Transcript 슬라이드 1 - Hanyang

Heart
- cardiomyocyte
- vascular endothelial cell
- smooth muscle cell
A heart attack = myocardial infarction
- occurs in nearly 1.1 million Americans each year.
- be the result of hypertension, chronic insufficiency in the blood supply to the heart
muscle caused by coronary artery disease, or a heart attack
- treatment: surgical procedures, mechanical assistance devices, drug therapy, and
organ transplantation
- Researchers are now exploring ways to
save additional lives by using replacement
cells for dead or impaired cells
- There is still no evidence that there are true
stem cells in the heart which can
proliferate and differentiate.
What is the evidence that a stem cell
therapy approach to restoring cardiac
function might work?
- a mouse or rat model of a heart attack to
study new therapies
Why Cell Therapy for Damaged Heart ?
“Adult” Cardiomyocytes
# No potential for regeneration after birth
# No capacity to reenter cell cycle in adult mammalian heart
Rumyantsev PP, Int Rev Cytol, 1977
# Cardiomyocytes respond to mitotic signals by cell hypertrophy
rather than by cell hyperplasia
Kodama H, et al., Circ Res, 1997
Pan J, et al., Circ Res, 1997
“Therefore”
Loss of cardiomyocyte will result in permanent reduction of
number of functioning units in myocardium
Classification of Etiology of Heart Failure
Contractile Dysfunction
Pressure Overload
Volume Overload
Inadequate Filling
Ischemic heart disease
Cardiomyopathies (dilated)
Aortic stenosis
Hypertension
Pulmonary hypertension
Aortic regurgitation
Mitral regurgitation
Ventricular septal defect
Amyloidosis
Constrictive pericarditis
Arrhythmia
Myocytes Changes in Heart Failure
Reduced ATP Supply
Reduced
responsiveness
to catecholamines
Coronary Atherothrombosis &
Myocardial Infarction
Reduced Myofilament Volume
Contractile Dysfunction
“Exhausted phase”
Myocyte dropout
Dilated
Cardiomy
opathy
Myocardial
Infarction
 “Decreased Contractility”
Drug Therapy
Surgical Procedures
Mechanical Assistant Device
Organ Transplantation
Death
Cell Therapy might be an Alternative
for a Damaged Heart !!!
Then, what do we consider for Heart
Cell Transplantation ?
Cell Source ?
Advantages over organ transplantation
Sufficient Amount ?
1. Transplantation of one cell type
2. Earlier angiogenesis
3. Less traumatic
4. Implantation at a specific site
Survive ?
Integrate ?
Improve Heart Function ?
Transplantation with Non-CMC is O.K. !!!….?
Skeletal myoblast (Satellite cell) Koh GY, et al., J Clin Invest, 1993
Taylor DA, et al., Nature Med, 1998
Yoon PD, et al., Tex Heart Inst J, 1995
Chiu RCJ, et al., Ann Thorac Surg, 1995
Fetal enteric smooth muscle cellLi R-K, et al., J Moll Cell Cardiol, 1999
Kumar A, et al., Natl Acad Sci U S A, 1997
Fetal skin fibroblast
Sakai T, et al., J Thorac Cardiolvasc Surg, 1999
Skeletal Myoblast (C2C12) Graft in Heart
“Satellite cell”
Comparison of LV Remodeling
after 3-Type-Cell Transplantation
Koh GY, et al., J Clin Invest, 1993
CMC
LV volume index*
Scar thickness index
Scar area index
Transplantation area/scar area**
SMC
Fibroblast
1.00  0.04
1.13  0.25
0.91  0.32
0.84  0.20
1
2.10  0.68
2.40  0.861
0.78  0.08
1.27  0.282
1.37  0.343
37.0  8.9
32.2  4.8
51.3  14.64
Sakai T, et al., J Thorac Cardiolvasc Surg, 1999
Cardiac -Actin
# Role of Actin
Contractility
Maintenance of cytoskeleton
Cell division / Cell motility
# At least 6 isoforms in higher vertebrates (4 muscular/2 non-muscular)
# Remarkable conservation of muscle actins along with their tissue & developmental specificity
Cardiac -actin-deficient mice
Kumar A, et al., Proc Natl Acad Sci USA, 1997
Low survival to term / Death within 2 weeks after birth
Homozygous mutants with increased expression of vascular smooth muscle
& skeletal -actin : Insufficient to maintain myofibrillar integrity
Rescue with enteric smooth muscle -actin
: Extremely hypodynamic /Considerably enlarged / Hypertrophied
“Therefore” Alteration in actin composition in heart are
associated with severe structural & functional perturbations.
