수혈용적혈구생산기술의개발은약 10년의짧은역사이지만아주빠르게발전하고있다. 제대혈이나말초혈액의 CD34+ 세포로부터성숙적혈구를만드는기술로시작하였고대량생산의한계와고가의생산비용이문제가되면서배아줄기세포와역분화줄기세포로부터적혈구를생산하고자하는기술로세포원이확대되었다. 즉모든수혈혈액을대체하겠다는막연한기대로부터가능한최대의기술개발범위내에서희귀혈액형보유자나적합한혈액을쉽게찾기어려운동종면역환자, 그리고 O형 D음성만능공혈혈액을생산하고자하는연구의목표도더욱뚜렷해졌다. 배양방법및분화기술도발전하면서만능줄기세포로부터성숙적혈구까지의생산에사용되는무혈청배지가개발되고기질세포가없이도적혈구를만드는연구도가시적인결과를보여주고있다. 단줄기세포기반인공혈액을임상에적용을하기위해서는대량생산이중요한기술이며, 탈핵에도좀더많은연구가필요할것이다. 그러나인공혈액개발에대해전세계적으로경쟁이본격화되고있으며기존의화학물질기반대체인공혈액의임상시험이모두중단되어세포기반의혈액대체물질개발이현재로서는유일한대안이다. 또한혈액은수출수입이안되는생물자원이 므로줄기세포기반인공혈액생산기술에있어국내자체기술개발만이제조기술에대한자율권확보임을생각할때혈액대체물질개발은단순히한기업및연구기관이해결해야할과제가아니라, 원천기술개발을위한국가장기프로젝트로지원되야할연구개발주제이며, 국내연구자들의관심과참여가그어느때보다절실히필요한시기이기도하다. 요약수혈은적혈구, 백혈구, 혈소판등살아있는세포가환자에게주입된다는점에서인류역사에서가장오래된세포치료제중의하나로평가될수있다. 그러나혈액은헌혈로서만충당할수있는제제로최근에는혈액부족현상이발생하고있으며, 특히수혈로인한감염전파문제는새롭게계속나타나는신종바이러스전염병이보고될때마다수혈의위험성이강조되고있다. 이런문제를극복하기위한인공혈액에대한연구는 1960 년대중반부터시작되었다. 산소를운반하는기능인헤모글로빈으로구성된혈색소기반산소운반체와과불화탄소 (perfluorocarbon) 를이용한산소운반체가개발되었으나임상시험진행중사망에이르는다양한부작용이보고되면서개발이거의중단되었다. 이후줄기세포가새로운의료의치료패러다임으로소개되면서줄기세포를이용하여수혈용적혈구를생산하고자하는연구가진행되었고제대혈과 G-CSF 가동말초조혈모세포로부터 CD34+ 세포를분리하여적혈구를생산하는 1단계프로토콜은완성되었다. 그러나수혈용으로사용하기위해서는대량생산및고가의생산비용이필요한점등모든헌혈혈액을대체하기에는역부족인점이지적되면서만능줄기세포인배야줄기세포나역분화줄기세포로부터적

10 Korean J Blood Transfus Vol. 27, No. 3, , Dec 혈구를생산하는연구로전환하게되었다. 두만능줄기세포로부터만들어지는적혈구의수혈대상은첫째, 희귀혈액형보유자나복합항체보유로적합한혈액을쉽게찾을수없는환자에대한자가혈액생산과둘째 O형 D음성혈액형을가진만능공혈자로부터얻은말초혈액에서역분화줄기세포를제작하고여기서부터적혈구로분화시키는기술개발로만능인공혈액을생산하는연구까지진행되고있다. 물론아직까지완벽한수혈용적혈구의생산단계에는이르지못했으나앞으로역분화줄기세포로확대된줄기세포기반적혈구생산은생각보다빠르게발전할것이며, 안전성과가격문제가해결된다면, 수년내에그결과를기대할수있을것이다. References 1. WHO. Blood safety and availability. int/mediacentre/factsheets/fs279/en/ [Online] (last visited on 25 November 2016). 2. FDA. Fatalities reported to FDA following blood collection and transfusion annual summary for fiscal year BiologicsBloodVaccines/SafetyAvailability/Re portaproblem/transfusiondonationfatalities/ UCM pdfFDA [Online] (last visited on 25 November 2016). 3. Park KU, Kwon S, Kin SW, Lim YA. Long term prospects for the blood supply and demand. Korean J Blood Transfus 2006;17: Chang TMS. From hemoglobin based oxygen carrier to oxygen therapeutics, blood substitutes, nanomedicine and artificial cells. In: Kim HW, Greenburg AG. Hemoglobin-based oxygen carriers as red cell substitutes and oxygen therapeutics. Heidelberg: Springer Berlin Heidelberg 2013: Castro CI, Briceno JC. Perfluorocarbon-based oxygen carriers: review of products and trials. Artif Organs 2010;34: Neildez-Nguyen TM, Wajcman H, Marden MC, Bensidhoum M, Moncollin V, Giarratana MC, et al. Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo. Nat Biotechnol 2002;20: Giarratana MC, Kobari L, Lapillonne H, Chalmers D, Kiger L, Cynober T, et al. Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nat Biotechnol 2005;23: Business Wire. Arteriocyte awarded DARPA funding for company s blood pharming technology /en/ [Online] (last visited on 25 November 2016). 9. Miharada K, Hiroyama T, Sudo K, Nagasawa T, Nakamura Y. Efficient enucleation of erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells. Nat Biotechnol 2006;24: Fujimi A, Matsunaga T, Kobune M, Kawano Y, Nagaya T, Tanaka I, et al. Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages. Int J Hematol 2008;87: Baek EJ, Kim HS, Kim S, Jin H, Choi TY, Kim HO. In vitro clinical-grade generation of red blood cells from human umbilical cord blood CD34 + cells. Transfusion 2008;48: Baek EJ, You J, Kim MS, Lee SY, Cho SJ, Kim E, et al. Enhanced production of red blood cells in suspension by electrostatic interactions with culture plates. Tissue Eng Part C Methods 2010;16: Kim HO. In-vitro stem cell derived red blood cells for transfusion: are we there yet? Yonsei Med J 2014;55: Giarratana MC, Rouard H, Dumont A, Kiger L, Safeukui I, Le Pennec PY, et al. Proof of

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untitled 대한수혈학회지 : 제 26 권제 1 호, 201 The Korean Journal of Blood Transfusion Vol. 26, No. 1, 18-2, April 201 http://dx.doi.org/10.1794/kjbt.201.26.1.18 pissn 1226-9336 eissn 2383-6881 Original Article 조혈모세포로부터적혈구분화에사용되는무혈청배지의성능비교