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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
# 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, 1997Pan J, et al., Circ Res, 1997
“Therefore” Loss of cardiomyocyte will result in permanent reduction of number of functioning units in myocardium
“Adult” Cardiomyocytes
Why Cell Therapy for Damaged Heart ?
Classification of Etiology of Heart Failure
Contractile Dysfunction Ischemic heart diseaseCardiomyopathies (dilated)
Pressure Overload Aortic stenosisHypertensionPulmonary hypertension
Volume Overload Aortic regurgitationMitral regurgitationVentricular septal defect
Inadequate Filling AmyloidosisConstrictive pericarditisArrhythmia
Coronary Atherothrombosis & Myocardial Infarction
Myocytes Changes in Heart Failure
Reducedresponsivenessto catecholamines
“Exhausted phase”
Myocyte dropout
Reduced Myofilament Volume
Reduced ATP Supply
“Decreased Contractility”Dilated Cardiomy
opathy
Myocardial Infarction
Contractile Dysfunction
Death
Drug TherapySurgical Procedures
Mechanical Assistant DeviceOrgan Transplantation
Cell Therapy might be an Alternative for a Damaged Heart !!!
Advantages over organ transplantation
1. Transplantation of one cell type 2. Earlier angiogenesis 3. Less traumatic 4. Implantation at a specific site
Cell Source ?
Sufficient Amount ?
Survive ?
Integrate ?
Improve Heart Function ?
Then, what do we consider for Heart Cell Transplantation ?
Transplantation with Non-CMC is O.K. !!!….?
Skeletal myoblast (Satellite cell) Koh GY, et al., J Clin Invest, 1993Taylor DA, et al., Nature Med, 1998Yoon PD, et al., Tex Heart Inst J, 1995Chiu RCJ, et al., Ann Thorac Surg, 1995
Fetal enteric smooth muscle cell Li R-K, et al., J Moll Cell Cardiol, 1999Kumar A, et al., Natl Acad Sci U S A, 1997
Fetal skin fibroblast Sakai T, et al., J Thorac Cardiolvasc Surg, 1999
Koh GY, et al., J Clin Invest, 1993
Skeletal Myoblast (C2C12) Graft in Heart
“Satellite cell”
CMC SMC Fibroblast
LV volume index* 1.00 0.04 0.91 0.32 0.84 0.20Scar thickness index 1.13 0.25 2.10 0.681 2.40 0.861
Scar area index 0.78 0.08 1.27 0.282 1.37 0.343
Transplantation area/scar area** 37.0 8.9 32.2 4.8 51.3 14.64
Comparison of LV Remodelingafter 3-Type-Cell Transplantation
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 ?
TEM analysis offetal CMC grafts
Soonpaa MH, et al., Science, 1994
developed Intercalated Discsbetween host & engrafted fetal CMC
Graft of Fetal Cardiomyocytes from Transgenic Mice
Contraction as early as 7 days after transplantation Long-axis length on day 21 after transplantation
0.60 ± 0.01 cm in relaxed state0.21 ± 0.02 cm in contracting state
Fractional Shortening = 35%
Evaluation of Contractility
Relaxed
FS = 75%
FS = 35%
Green Red
Coculturedneonatal &adult CMC
Green Red Green Red
Rhodamine filterLucifer yellow filter
Electromechanical Coupling ofNeonatal & Adult CMC In Vitro
Reinecke H, et al., Circulation, 1999
Fate of Transplanted Fetal CMC
After transplantation Transplant tissue (mm2) Scar tissue (mm2)
8 wk 20.7 ± 6.9 90.4 ± 25 24 wk 6 ± 6 162 ± 46
Li R-K, et al., Circulation, 1997
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
# 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 syringeCenter / Periphery of infarct zone
: No evidence of rejection at 4 weeks after transplantation
Li R-K, et al., Thorac Cardiovasc Surg, 2000
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 calciumAction 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 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)
Genetically Selected CMC from Differentiating ES Cells
Klug MG, et al., J Clin Invest, 1996
Relative CMC Content in Non-selected, Physically Selected, & G418 Selected Cultures of Differentiating ES cells
Sarcomeric myosin Sarcomeric myosin Percent Preparation positive cells negative cells CMC
No selection 11 2,000 0.55 Physical isolation 68 2,000 3.4 G418 selection 791 3 99.6
Klug MG, et al., J Clin Invest, 1996
G418 Selection of ES-derived CMC In Vitro & Formation of Intracardiac Grafts
Non-selected culture
G418-selected culture
Titin -actin
Desmin Sarcomeric myosin
Dystrophin
Recipient
Donor
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
Makino S, et al., J Clin Invest, 1999
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
Troponin I staining
BM cells in culture before 5-AzacytidineSpindle-like mesenchymal stem cells
BM cells cultured with 5-AzacytidineForming network of myotubules
Tomita S, et al., Circulation, 1999
Tomita S, et al., Circulation, 1999
BrdU-labeled BM transplant inLV free wall scar
Troponin I
Capillary with some RBC
Tomita S, et al., Circulation, 1999
Transplanted BM cellsstimulated angiogenesis !!
MorphologicalAnalysis
LV FunctionAnalysis
Tomita S, et al., Circulation, 1999
5-aza BMC
5-aza BMC
Scar area
LV size
Scar thickness
Derivation & potential applications of BM stroma-derived CMG cell lines
5-Azacytidine
Bone MarrowHematopoietic Cells
Transfect with cDNAexpression libraries toidentify cardiomyocyte
determining genes
Inject into myocardiumfor cell replacement therapy
in patients with cardiomyopathy
Genetically modify forcardiomyoctic cell
replacement therapies
Bone MarrowStromal Cells
ImmortalizedBone Marrow
Stromal Cells (CMG cells)
BeatingCardiomyocytes
Passage4 monthsin culture
Leiden JM, J Clin Invest, 1999
Locally Delivered BM Cells Can Regenerate do novo Myocardium
Orlic D, et al., Nature, 2001
Injection of male Lin-c-kit+
BM cells inperi-infarcted LVof 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
BM cell migration to damaged area
Proliferation & differentiation
Cytoplasmic proteinCardiac myosin
-Sarcomeric actinConnexin 43
Nuclear proteinCsx/Nkx2.5
MEF2GATA-4
Functional competence
Unknown molecular “Signal(s)”
Neovascularization of Ischemic Myocardiumby Systemic Injection of hBM-derived Angioblasts
Kocher AA, et al,. Nature Med 2001IV injection of G-CSF-mobilized CD34+ hBMC
in rat tail
AMI model
Saline CD34+ hBMC
Saline CD34+ hBMCFactor VIII
Human CD34+ Human CD31 Rat CD31
Vasculogenesis Angiogenesis
Regeneration of Ischemic Cardiac Muscle & Vascular Endotheliumby Transplanted Bone Marrow Stem Cells
Jackson KA, et al., J Clin Invest, 2001
Lethal irradiationSP cells (CD34-c-kit+Sca-1+)
marked with LacZ gene
10 weeksafter transplantation
for 60 min
2 or 4 weeks after injury
M
F
(-) controlfor LacZ
(+) controlfor LacZ
-actin
-actin(+) controlfor CD45CD45 (-)
LacZ
Flt-1
ICAM-1
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.