O.K. , Cardiomyocytes may be the Best Donor Cells !
Fetal / Neonatal / Adult ?
Xenogenic / Allogenic / Autologous ?
Graft of Fetal Cardiomyocytes
from Transgenic Mice
Evaluation of Contractility
Relaxed
TEM analysis of
fetal CMC grafts
FS = 75%
Soonpaa MH, et al., Science, 1994
developed Intercalated Discs
between host & engrafted fetal CMC
FS = 35%
Contraction as early as 7 days after transplantation
Long-axis length on day 21 after transplantation
0.60 ± 0.01 cm in relaxed state
0.21 ± 0.02 cm in contracting state
Fractional Shortening = 35%
Electromechanical Coupling of
Neonatal & Adult CMC In Vitro
Fate of Transplanted Fetal CMC
Li R-K, et al., Circulation, 1997
Reinecke H, et al., Circulation, 1999
After transplantation
8 wk
24 wk
Green
Transplant tissue (mm2) Scar tissue (mm2)
20.7 ± 6.9
6±6
90.4 ± 25
162 ± 46
Red
Cocultured
neonatal &
adult CMC
Green
Red
Green
Red
Rhodamine filter Lucifer yellow filter
Lymphocyte infiltration surrounded cardiac tissue formed by
transplanted CMC despite use of cyclosporin A
“Allograft Rejection”
Autologous Cardiomyocyte Transplantation
In Adult Swine Model of Myocardial Infarction
Li R-K, et al., Thorac Cardiovasc Surg, 2000
# 16 adult female Yorkshire swine (controls=8)
# Generation of MI by Intraluminal coil occlusion of d-LAD
# Sampling & culture of CMC by interventricular septal biopsies
# Cell labeling with BrdU to identify transplanted cells
# Cell transplantation after 4-week culture
Cell suspension : 2 ml (107 cells/ml)
Injection with tuberculin syringe
Center / Periphery of infarct zone
: No evidence of rejection at 4 weeks after transplantation
Stem Cells are Versatile: Can Stem Cell Repair a Damaged Heart ?
Generation of CMC from Embryonic Stem Cells
Embryonic stem (ES) cells
Totipotent cell line derived from inner cell mass of blastocysts
Cardiogenic induction during ES differentiation
Appearance of spontaneously and rhythmically contracting myocytes
Expression of - & -MHC,  -tropomyosin, MLC-2v, ANF, & …..
Normal contractile sensitivity to calcium
Action potentials typical for atrial, ventricular, & conduction system CMC
Cell cycle withdrawal & multinucleation
Prerequisite for donor cells
Likely resulting in teratoma formation
 Require generation of essentially pure CMC cultures
Genetically Selected CMC from Differentiating ES Cells
Klug MG, et al., J Clin Invest, 1996
# Genetically modified murine ES cell lines
Transfection with “Fusion gene” (MHC-neor)
-cardiac MHC promoter
cDNA encoding aminoglycoside phophotransferase
Expression of fusion gene in ES-derived CMC
Selected with G418 after in vitro differentiation
# Recipient : Heart of adult dystrophic mice (mdx mice)
Relative CMC Content in Non-selected, Physically Selected, & G418 Selected Cultures
of Differentiating ES cells
Preparation
No selection
Physical isolation
G418 selection
Sarcomeric myosin Sarcomeric myosin
positive cells
negative cells
11
68
791
Percent
CMC
2,000
2,000
0.55
3.4
3
99.6
G418 Selection of ES-derived CMC In Vitro & Formation of Intracardiac
Grafts
Dystrophin
Non-selected culture
G418-selected culture
Recipient
Titin
-actin
Desmin
Sarcomeric myosin
Donor
Klug MG, et al., J Clin Invest, 1996
Generation of CMC from Bone Marrow Stromal Cells
Marrow stromal cells
Pluripotential differentiating into bone, muscle, fat, tendon, or cartilage
Differentiate into CMC ?
