줄기세포와재생의학 (Stem Cells & Regenerative Medicine) Prof. Dong-Wook HAN Department of Nanomedical Engineering, College of Nanoscience & Nanotechnology, Pusan National University
contents 1. 줄기세포의정의 (Definition of Stem cells) 2. 줄기세포의종류 (Type of Stem cells) 3. 줄기세포의확립 (Establishment of Stem Cells) 4. 줄기세포의재생의학적응용 (Application of Stem Cells to Regenerative Medicine)
1. 줄기세포란무엇인가?
줄기세포란? 여러종류의싞체조직으로분열핛수있는능력을 가짂세포, 즉 ' 미분화 ' 세포 ( 분열 분화 ) 특징 : 자가복제, 빠른증식, 세포표면에 indicator ( 분화가가능하다는표식 )
Stem
2. 줄기세포의종류
줄기세포의종류 (1) ( 상실배 ) ( 배반포 ) inner cell mass ( 내부세포 ) 분화능력에따라 Totipotent Pluripotent Multipotent Unipotent
Totipotent ( 전분화능 ) : produce all the differentiated cells Pluripotent ( 만능분화능 ) : differentiate into any of the three germ layers Unipotent ( 단일분화능 ) : differentiate into only one type of tissue/cell type
Unipotent Stem Cells
줄기세포 vs. 전구세포
수정란접합체 배반포 내부세포 낭배
내배엽 / 중배엽 / 외배엽 Endoderm : 갑상선, 흉선, 기관지, 폐, 갂, 췌장등 Mesoderm : 골수, 근육, 결합조직 ( 뼈, 연골, 혈액 ), 싞장, 심장, 혈관계등 Ectoderm : 피부, 싞경, 각막등 Pluripotent cells 는삼배엽으로모두분화가가능
동물의발생 (animal development) 수정 난할 ( 포배기 ) 성체 낭배형성 기관형성 Developmental Stages of a Frog
Mouse embryo 의 cleavage 단계 2-cell stage 4-cell stage Early 8-cell stage Compacted 8-cell stage Morula Blastocyst
줄기세포의종류 (2) 추출시점에따라 배아줄기세포 (embryonic SCs) 성체줄기세포 (adult SCs) 제대혈줄기세포 (cord blood SCs)
(1) 배아줄기세포 (Embryonic Stem Cells, ESCs) 포배기 ( 수정후 4-5 일 ), inner cell mass 형성 pluripotency 50 ~ 150여개의세포로구성 이후, 200여개이상의세포로분화가능
(1) 배아줄기세포 (Embryonic Stem Cells, ESCs)
( 난할, 포배기 ) ( 배반포 ) ( 내부세포 ) Human Blastocyst Showing Inner Cell Mass & Trophectoderm ( 할구세포 )
Embryonic Stem Cells A human embryo in the blastocyst stage of development Embryoid Bodies In Vivo In Vitro
배아줄기세포 (ESCs) 의문제점 윢리적논란 : 배아의파괴 특별핚싞호메커니즘및조절능력부재 기형종 (Teratoma) 의발생
(2) 성체줄기세포 (Adult Stem Cells, ASCs) 분화가끝난성체조직에존재 ( 뇌 ) 골수, 중갂엽, 뇌 ( 중추싞경계 ) ( 골수 ) 표피층, 근육, 싞경등으로분화
Bone Marrow Stem Cells Mesenchymal Stem Cells Mesenchymal stem cells are found arrayed around the central sinus in the bone marrow (also called marrow stromal cells). Bone Marrow Hematopoietic Stem Cells Hematopoietic stem cells give rise to the three classes of blood cells that are found in the circulation: white blood cells (leukocytes), red blood cells (erythrocytes), and platelets (thrombocytes).
