Hyo-Soo Kim, MD/PhD Cardiovascular Center, Seoul National University Hospital · 2005. 3. 31. ·...
Transcript of Hyo-Soo Kim, MD/PhD Cardiovascular Center, Seoul National University Hospital · 2005. 3. 31. ·...
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Therapeutic Vasculogenesis using Stem Cells :
Hyo-Soo Kim, MD/PhD
Cardiovascular Center,
Seoul National University Hospital
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Content1. Background : Stem cell
: Vasculogenesis
2. State of art “EPC” : Animal experiments
: Clinical applications
3. Problem solving : Mobilization
: Genetic engineering
: Homing
4. Future applications
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Stem cell
• Self-renewal for life-time
• Differentiation to generate specialized cellpluripotent : various types of cells from three germ layersunipotent : generate only one types of cells
• Cells from embryo (embryonic stem cell)fetus (embryonic germ cell)adult (adult stem cell)
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Mechanism of self renewal in stem cell
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Three kinds of stem cell(1) Embryonic stem cells
1.Source : inner cell mans of blastocyst(5 day embryo)2.Pluripotent
(2) Embryonic germ cells1.Source : primordial germ cells of gonadal ridge
of the 5~10 week fetus2.Pluripotent
(3) Adult stem cells1.Sources : bone marrow, blood, eye(cornea, retina)
brain, GI tract, liver, pancreasskeletal muscle, skin, dental pulp
2.pluri~unipotent3.Undifferentiated cells in differentiated adult tissue
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Differentiation potential of stem cells
1) Totipotent (totus in Latin=entire, total)fertilized egg generate all cells and tissues
200 kinds of cells in adult mammals
2) Pluripotent (plures in Latin=several, many)emb stem cell generate all three germ layersemb germ cell endoderm, mesoderm & ectoderm
3) Unipotent (uni in Latin=one)adult stem cell generate one cell type in “normal condition”
(but, in severe damage, adult stem cell may be pluripotent.)
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Differentiation induction of stem cells
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Similarity of adult and embryonic stem cell
1. ability to self-replicateand to generate specialized cells
2. embryonic stem cell differentiated cellsadult stem cell undergo homing
3. same immunologic characteristics
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Differences between embryonic and adult stem cell
embryonic stem cell adult stem cell
1) Source inner cell mass ES cell many adult tissuesfetal gonads EG cell
2) Potentiality pluripotent unipotent or pluripotentgenerate many cell typeof three germ layers
3) Replication replication without differentiation difficult to proliferate in vitrofor many generations without differentiation
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Plasticity of adult stem cell
(1) Some adult stem cell can be genetically reprogrammedto generate specialized cells of different tissues
(2) Adult stem cell : unipotent pluripotent
(3) Hematopoietic stem cellusually generate blood cells (mesodermal)sometimes generate neurons (ectodermal)
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Clonality
Clonally-derived cell or cell to exhibit clonality
: generated by the division of a single cell: genetically identical to mother cell: human pluripotent stem cells - clonal: adult stem cells - clonality(?)
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Progenitor (precursor) cell vs stem cells
(1) Partly specialized (1) not specialized(2) Committed (2) not committed(3) Not self-renewal (3) self renewal(4) Differentiation into more (4) pluripotent
specialized cells(5) Examples
endothelial progenitor cells for EClymphoid progenitor cells for lymphocytes
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Adult Stem Cell[1] Two characteristics
1. self replication throughout life time
2. differentiation to restore damaged tissue
[2] Population ; rare (HSC = 1 in 10000~15000 cells in BM)
[3] Distribution ; dispersed in various tissues
1. bone marrow 2. peripheral blood
3. brain 4. spinal cord
5. blood vessel 6. skeletal muscle
7. liver 8. pancreas
9. epithelium of GI tract 10. Epithelium of skin
11. cornea 12. retina
[4] Origin ; unclear
= ? cells set aside during fetal development and restrained from differentiating
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Adult Stem Cell Plasticity
[1] Plasticity = transdifferentiation, unorthodox differentiation
[2] Adult stem cell can differentiate into different types of cells
with the same primary germ layer or
with the different germ layer
[3] For examples
1. bone marrow stem cell . Cardiac muscle
(mesodermal) . Skeletal muscle
. Liver
(mesodermal)
. Neural tissue
(ectodermal)
2. neural stem cell from adult brain . Hematopoietic cells
(ectodermal) (mesoderamal)
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New therapeutic options for Ischemic Heart DiseasePCI
CABGDrug
IHD
Gene therapy for angiogenesis
• Naked DNA or Adenoviral vector
• VEGF, FGF, HIF1-α
• Phase 1 study ; feasible
• Phase 2 study ; marginal benefit over placebo
Cell therapy for vasculogenesis
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Mechanisms of new vessel formation
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Two possible sources of new vessel formation
Angiogenesis Vasculogenesis
ischemic area
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Vasculogenesis in EmbryoPrimitive vascular
plexus Vascular
remodellingES cell Hemangioblast Blood island
angioblast
hematopoietic stem cell
Blood cells
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Embryo Adult Origin of Endothelial CellTotipotentStem cell(ES cell)
PluripotentStem cell
DeterminedStem cell
CommittedProgenitorcell
PrecursorCell
Differentiatedcell
NeuralStem cell
MesenchymalStem cell
HematopoieticStem cell
LymphoidProgenitor cell
MyeloidProgenitor cell
?
