Stem cell therapy in cardiac practice Dr Sajeer K T Senior resident, Department of Cardiology, Govt. Medical College, Kozhikode.

Background Cardiovascular disease ➔ leading cause of morbidity and mortality worldwide Despite advances in Mx and cath-based therapy for AMI 1-year mortality: 13% 5-year prognosis for patients with HF: 50%. LV systolic dysfunction: major determinant of prognosis associated with significant loss of cardiomyocytes (Irreversible ➔heart post mitotic organ) Abdul M. Mozid et al. British Medical Bulletin 2011; 98: 143–159

Stem cell therapy Clinical trials focused on 3 main situations: Acute MI (with the hope of preventing LVSD) Chronic heart failure secondary to previous MI DCM (non ischemic cardiomyopthy) Areas of discussion: 1. Stem cell types used in cardiac repair 2. Methods of cell delivery in clinical practice 3. Clinical trial evidence to date

Stem cell ? Stem cell a cell with a unique capacity to produce unaltered daughter cells (self-renewal) & to generate specialized cell types (potency) Self-renewal symmetric division: two stem cells two cells destined for differentiation asymmetric division: one stem cell and one differentiating cell

Signature characteristic of the stem cell

Types of stem cells Stem cells : 2 types Somatic stem cells : BM, brain, liver, skeletal & dermal tissue in vitro proliferative ability is limited Pluripotent stem cells : Embryonic stem cells, embryonic germ cells embryonic carcinoma cells, induced pluripotent stem (iPS) Exhibit pluripotency

Traditional view ➲ Current evidence heart is a terminally differentiated post mitotic organ Current evidence: heart is not a terminally differentiated post mitotic organ 4 sources of cells : for new cardiomyocytes after birth 1. Adult cardiomyocytes (mononucleated) 2. Bone marrow–derived cardiac stem or progenitor cells 3. Embryonic epicardium derived cells 4. Niches of cardiac stem or cardiac progenitor cells (CPCs) Stem Cells  2007; 25:589. Cell 2009 138;257–270.

Different cell types investigated for use in cardiac repair Abdul M. Mozid et al. British Medical Bulletin 2011; 98: 143–159

Heart - self-renewing organ Myocyte regeneration occurs throughout organism lifespan Cardiac niches contain stem cells ➔ after activation ➔ give rise to myocytes and vascular structures Piero Anversa, Jan Kajstura, Annarosa Leri. Circulation. 2006;113:1451-1463

Cardiac Stem or Progenitor cells (CSC or CPC) niche of primitive cells present in human heart possess the ability to form cardiomyocytes, endothelial cells (ECs), smooth muscle cells (SMCs) Different classes of CPCs : based on cell surface markers c-Kit+ cells 2. Sca-1+ cells 3. side population cells (SP) 4. cells expressing the protein Islet-1 (transcription factor)

Cardiac progenitor cell classes c-kit positive cell Sca-1 positive cell SP cells Cardiospheres Epicardial progenitors

Hierarchy of CPC growth and differentiation Asymmetrical division of a CSC ➨ a daughter CSC and a daughter cardiac progenitor (CPg) CPg gives rise to - Myocyte progenitor (MPg )& precursor (MPr) - EC progenitor (EPg) & precursor (EPr) - SMC progenitor (SMPg) & precursor (SMPr) Precursors ➨ transient amplifying cells ➨ divide and differentiate into mature myocytes, ECs, and SMCs. CSCs are lineage-negative cells : express only c-kit, MDR1, or Sca-1. Progenitors express stem cell antigens and transcription factors of cardiac cells but do not exhibit specific cytoplasmic proteins Precursors possess stem cell antigens, transcription factors, membrane& cytoplasmic proteins typical of myocytes, ECs, and SMCs Cardiac niches contain quiescent stem cells that undergo asymmetric division, forming a daughter CPC (self-renewal) and a daughter-committed cell. Following activation, daughter-committed cells leave the niche area and give rise to rapidly proliferating transit-amplifying cells (growth and differentiation), which progress into fully mature myocytes, SMCs, and ECs (functional competence). CPCs in the niches are connected structurally and functionally to the supporting cells by gap and adherens junctions made by connexins and cadherins, respectively. Myocytes and fibroblasts act as supporting cells.

Hierarchy of CPC growth and differentiation Cardiac niches contain quiescent stem cells that undergo asymmetric division, forming a daughter CPC (self-renewal) and a daughter-committed cell. Following activation, daughter-committed cells leave the niche area and give rise to rapidly proliferating transit-amplifying cells (growth and differentiation), which progress into fully mature myocytes, SMCs, and ECs (functional competence). CPCs in the niches are connected structurally and functionally to the supporting cells by gap and adherens junctions made by connexins and cadherins, respectively. Myocytes and fibroblasts act as supporting cells.

