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.