Therapeutic Vasculogenesis using Stem Cells :

Hyo-Soo Kim, MD/PhD

Cardiovascular Center,

Seoul National University Hospital

Content1. Background : Stem cell

: Vasculogenesis

2. State of art “EPC” : Animal experiments

: Clinical applications

3. Problem solving : Mobilization

: Genetic engineering

: Homing

4. Future applications

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)

Mechanism of self renewal in stem cell

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

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.)

Differentiation induction of stem cells

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

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

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)

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(?)

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

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

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)

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

Mechanisms of new vessel formation

Two possible sources of new vessel formation

Angiogenesis Vasculogenesis

ischemic area

Vasculogenesis in EmbryoPrimitive vascular

plexus Vascular

remodellingES cell Hemangioblast Blood island

angioblast

hematopoietic stem cell

Blood cells

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

?

?

Content1. Background : Stem cell

: Vasculogenesis

2. State of art “EPC” : Animal experiments

: Clinical applications

3. Problem solving : Mobilization

: Genetic engineering

: Homing

4. Future applications

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.

Hindlimb Ischemia Model of Nude Mouse

Analysis of salvage of ischemic limb

21 days

limb salvage tip necrosis limb necrosishindlimb ischemia

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

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

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.

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

Tateishi-Yuyama E, Matsubara H, Murohara T,, Imaizumi T. Lancet 2002;360:427-35.

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.

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

Content1. Background : Stem cell

: Vasculogenesis

2. State of art “EPC” : Animal experiments

: Clinical applications

3. Problem solving : Mobilization

: Genetic engineering

: Homing

4. Future applications

3차원적 새 접근법

Mobilization(동원)

Homing(회귀)

Adv-GSK , Ex Vivo modification

골 수 허혈 심근

Genetic Engineering(유전적 변환)

Aspect 1. Mobilization : previous studies

골수

SDF-1

말초혈액

SDF-1SCFVEGF G-CSFGM-CSF

EPC

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

In vivo incorporation of EPC in vessels of muscle

Proliferative activity assay of EPC

In vitro incorporation of EPC on HUVEC

Homing to Ischemic Tissue

혈관주사

근육주사

E-selectin ICAM-1 VCAM-1

VEGF Akt

Aspect 3. Homing : for convenient delivery

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

Content1. Background : Stem cell

: Vasculogenesis

2. State of art “EPC” : Animal experiments

: Clinical applications

3. Problem solving : Mobilization

: Genetic engineering

: Homing

4. Future applications

Application of EPC to Artificial Vessel

Application of Stem Cell for Angioplasty

BallooningEC regeneration Foam cell infiltration

Ballooning+

Radiation

Ballooning+

Radiation+

GM-CSF

Application of Stem Cell for Angioplasty

CD31 CD34대조군

골수 줄기세포

동원 (GM-CSF) 말초 혈중 줄기세포 증가 형광표지된 골수-유래 세포에의한 내피 재생

Application of Stem Cell for Angioplasty

Peripheral blood

& injured artery

Bone Marrow

GM-CSF

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)