Cardiac Stem Cells and Myocardial Diseases Role of cardiac stem cells in the advanced stages of HF: - to modulate endogenous CPCs to regenerate cardiac muscle and to create new blood vessel formation Ventricular remodeling ➔ progressive chamber dilation and thinning of the walls Myocardial regeneration ➔ reverse this process ➔ transforming a dilated failing heart into a normal, functionally competent organ

Bone marrow stem cells Bone marrow–derived stem cells: - best studied cells - used in clinical trials in MI and/or idiopathic DCM Bone marrow progenitor cells: - hematopoietic stem cells (HSCs) - side population cells (SP cells) (expression of the Abcg2 transporter and allowing export of Hoechst dye) - mesenchymal stem cells (MSCs) - multipotential adult progenitor cells (MAPCs)

Inducible Pluripotent Stem Cells (iPSCs) Adult stem cells ➔ successfully reprogrammed back to an undifferentiated pluripotent state (by inserting 4 genes: Oct3/4, Sox2, KL4 and c-Myc into differentiated somatic cells) Morphological phenotype of ES cells Same differentiation potential as ES cells ( in vivo and in vitro) - able to form all three germ layers Functioning cardiomyocytes ➔ produced from iPSCs

Inducible Pluripotent Stem Cells (iPSCs) Generation of iPS cells from somatic cells Reprogramming factors ➨ introduced in vitro Established iPS cells ➨ differentiate into various cell types Circulation. 2010;122:80-87

iPS demonstrate pluripotent features Circulation. 2009;120:408-416

Cell types and mode of delivery of cells for cardiac repair A : Cell types used for cardiovascular repair B: Delivery strategies used in the clinical setting for cell therapy EPCs = endothelial progenitor cells iPS cells = induced pluripotent SCs MSCs = mesenchymal stem cells SP cells = side population cells

Summary of cell types used in clinical trials and future perspective Circulation. 2010;121:325-335

Method of delivery of stem cells Intracoronary infusion of cells: - most popular mode of cell delivery - after AMI Intramyocardial injection: - performed in patients with chronic heart failure secondary to IHD - transendocardial injections (catheter-based) - transepicardial injections (during open heart surgery)

Mechanisms of action Progenitor cells: improve functional recovery of infarcted or failing myocardium 1. Direct or indirect improvement of revascularization 2. Paracrine factors released by progenitor cells may inhibit cardiac apoptosis, affect remodeling, or enhance endogenous repair (eg: by tissue-resident PCs) 3. Differentiation into cardiomyocytes may contribute to cardiac regeneration.

Homing of BM-derived stem cells to the myocardium - fate of bone marrow-derived stem cells is determined by the microenvironment that they enter

Mobilization and homing Homing is mediated by : Adhesion Transmigration Invasion

Prerequisite for cell-based therapies Summary of prerequisite for cell-based therapies Circulation. 2010;121:325-335

Stem cell therapy in acute MI

Stem cell therapy in acute myocardial infarction Most of the trials used intracoronary delivery of BMSCs following successful stenting of the infarct-related artery Surrogate markers used to assess efficacy of cell therapy: - Improvements in the LVEF - Reduction in size of scar tissue - Reduction in cardiac volume Post infarction heart failure: - results from ventricular remodeling processes - characterized by progressive expansion of the infarct area and dilation of the LV cavity

Major goal to reverse LV remodeling: - enhancement of regeneration of cardiac myocytes - stimulation of neovascul. within the infarct area 4 main randomized controlled trials (RCTs) - published with positive findings 1. TOPCARE-AMI ( Circulation- 2002) 2. BOOST trial ( Lancet - 2004) 3. REPAIR-AMI trial ( EJM - 2006) 4. FINCELL (Eur Heart - 2008)

Randomized control trials of intracoronary BMSC therapy following acute myocardial infarction

Birgit Assmus et al. Circulation. 2002;106:3009-3017 TOPCARE-AMI :Transplantation of Progenitor Cells and Regeneration Enhancement in AMI Birgit Assmus et al. Circulation. 2002;106:3009-3017

LV Function Assessed by Analysis of LV Angiography in the Cell Therapy Group Demonstrated potential beneficial effect of BSMC therapy following AMI with improvement in the LVEF from 51.6+ 9.6% to 60.1+8.6% at 4 months