5-Azacytidine
Cytosine analogue
Alteration of expression of certain genes that may regulate differentiation
Cardiomyocytes can be generated from marrow stromal cells in vitro
Makino S, et al., J Clin Invest 1999; 103: 697-705
Autologous transplantation of bone marrow cells improve damaged heart function
Tomita S, et al., Circulation 1999; 100[suppl II]: II-247-II-256
Cardiomyogenic Cell (CMG) Before & After 5-Azacytidine Treatment
Phase-contrast photograph
Makino S, et al., J Clin Invest, 1999
Action Potential of CMG Myotubes
a Sinus node-like AP
b Ventricular CMC-like AP
BM cells in culture before
5-Azacytidine
Spindle-like mesenchymal
stem cells
BM cells cultured with
5-Azacytidine
Forming network of
myotubules
Troponin I staining
Makino S, et al., J Clin Invest, 1999
Tomita S, et al., Circulation, 1999
BrdU-labeled BM transplant in
LV free wall scar
Tomita S, et al., Circulation, 1999
Transplanted BM cells
stimulated angiogenesis !!
Capillary with some RBC
Troponin I
Tomita S, et al., Circulation, 1999
LV Function
Analysis
Scar area
Scar thickness
5-aza BMC
Morphological
Analysis
LV size
5-aza BMC
Tomita S, et al., Circulation, 1999
Derivation & potential applications of BM stroma-derived CMG cell lines
Inject into myocardium
for cell replacement therapy
in patients with cardiomyopathy
Genetically modify for
cardiomyoctic cell
replacement therapies
Bone Marrow
Hematopoietic Cells
Passage
4 months
in culture
Bone Marrow
Stromal Cells
5-Azacytidine
Immortalized
Bone Marrow
Stromal Cells (CMG cells)
Transfect with cDNA
expression libraries to
identify cardiomyocyte
determining genes
Beating
Cardiomyocytes
Leiden JM, J Clin Invest, 1999
Locally Delivered BM Cells Can Regenerate do novo Myocardium
Orlic D, et al., Nature, 2001
Injection of male Linc-kit+
BM cells in
peri-infarcted LV
of female mice
Lin-c-kit+
Border Zone
Regenerating Myocardium
Proposed Scheme for Lin-c-kit+ Cell Differentiation in Cardiac Muscle
Orlic D, et al., Nature, 2001
Transplanted BM cells
Infarcted myocardium
Unknown molecular “Signal(s)”
BM cell migration to damaged area
Proliferation & differentiation
Cytoplasmic protein Nuclear protein
Cardiac myosin
-Sarcomeric actin
Connexin 43
Csx/Nkx2.5
MEF2
GATA-4
Functional competence
Neovascularization of Ischemic Myocardium
by Systemic Injection of hBM-derived Angioblasts
Kocher AA, et al,. Nature Med 2001
IV injection of G-CSFmobilized CD34+
hBMC
in rat tail
AMI model
Saline
CD34+ hBMC
Saline
CD34+ hBMC
Factor VIII
Vasculogenesis
Human CD34+
Human CD31
Angiogenesis
Rat CD31
Regeneration of Ischemic Cardiac Muscle & Vascular Endothelium
by Transplanted Bone Marrow Stem Cells
Jackson KA, et al., J Clin Invest, 2001
M
Lethal irradiation
SP cells (CD34-c-kit+Sca-1+)
marked with LacZ gene
(-) control (+) control
for LacZ
for LacZ
F
10 weeks
after transplantation
for 60 min
LacZ
-actin
Flt-1
2 or 4 weeks after injury
ICAM-1
-actin
CD45 (-)
(+) control
for CD45
Conclusions
Adult & embryonic stem cells may be able to replace
damaged heart muscle and establish new blood vessels
to supply them.
Functional role of adult (hematopoietic) stem cell may
be ultimately determined by their migration into heart,
and their exposure to locally generated signals at injured
sites.