Intravenously Delivered Mesenchymal Stem Cells: a transformative strategy for cardiac stem cell therapeutics Stephen E. Epstein, MD Dror Luger, PhD Michael J Lipinski MD MedStar Heart and Vascular Institute Washington Hospital Center

Stephen Epstein, MD I have a relevant financial relationship:   I have a relevant financial relationship: Conflict of Interest Chairman, Scientific Advisory Committee, CardioCell Equity holder in CardioCell, LLC

Cellular and cytokine response to AMI

Progressive deterioration in LV function and adverse remodeling post AMI (murine AMI model) LVEF ** LVEDV LVESV

Cellular and cytokine response to AMI Hypothesis: The inflammatory responses of some patients are excessive and persistent, leading to progressive inflammation-induced myocardial dysfunction.

Evidence that inflammation plays an important role in the progressive decrease in LV function occurring following AMI…

Effects of AMI on NK cells in the spleen and myocardium. Splenic NK cells (%) P<0.001 P=0.048 P=0.045 NK cells in the Spleen 7 days post AMI AMI increases NK cells in the spleen and myocardium. NK cells are major orchestrators of inflammation, having effects on many cellular players involved in inflammation. Luger, Lipinski, Epstein et al. 2017

If inflammation plays a key causal role in the progressive myocardial dysfunction that occurs following AMI, then decreasing NK cells—a key marker of inflammation—should prevent the progressive myocardial dysfunction…

Effects of NK cell depletion (via an anti NK cell antibody) on LV infarct size, LV function, and adverse remodeling A LV Infarct (%) Control NK Depletion B Ejection Fraction (%) Baseline Day 7 Day 20 C D NK cell depletion decreases infarct size and improves both LV function and adverse remodeling.

Pathways by which persistent inflammation causes progressive functional deterioration of the myocardium in patients with AMI or with cardiomyopathy Increased interstitial collagen deposition ROS microvascular-induced ischemia Microvascular dysfunction Parenchyma loss Apoptosis replacement fibrosis Inflammatory and immune responses Matrix metalloproteinases Heart Failure reactive fibrosis Decreased energy availability Progressive parenchyma Dysfunctional parenchyma Mitochondrial dysfunction

Implications of the concept that inflammation contributes to progressive myocardial dysfunction…

Immunomodulatory activities of MSCs Aggarwal S, Pittenger MF. Blood. 2005 Feb 15;105(4):1815-22.

The prevailing view of stem cell-mediated mechanisms responsible for cardiac benefit = local myocardial effects: The cells engraft in damaged myocardium, then either– transdifferentiate into functional myocardium, stimulate resident myocardial stem cells to expand, or secrete substances leading to myocardial healing. This perspective implies that the greater the number of engrafted cells in the myocardium the greater the cardiac benefit. Because only a small percentage of intravenously administered stem cells engraft in the myocardium, invasive catheter-based strategies have been uniformly adopted (intracoronary or transendocardial injection).

Given that MSCs secrete, through paracrine mechanisms, multiple cytokines that have anti-inflammatory activities…could the intravenous administration of MSCs, through systemic anti-inflammatory effects, improve cardiac outcomes post AMI and in ischemic CM?

IV administered MSCs decrease NK cells in the spleen Splenic NK cells (%) NK cells in the Spleen P<0.001 P=0.048 P=0.045 7 days post AMI

IV administered MSCs decrease neutrophils in the heart Naïve MI MI+MSCs 7 days post AMI Heart Neutrophils (%) Neutrophils Neutrophils are immediate responders to injury and contribute importantly to adverse myocardial remodeling post MI.

IV administered MSCs improve LV function following AMI = Control = MSC treated Intravenously administered MSCs improve, but not significantly, LV function and adverse remodeling post AMI.

Hypothesis: MSCs improve LV outcomes, but only in mice with marked LV dysfunction caused by large infarcts.

Acute Myocardial Infarction: Effects of MSCs in small vs. large infarcts 21 days post AMI 40 20 60 Ejection Fraction (%) at Day 20 <25% ≥25% Control <25% ≥25% MSC-treated Infarct size Pre-Infarct value P=0.10 P=0.93 EF P=0.03 P=0.46 End Diastolic Volume (µL) at Day 20 100 50 150 <25% ≥25% Control <25% ≥25% MSC-treated Infarct size Pre-Infarct value LVEDV End Systolic Volume (µL) at Day 20 100 50 150 <25% ≥25% Control <25% ≥25% MSC-treated Infarct size Pre-Infarct value P=0.04 P=0.53 LVESV

Mice with Ischemic Cardiomyopathy = Control = MSCs

CardioCell Phase IIa Clinical Trial Safety and Preliminary Efficacy of Intravenous Allogeneic MSCs in Patients With Non-ischemic Heart Failure All subjects MSCs Placebo Day 0 Randomization Day 90 Crossover Day 180 Butler J, Epstein SE, Lipinski MJ, Gheorghiade M, et al. Circ Res. 2017;120:332-340

Significantly improved six minute walk test, IV administered MSCs: Significantly improved six minute walk test, Significantly improved clinical functional assessment, Significantly improved NYHA functional class. In ad-hoc exploratory analyses, there were significant reductions in LVEDV (p=0.04) and LVESV (p=0.02) and increases in LVEF (p=0.02) within the MSC group. No such changes occurred in the placebo group. Importantly, improvement in LVEF was associated with the number of circulating NK cells… Butler J, Epstein SE, Lipinski MJ, Gheorghiade M, et al. Circ Res. 2017;120:332-340

IV administered MSCs decreased blood NK cells. The magnitude of NK cell decrease correlated with the magnitude of the increase in LVEF. The greater the reduction in NK cells produced by IV MSCs, the greater the improvement in LVEF. Butler J, Epstein SE, Lipinski MJ, Gheorghiade M, et al. Circ Res. 2017;120:332-340

Conclusions IV administered MSCs: Inhibit progressive myocardial deterioration that occurs following AMI and during chronic heart failure in murine models. The improved LV outcomes are causally related to potent systemic anti-inflammatory effects. Improve clinical outcomes and LV function in a Phase IIa clinical study.

These data suggest that MSCs, contrary to existing concepts: Do not have to be delivered directly to the heart to improve cardiac function. Rather, MSCs, delivered intravenously to patients with AMI or ICM/NICM, by modulating excessive inflammatory responses, may improve cardiac function and patient outcomes. Thus, invasive delivery procedures can be avoided. The validity of what could be a transformative concept to the stem cell field awaits the results of pivotal clinical trials.