Birgit Assmus et al. Circulation. 2002;106:3009-3017 TOPCARE-AMI :Transplantation of Progenitor Cells and Regeneration Enhancement in AMI Left panel (A): LV angiography before CPC therapy Right panel (B): at 4-month follow-up Birgit Assmus et al. Circulation. 2002;106:3009-3017

Birgit Assmus et al. Circulation. 2002;106:3009-3017 TOPCARE-AMI :Transplantation of Progenitor Cells and Regeneration Enhancement in AMI Echocardiographic wall motion score index at rest (initial basal) and during low-dose dobutamine stimulation (initial 10 g dobutamine) at baseline before progenitor cell therapy and at rest at 4-month follow-up (follow-up basal). Birgit Assmus et al. Circulation. 2002;106:3009-3017

Birgit Assmus et al. Circulation. 2002;106:3009-3017 TOPCARE-AMI :Transplantation of Progenitor Cells and Regeneration Enhancement in AMI Birgit Assmus et al. Circulation. 2002;106:3009-3017

BOne marrOw transfer to enhance ST-elevation infarct regeneration (BOOST trial) Lancet 2004; 364: 141–48

BOne marrOw transfer to enhance ST-elevation infarct regeneration (BOOST trial) Lancet 2004; 364: 141–48

BOne marrOw transfer to enhance ST-elevation infarct regeneration (BOOST trial) Lancet 2004; 364: 141–48

BOne marrOw transfer to enhance ST-elevation infarct regeneration (BOOST trial) Lancet 2004; 364: 141–48

BOne marrOw transfer to enhance ST-elevation infarct regeneration (BOOST trial) Representative colour-coded images showing systolic wall motion at baseline and 6 months' follow-up in two patients. Both patients had A/C AWMI. Bright colours: good systolic wall motion. Dark colours : poor wall motion (expressed in mm). Note improved functional recovery in patient treated with BMCs. Lancet 2004; 364: 141–48

Enrollment and outcomes Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial Enrollment and outcomes N Engl J Med 2006;355:1210-21.

Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial N Engl J Med 2006;355:1210-21.

Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial P value Placebo n=92 BMC n=95 N Engl J Med 2006;355:1210-21.

Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial N Engl J Med 2006;355:1210-21.

Interaction between baseline LVEF and the absolute change in LVEF Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial Panel A: Interaction between baseline LVEF and the absolute change in LVEF Panel B: Interaction between the timing of intracoronary infusion of BMC or placebo after reperfusion therapy and the absolute change in LVEF N Engl J Med 2006;355:1210-21.

Reinfusion of Enriched Progenitor Cells and Infarct Remodeling in Acute Myocardial Infarction (REPAIR-AMI) trial Conclusion Intracoronary administration of BMC is associated with improved recovery of left ventricular contractile function in patients with acute myocardial infarction N Engl J Med 2006;355:1210-21.

- FINCELL Investigators Effects of I/C injection of mononuclear BMCs on LV function, arrhythmia risk profile, and restenosis after lytic therapy of AMI - FINCELL Investigators Aim: To assess the efficacy and safety of bone marrow cell (BMC) therapy after thrombolytic therapy of an acute STEMI Methods: Patients with STEMI treated with thrombolysis followed by PCI (2–6 days after STEMI ) Intracoronary BMCs (n= 40) placebo medium (n= 40) (collected & prepared 3–6 h prior PCI) Injected into the infarct artery immediately after stenting European Heart Journal (2008) 29, 2723–2732

- FINCELL Investigators Efficacy assessed by : Effects of I/C injection of mononuclear BMCs on LV function, arrhythmia risk profile, and restenosis after lytic therapy of AMI - FINCELL Investigators Efficacy assessed by : measurement of global LVEF - LV angiography - 2-D echo Safety by measuring arrhythmia risk variables Restenosis of the stented vessel by IVUS At 6 months: BMC group : greater absolute increase of global LVEF than placebo (measured by angiography or 2-D echo) No differences observed between the groups in - adverse clinical events - arrhythmia risk variables - MLD of stented coronary lesion European Heart Journal (2008) 29, 2723–2732

Effects of I/C injection of mononuclear BMCs on LV function, arrhythmia risk profile, and restenosis after lytic therapy of AMI

Effects of I/C injection of mononuclear BMCs on LV function, arrhythmia risk profile, and restenosis after lytic therapy of AMI - FINCELL Investigators Conclusion : Intracoronary BMC therapy is associated with an improvement of global LVEF and neutral effects on arrhythmia risk profile and restenosis of the stented coronary lesions in patients after lytic therapy of STEMI European Heart Journal (2008) 29, 2723–2732