?
EndothelialProgenitor Cell
(EPC)
OsteoblastChondroblast
MyoblastCardiomyoblast
Pre-adipocyteFibroblast
Neurons
Astrocyte
Oligodendrocyte
T-lymphocyte
B-lymphocyte
NK cell
Granulocytes
Monocyte
Erythrocyte
Platelet
Dendritic cell
Endothelial cell OsteocyteChondrocyte
Myocyte
Cardiomyocyte
Adipocyte
Fibroblast
SMC
Hemangioblast
Angioblast
Flk(+) Cell
?
?
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Content1. Background : Stem cell
: Vasculogenesis
2. State of art “EPC” : Animal experiments
: Clinical applications
3. Problem solving : Mobilization
: Genetic engineering
: Homing
4. Future applications
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Discovery of Endothelial Progenitor Cell (EPC)
EM 사진 : 혈관의 상해부위에
집락되는 세포들
Asahara Isner
1) Interesting pattern of endothelial regeneration after denudation injury
• Not migration from the adjacent intact endothelium
• But regenerating islands form in the denuded area
2) Vascular cells and blood cells share
the same origin (hemangioblast) and surface marker (CD34+).
Hypothesis : There is endothelial progenitor cell in the circulating CD34 cells in peripheral blood.
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Hindlimb Ischemia Model of Nude Mouse
Analysis of salvage of ischemic limb
21 days
limb salvage tip necrosis limb necrosishindlimb ischemia
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Laser Doppler perfusion imager : non-invasive evaluation of blood flow
(RU)
1000
Index of neovascularization :
500perfusion of ischemic limb=
perfusion of non-ischemic limb0
decreased blood flow in ischemic limb (4 %)
improved blood flow (59 %)0 % : no flow
100 % : normalChoi, Korean Circulation Society 2002
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Source of EPC (endothelial progenitor cell)number of CD34 (+) cell
in mononuclear cells
autolograftperipheral blood 0.1 ~ 0.2%
amnion
fetusplacenta
cord bloodallograft2 %
autograft, allograft
2 ~ 3 %bone marrow
Murohara, JCI 2000
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국외 현황 신생혈관조성 세포이식 요법
내피세포-전구세포 (Endothelial Progenitor Cell : EPC)의 발견Asahara T, et al. Science. 1997;275:964-967.Isolation of Putative Progenitor Endothelial Cells for Angiogenesis.
EPC의 source Murohara T. TCM 2001;11(8):303-307.Therapeutic Vasculogenesis Using Human Cord Blood-Derived Endothelial Progenitors. •말초혈액 : 0.2% of MNC •제대혈액 : 2% of MNC •골수 : 3% of MNC
동물실험
•EPC (말초혈액, 제대혈액, 골수)•마우스, 래트, 토끼, 돼지의 허혈하지, 허혈심근•정맥 주사, 심강내 주사, 근육 주사 신생혈관 조성 효과
임상시도 (세계 최초)•허혈하지 질환환자에게 골수-유래 세포를 근육주사하여 신생혈관조성을 도모함.•Tateishi-Yuyama E, Matsubara H, Murohara T,, Imaizumi T. Lancet 2002;360:427-35.
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Tateishi-Yuyama E, Matsubara H, Murohara T,, Imaizumi T. Lancet 2002;360:427-35.
Cell processing and centrifuge into 30 ml, total 1×109 cells
Autologous bone marrow aspiration, 500 ~ 700 ml
Intramuscular injection into 40 sites, 0.75 ml each
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Tateishi-Yuyama E, Matsubara H, Murohara T,, Imaizumi T. Lancet 2002;360:427-35.