중간엽줄기세포 (Mesenchymal SCs, MSCs) MSC Proliferation Osteogenesis Chondrogenesis Myogenesis Marrow stroma Tendogenesis/ Ligamentagenesis Other Transitory Osteoblasts Transitory Chondrocytes Myoblasts Transitory Stroma Cell Transitory Fibroblast Osteoblasts Chondrocytes Myoblast Fusion ECM Unique Micro-niche Osteocyte Hypertrophic Chondrocyte Myotube Stromal Cells BONE CARTILAGE MUSCLE MARROW T/L Fibroblast TENDON/ LIGAMENT Adipocytes, Dermal and other cells CONNECTIVE TISSUE
지방 - 유래줄기세포 (Adipose-Derived SCs, ADSCs) Epidermis Dermis Liposuction aspiration Adipocyte Ficoll density centrifugation Subcutaneous Fat Erythrocyte ADSC Osteoblasts Chondrocytes Cardiomyocytes Differentiation inducing Factor Shaking Centrifuge Filtration Adipocytes Endothelial cells Neural cells Multi-lineage differentiation Expansion ADSC
치아줄기세포 (Dental SCs, DSCs) Tooth Enamel Dentin Stem Cell Source Baby teeth (age 6-12) Wisdom teeth (age 16-20) Permanent teeth (over age 20) Stem Cells DPSC, PDLSC, DP/GSC DPSC, DP/GSC DPSC, PDLSC Pulp Periodontal Ligament Alveolar Bone Dental Stem Cells Dental Pulp Stem Cell: DPSC Periodontal Ligament Stem Cell: PDLSC Dental Papillar/Germ Stem Cell: DP/GSC Multi-Differentiation Osteoblasts Chondrocytes Adipocytes Endothelial cells Neural cells
(3) 제대혈줄기세포 (Cord Blood Stem Cells) 태반 (placenta) 과탯줄 (umbilical cord) 부터나온혈액 홗발핚증식력의조혈줄기세포 (Hematopoietic SCs) 가존재 수량및종류의핚계 현재홗발히연구중
제대혈줄기세포 (Umbilical Cord Blood SCs, UCB SCs) Umbilical Cord Blood Ficoll Density Centrifugation Buffy Coat Layer Mononuclear Cell Removal Adherent Fibroblastoid Spindle Shaped Cells Alizarin Red Staining (Mineralization) Differentiation inducing Factor UCB-SCs Osteogenic Differentiation
성체줄기세포 (ASCs) 의문제점 윢리적인논란에서비교적자유로운이점을가지지만대부분이다분화성 (Multipotent) 능력을지니고있어분화에제핚적 짧은수명 ( 다음그림참고 ) 낮은수득윣 실제임상적용이어려움
3. 줄기세포의확립
줄기세포획득방법 1 체세포핵치홖 2 Cell fusion 3 Cell culture
2N 4 직접 재프로그래밍 (v) 2N 5 체세포역분화 ( 핵추출물 ) 만능성세포의추출물을분화된체세포에도입 미분화마커유전자가재발현 만능성의일부능력만재획득 일차세포에만부분적으로적용 특정전사인자발견
만능유도줄기세포 (Induced Pluripotent Stem Cells, ips Cells) 이미분화된세포들이인위적인역분화 (Dedifferentiation) 과정을통해만능분화능력을가지도록유도된세포 Dedifferentiation 시계를거꾸로돌려분화가다 끝난세포를줄기세포로되돌리 는것
ips 세포의장점 난자와배아가불필요 윢리, 법, 사회 (ELSI, Ethical, Legal, Social Issues) 및생명, 종교적논쟁에서자유로움 홖자의체세포를이용 홖자면역적합형세포치료제개발가능 어떤장기의세포도역분화될수있음 Pluripotent
ips 세포의유도방법 1: 전사인자 2006년일본 Kyoto 대학의 Shinya Yamanaka 교수가세계최초로성공 mouse의섬유아세포 (fibroblasts) 에네가지전사인자 (Oct-4, Sox2, c-myc, Klf4) 적용
세포내전사 / 번역조절기작
ips 세포의유도방법 2: 전사인자 미국 Wisconsin 대학의 James Thomson 그룹 4개의유전자 (Nanog, Lin28, c-myc, Klf4) 이용 Oct-4와 Sox2 유전자가필수적인역핛 다른인자 : 세포상황에따른역분화효윣을결정 아직밝혀지지않은역분화유도인자들또핚상당수가있을것으로예상
ips 세포의유도방법 3: 전사인자 독일막스플랑크분자의학연구소의 Hans R. Schöler 박사연구짂 Oct-4 인자만단독으로인갂싞경세포에주입 인체의모듞세포로분화핛수있는 ips 확립
ips 세포의유도방법비교 S. Yamanaka 교수 (2000) Oct-4 Sox2 c-myc Klf4 내배엽, 중배엽, 외배엽으로분화가잘됨 문제점 : c-myc, Klf4 : 발암유전자동시에 4 가지유전자 : 돌연변이 H. R. Schöler 박사 (2009) Oct-4 만삽입 암, 돌연변이확률감소 배엽으로부터분화시킨세포이므로질병치료에이용가능
ips 세포유도매개체 : viral vector 바이러스를이용핚방법 문제점 Virus : 무작위로삽입, 돌연변이의가능성 c-myc : 분화과정에서종양유발가능성
ips 세포유도매개체 : plasmid 플라스미드 (plasmid) 를이용 : Non-viral Non-integrating episomal vecter 유전자내적인돌연변이 제거불가능
ips 세포유도매개체 : electroporation 전기충격방법 : 4 개의전사인자 (Oct-4, Sox2, c-myc, Klf4) 를직접도입 Liposome 매개방법 : 동일하지만효윣이떨어짐
ips 세포유도매개체 : Selenium, 34 Se 브로콜리식물성분에서찾은천연화합물 셀레늄의농도를적절히조절 역분화유발유전자의주입없이줄기세포로역분화 ( 부산대의대강수경교수팀 : 사람지방세포 줄기세포역분화 ) 모듞세포내에역분화유발유전자가내재 셀레늄이잠자는유전자를깨워홗성화
Stem Cells - summary
4. 재생의학 (Regenerative Medicine)
Prospect of Regenerative Medicine Organ Damage - Disease State - Medical Approach Pharmacol. Management Organ Transplantation Kidney, BM, Liver Heart, Pancreas, etc. 1984 신장이식 1987 국내최초췌장이식 1990 국내최초뇌사자장기이식 1994 골수이식센터 1994 간이식성공 1996 국내최고령자간이식 Regeneration of Organ Cell Therapy By by Stem Cell CELL
1. Labile cells Epithelial, hemopoietic system 2. Stable cells Endocrine glands, endotheliums, liver cells 3. Permanent cells Neurons, cardiac myocytes 조직공학 / 재생의학에적용가능성? Stem Cell!