RCTs with neutral findings LEUVEN-AMI study1: - No changes in global LVEF after BMSC infusion ASTAMI trial2 : - No significant effect on the LVEF, LV volumes, or infarct size HEBE trial3 : - No changes in global or regional LV systolic function after BMSC therapy 1Janssens et al. Lancet 2006;367:113–21 2 Lunde K et al. N Eng J Med 2006;355:1199–209 3Alexander Hirsch et al. Eur Heart J 2010

Reasons for the inconsistent findings: 1. Variations in the number of cells delivered 2. Timing of delivery after AMI 3. Differences in the cell isolation protocol

Stem cell therapy in chronic ischemic heart failure

Stem cell therapy in chronic ischemic heart failure Skeletal myoblasts and BMSCs : 2 cell types investigated in chronic heart failure & severely impaired LV function secondary to previous MI Skeletal myoblasts transplantation: : initially investigated in patients undergoing open heart surgery Myoblasts are obtained from culture of a prior muscle biopsy Injected in to epicardium at the time of surgery

Clinical trials of stem cell therapy for chronic ischemic heart failure

1st randomized placebo-controlled study of Myoblast transplantation The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) Trial 1st randomized placebo-controlled study of Myoblast transplantation Patients with LVSD secondary to previous MI and who required CABG Cells ➔ injected into epicardium within scarred areas during open heart surgery Circulation. 2008;117:1189-1200

The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) Trial Circulation. 2008;117:1189-1200

B: Time to first ventricular arrhythmia The Myoblast Autologous Grafting in Ischemic Cardiomyopathy (MAGIC) Trial A : Time to first MACE B: Time to first ventricular arrhythmia study was prematurely stopped no improvement in regional or global LVEF higher number of arrhythmic events Circulation. 2008;117:1189-1200

STAR-heart study The acute and long-term effects of intracoronary Stem cell Transplantation in 191 patients with chronic heARt failure: the STAR-heart study only limited data available on the effectiveness of BMC’s in chronic HF largest study to date of BMSC therapy in chronic ischaemic HF investigated ventricular hemodynamics, geometry, and contractility as well as the long-term clinical outcome of BMC treated patients with reduced LVEF due to chronic ischaemic cardiomyopathy Bodo-Eckehard Strauer et al. European Journal of Heart Failure (2010) 12, 721–729

STAR-heart study

STAR-heart study Bodo-Eckehard Strauer et al. European Journal of Heart Failure (2010) 12, 721–729

EF over time in the BMC group compared with the control group STAR-heart study EF over time in the BMC group compared with the control group Bodo-Eckehard Strauer et al. European Journal of Heart Failure (2010) 12, 721–729

STAR-heart study Effect of BMC therapy on survival in patients with chronic ischaemic cardiomyopathy Bodo-Eckehard Strauer et al. European Journal of Heart Failure (2010) 12, 721–729

STAR-heart study

STAR-heart study-conclusion 5-year follow-up intracoronary BMSC therapy ➔ associated with significant improvement in the LVEF and exercise capacity significant decrease in long-term mortality Bodo-Eckehard Strauer et al. European Journal of Heart Failure (2010) 12, 721–729

dilated cardiomyopathy Stem cell therapy in dilated cardiomyopathy

Stem cell therapy in dilated cardiomyopathy TOPCARE-DCM Transplantation of Progenitor Cells and Functional Regeneration Enhancement Pilot Trial in Patients with Non-ischemic DCM A Pilot Trial : to assess potential effects of selective intracoronary bone marrow– derived progenitor cell infusion in patients with non-ischemic DCM Intracoronary administration of BMCs ➔ shown to improve coronary micro vascular function in IHD Coronary micro-vascular dysfunction is implicated in the pathogenesis and prognosis of noni-schemic DCM Ulrich Fischer-Rasokat et al. Circ Heart Fail. 2009;2:417-423

TOPCARE-DCM Individual changes of the extent of hypokinetic area (A), severity of hypokinesia (B), and ejection fraction (C) between baseline and 3-month follow-up

TOPCARE-DCM Conclusion: Intracoronary administration of BMC seems to be associated with improvements in cardiac contractile and micro vascular function in patients with DCM Individual changes of the adenosine-induced minimal vascular resistance index of the BMC-treated LAD for all 24 patients between baseline and 3-month follow-up

Summary and future directions Past decade has seen an explosion in clinical studies investigating the safety and efficacy of stem cell therapy for heart diseases. Safety of stem cell therapy has been demonstrated uniformly in the vast majority of the studies. Beneficial effects of cell therapy have been demonstrated : AMI, chronic ischaemic HF and DCM. Need for larger RCTs with longer term follow-up assessing morbidity and mortality as primary outcome measures.

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