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, 1997 Pan 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 DysfunctionIschemic heart disease Cardiomyopathies (dilated) Pressure OverloadAortic stenosis Hypertension Pulmonary hypertension Volume OverloadAortic regurgitation Mitral regurgitation Ventricular septal defect Inadequate FillingAmyloidosis Constrictive pericarditis Arrhythmia
Death Drug Therapy Surgical Procedures Mechanical Assistant Device Organ 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, 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 fibroblastSakai 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 miceKumar 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 of fetal CMC grafts Soonpaa MH, et al., Science, 1994 developed Intercalated Discs between 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 state 0.21 ± 0.02 cm in contracting state Fractional Shortening = 35% Evaluation of Contractility Relaxed FS = 75% FS = 35%
Green Red Cocultured neonatal & adult CMC Green Red Rhodamine filterLucifer yellow filter Electromechanical Coupling of Neonatal & Adult CMC In Vitro Reinecke H, et al., Circulation, 1999 Fate of Transplanted Fetal CMC After transplantationTransplant tissue (mm 2 ) Scar tissue (mm 2 ) 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 (10 7 cells/ml) Injection with tuberculin syringe Center / 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 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 modified murine ES cell lines Transfection with “Fusion gene” (MHC-neo r ) -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 myosinSarcomeric myosinPercent Preparation positive cells negative cells CMC No selection 11 2,000 0.55 Physical isolation 68 2,000 3.4 G418 selection791 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-Azacytidine Spindle-like mesenchymal stem cells BM cells cultured with 5-Azacytidine Forming network of myotubules Tomita S, et al., Circulation, 1999
BrdU-labeled BM transplant in LV free wall scar Troponin I Capillary with some RBC Tomita S, et al., Circulation, 1999 Transplanted BM cells stimulated angiogenesis !!
Morphological Analysis LV Function Analysis Tomita S, et al., Circulation, 1999 5-aza BMC Scar area LV size Scar thickness
Derivation & potential applications of BM stroma-derived CMG cell lines 5-Azacytidine Bone Marrow Hematopoietic Cells Transfect with cDNA expression libraries to identify cardiomyocyte determining genes Inject into myocardium for cell replacement therapy in patients with cardiomyopathy Genetically modify for cardiomyoctic cell replacement therapies Bone Marrow Stromal Cells Immortalized Bone Marrow Stromal Cells (CMG cells) Beating Cardiomyocytes Passage 4 months in 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 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 cellsInfarcted myocardium BM cell migration to damaged area Proliferation & differentiation Cytoplasmic protein Cardiac myosin -Sarcomeric actin Connexin 43 Nuclear protein Csx/Nkx2.5 MEF2 GATA-4 Functional competence Unknown molecular “Signal(s)”
Neovascularization of Ischemic Myocardium by Systemic Injection of hBM-derived Angioblasts Kocher AA, et al,. Nature Med 2001 IV injection of G-CSF- mobilized CD34 + hBMC in rat tail AMI model SalineCD34 + hBMC SalineCD34 + hBMC Factor VIII Human CD34 + Human CD31Rat CD31 Vasculogenesis Angiogenesis
Regeneration of Ischemic Cardiac Muscle & Vascular Endothelium by Transplanted Bone Marrow Stem Cells Jackson KA, et al., J Clin Invest, 2001 Lethal irradiation SP cells (CD34 - c-kit + Sca-1 + ) marked with LacZ gene 10 weeks after transplantation for 60 min 2 or 4 weeks after injury M F (-) control for LacZ (+) control for LacZ -actin (+) control for CD45 CD45 (-) 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.