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saline BM-MNC before
BM-MNC implantation
24 weeks afterBM-MNC
implantationLaser Doppler
perfusion image
knee
upper tibia
beforeBM-MNC
implantation
lower tibia
ankle
28 days afterBM-MNC
implantation
Tateishi-Yuyama E, Matsubara H, Murohara T,, Imaizumi T. Lancet 2002;360:427-35.
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첫째, 추출 가능한 EPC의 양적 부족•말초혈액 : 0.2% of MNC •제대혈액 : 2% of MNC •골수 : 3% of MNC
둘째, EPC의 신생혈관에로의 참여율 저조•1 ~ 5 % of endothelial cells in new vessels
세포이식 요법
한계점
첫째, Mobilization (EPC 의 동원 향상)
둘째, Genetic Engineering (EPC 기능 향상)
셋째, Homing (표적 장기로의 회귀 향상)
즉,
[1] 보다 많은 세포를 골수로부터 말초혈액으로 동원시킨 후,
[2] 이 세포들을 유전적으로 변화시켜 증식능, 분화능을 강화하고,
[3] 강화된 세포들을 표적장기에 선택적으로 회귀시킴.
극복 가능성 모색
세포+유전자 이식 요법
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Content1. Background : Stem cell
: Vasculogenesis
2. State of art “EPC” : Animal experiments
: Clinical applications
3. Problem solving : Mobilization
: Genetic engineering
: Homing
4. Future applications
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3차원적 새 접근법
Mobilization(동원)
Homing(회귀)
Adv-GSK , Ex Vivo modification
골 수 허혈 심근
Genetic Engineering(유전적 변환)
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Aspect 1. Mobilization : previous studies
골수
SDF-1
말초혈액
SDF-1SCFVEGF G-CSFGM-CSF
EPC
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Other GF
GSKMAPK-APK
Dsh
PI-3 K pathway
Integrin
VEGFAspect 2. Genetic modification : previous studies
ECMR
ILK
Akt
Axin/Axil
Wnt pathway
Frz
Ras/Raf P70 S6K
MAPK pathway
sFRP
R R
β-catenin
Survival Proliferation Migration
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In vivo incorporation of EPC in vessels of muscle
Proliferative activity assay of EPC
In vitro incorporation of EPC on HUVEC
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Homing to Ischemic Tissue
혈관주사
근육주사
E-selectin ICAM-1 VCAM-1
VEGF Akt
Aspect 3. Homing : for convenient delivery
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Integration : Mobilization – Genetic Engineering – HomingOptimal protocol for vasculogenesis stem cell therapy
PigMouse, Rat Human
Ischemic heart (MI) model Ischemic heart / limb patientsHind-limb ischemia model
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Content1. Background : Stem cell
: Vasculogenesis
2. State of art “EPC” : Animal experiments
: Clinical applications
3. Problem solving : Mobilization
: Genetic engineering
: Homing
4. Future applications
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Application of EPC to Artificial Vessel
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Application of Stem Cell for Angioplasty
BallooningEC regeneration Foam cell infiltration
Ballooning+
Radiation
Ballooning+
Radiation+
GM-CSF
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Application of Stem Cell for Angioplasty
CD31 CD34대조군
골수 줄기세포
동원 (GM-CSF) 말초 혈중 줄기세포 증가 형광표지된 골수-유래 세포에의한 내피 재생
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Application of Stem Cell for Angioplasty
Peripheral blood
& injured artery
Bone Marrow
GM-CSF
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ContributorsIn cardiovascular laboratory, Clinical Research Institute,
Seoul National University Hospital
Stem cell
최진호
황경국
강현재
조현재
윤창환
허진
장서영
김지현
Gene therapy
김광일
배장환
박경우
김대희
박승정
양한모
조현주
유현정
Animal Experiment
양현주
전수인
Stem cellThree kinds of stem cellDifferentiation potential of stem cellsSimilarity of adult and embryonic stem cellDifferences between embryonic and adult stem cellPlasticity of adult stem cellClonalityAdult Stem CellMechanisms of new vessel formationVasculogenesis in EmbryoDiscovery of Endothelial Progenitor Cell (EPC)Hindlimb Ischemia Model of Nude MouseLaser Doppler perfusion imager : non-invasive evaluation of blood flowSource of EPC (endothelial progenitor cell)