Artificial Heart Prosthetics Vascular Grafts Dental Implant Artificial Cornea Bioprosthetic Heart Valve Biomedical Engineering & Medical Devices Artificial Skin Substitute Hip Joint Bone Cement
Tissue Engineering Tissue engineering is an interdisciplinary field at the intersection of engineering, and biology and medicine toward the development of biological substitutes that restore, maintain, or improve tissue function or a whole organ. Blood Vessel Tissue Engineering Liver Tissue Engineering Cartilage Tissue Engineering Bone Tissue Engineering Cardiac Tissue Engineering Muscular Tissue Engineering ETC
Tissue Engineered Construct Nature Clinical Practice Rheumatology (2006) 2, 373-382 Skeletal Muscle Cell Epithelial Porous polymer Tube Tissue Engineered Esophagus SEM image Porous PU Scaffold Subcutaneous implantation
Fat Bone Marrow Embryonic Stem Cell Bone Marrow-derived Mesenchymal Stem Cell Umbilical Cord Bloodderived Mesenchymal Stem Cell Adipose Tissue Derived Stem Cell Bone Tissue Engineering Culture System Cells & Osteogenic Culture Condition Biomaterials 3D Bone Construct Bone Regeneration
Why Do We Need Cells in Medicine? Osteoblast for Osteoporosis Chondrocyte for Osteoarthritis & degenerative arthritis Bone marrow for Leukemia & Chemotherapy Nerve cells for Parkinsons & Alzhiemer s disease Heart muscle cells for Heart disease Pancreatic islet cells for Diabetes Etc
Regenerative Medicine
Cell Sources Somatic Cell Somatic Stem Cell Embryonic Stem Cell Autologous Limited expansion Very limited application High proliferation Multipotency Autologus Donor age dependent Limited application Unlimited Self-Renewal Pluripotency Immunogenesity Teratoma Difficulty in derivation Oct4 Primary Isolated Skeletal Muscle Cells Umbilical Blood Stem Cells Human Embryonic Stem Cells
Cells? Somatic Cells (Adult Cells) Osteoblasts Chondrocytes Dermal Fibroblasts Hepatocytes Neurons Odontoblasts Cardiomyocytes Etc Somatic Stem Cells (Adult Stem Cells) Mesenchymal Stem Cells Haematopoietic Stem Cells Umbilical Cord Blood Stem Cells Peripheral Blood Stem Cells Cardiac Stem Cells Adipose Derived Stem Cells Dental Pulp Stem Cells Periodontal Ligament Stem Cells Etc Embryonic Stem Cells
What is Somatic Cell? Cardiomyocyte Neuron Chondrocyte
Somatic Cell in Regenerative Medicine Autologus Chondrocyte Implantation Autologus Artificial Skin Biopsy of healthy cartilage Injection Cultivation of chondrocytes Periosteal Patch
What is Somatic/Adult Stem Cell? Cardiac Stem Cells Hair Follicle Stem Cells Neural Stem Cells
Adult Stem Cell Transplant: Bone Marrow Stem Cells Adult Stem Cell Transplant: Peripheral Blood Stem Cells Umbilical Cord Blood Stem Cell Transplant 1X10 6 Cells/kg of body weight Yield: 2X10 6 Cells from 1 Blood Bag
Bone Marrow Transplantation In the 1950 s, bone marrow transplantation began to use for patients with malignant diseases, and the evidence of multipotent stem cells in bone marrow was later reported in 1968 by Friedenstein and his colleagues by establishing cells that are adherent, clonogenic potential, and fibroblastic
Bone Marrow Transplantation & Stem Cell Therapy
Bone Marrow-derived Mesenchymal Stem Cell
MSC-based Bone Tissue Engineering
MSC-based Cartilage Tissue Engineering
중간엽줄기세포를이용한세포치료법개발및조직공학적방법의기반기술개발연구의예 연골재생 Scaffold 혹은액상고분자 중갂엽줄기세포분리 Scaffold 내세포이동제어기술 3 차원배양및분화유도기술개발 세포치료법개발 조직공학기술
neuronal inducing factors including b- mercaptoethanol, epidermal growth factor, nerve growth factor and brain derived growth factor in DMEM/F12 media
Bone Marrow-derived Hematopoietic Stem Cell
MSC Differentiation Capacity
NATURE VOL 422 24 APRIL 2003 www.nature.com/nature
Diabetes 56:1810 1816, 2007
ips 세포의조직공학적응용 혈관평홗근세포를역분화시켜줄기세포로만듞후다시혈관평홗근세포로분화
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