Cell therapy: enhancing the therapeutic potential of cardiac progenitors for delivery post myocardial infarction. Rita Alonaizan

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1 Cell therapy: enhancing the therapeutic potential of cardiac progenitors for delivery post myocardial infarction Rita Alonaizan Department of Physiology, Anatomy & Genetics St Catherine s College Supervisor: Prof. Carolyn Carr Co-supervisor: Dr. Nicola Smart Start date: Hillary term 2016

2 Abstract Myocardial infarction (MI) is a leading cause of morbidity and mortality worldwide. Although cell therapy represents a promising approach for repair, poor survival and retention of the transplanted cells in the infarcted myocardium is a limitation to long-term improvement. Overcoming this to achieve the full potential of cell therapy holds high clinical importance. We hypothesise that mir-210 may improve the survival of the transplanted cells, thereby promoting further improvement in cardiac function. We found that mir-210 has a pro-survival and pro-angiogenic effect in vitro. We will assess if this translates in vivo in a mouse MI model. 2

3 Myocardial infarction (MI) is a leading cause of morbidity and mortality worldwide 1. In the UK there are nearly 200,000 hospital visits each year due to MIs (BHF). Apart from heart transplantation, current available compensatory treatments are unable to reverse the myocardial loss and deterioration of cardiac function caused by MI. However, cell therapy offers a promising approach for cardiac repair and regeneration. Different cell types are under evaluation for therapy post MI. However, despite promising results from pre-clinical models, clinical trials showed varied outcomes that may be attributed to the cell type and isolation, delivery method, and timing of administration 2,3. Mechanisms responsible for cardiac repair following cardiac progenitor cell administration remain under debate. Due to the significantly limited cell retention and differentiation in vivo, it has been suggested that the observed improvement is mostly due to cardiac progenitor cell production of paracrine factors that may stimulate endogenous regeneration or alter the tissue s response to injury 4. In my project, I aim to better define the progenitor cells found in the adult heart by detailed examination of their expression profile. I also aim to optimise cell therapy for cardiac repair by harnessing advances in the field of microrna therapeutics. This will be achieved by attempting to enhance the survival and retention of the progenitors, in the infarcted heart, as well as their angiogenic potential through mir-210 overexpression. Our group has previously shown that cardiac progenitors isolated as cardiosphere-derived cells (CDCs), an established protocol, enhance capillary density and cardiac function in a rat MI model 5. However, the CDC isolation protocol produces a highly variable population of cells. For example, it was noted that CDCs vary in their gene expression profile between different 3

4 isolations and in their expansion time, which can range from 5-84 days 6. In cell therapy, a consistent cell population with short expansion time is required. Therefore, in an attempt to optimise cardiac progenitor cell isolation, our group has developed a collagenase-trypsin (CT) protocol adapted from a study for isolating slowly adhering cells (SACs) from skeletal muscle 7. SACs were shown to have greater myogenic purity, in comparison to rapidly adhering cells (RACs). SACs had enhanced resistance in conditions of stress and higher therapeutic potential in mice post MI 8. We hypothesised that employing this technique to isolate SACs from cardiac tissue may result in a more homogenous and robust population. Indeed, the CT protocol generated a population with less variation in cell yield and expansion time (Malandraki-Miller, thesis). Moreover, we found that when CTs and CDCs were cultured without serum, thereby depriving the cells of essential nutrients, CTs had superior survival potential. To further characterise the CTs, I examined their expression profile by immunocytochemistry and found that they express the cardiac progenitor and mesenchymal markers ckit, Vimentin and CD44, among others (figure 1). However, the CT population was still found to be heterogeneous, as evident by, for example, some markers being expressed on a sub-population of cells and by some cells having higher expression of a marker than others. Therefore, implementing single-cell transcriptomics would uncover more information about this cell population and would answer two main questions needed to fully characterise these cells: (1) what sub-populations exist within this CT population? and (2) is there a combination of markers that uniquely defines each sub-population? Answering these questions may aid in the identification of a subpopulation with a superior therapeutic potential and markers that better define the progenitor population in the adult heart for future studies. Thereby, providing a 4

5 highly therapeutic population that is needed to improve the outcome of clinical trials. The hypoxia microrna mir-210 has been shown to be upregulated following MI 9 and to have a cytoprotective role in cardiomyocytes and a pro-angiogenic role in several ischemic conditions and was therefore selected for this study We found that mir-210 has a pro-survival effect in CTs and upregulates cell secretion of the pro-angiogenic factors VEGF and IGF-1. In addition, I uncovered a novel role for mir-210 in the regulation of mitophagy (figure 2). Mitophagy is a quality control process by which mitochondria are selectively targeted for degradation in autolysosomes, acidic vacuolar elements. This can have the beneficial effect of eliminating damaged mitochondria to maintain a healthy mitochondrial pool. Mitophagy can be triggered by certain stressors, such as hypoxia and nutrient starvation, to reduce the overall mass of mitochondria. This may reduce the generation of reactive oxygen species and the inefficient consumption of nutrients, thus promoting cell survival 13,14. Due to the ischemia and nutrient restriction found in the infarct region, it would be beneficial to pre-condition the cells for such an environment using a prosurvival and pro-angiogenic factor such as mir-210. Finally, we will assess if the pro-survival effect of mir-210 translates in vivo in a mouse MI model (figure 3). CTs will be isolated and expanded from adult mice that ubiquitously express green fluorescent protein (GFP). MI will be induced in wild type adult mice by permanent ligation of the left-anterior descending coronary artery. Mice will then immediately be injected with the GFP CTs carrying either mir-210 or a backbone as a control. In a group of mice, hearts will be collected at day 3 post MI to assess the retention of the injected cells by digesting the hearts into a single-cell dissociation and quantifying GFP CTs by flow cytometry. In 5

6 another group of mice, cardiac cine-magnetic resonance imaging (MRI) will be performed in vivo at days 7 and 42 to assess changes in cardiac function. Both the MI surgery and MRI will be performed by Prof. Carr. There is still great controversy about the identity of the cardiac progenitor. My project would shed some insight into the profile of a heterogeneous population of progenitors that exist in the adult heart by utilising single-cell transcriptome profiling. The results of the in vivo study would demonstrate if our cell isolation protocol generates a therapeutic population that can be used to alleviate MI. Moreover, if successful, it would identify mir-210 signalling or the mitophagy pathway as potential therapeutic targets in future cell therapy clinical trials. 6

7 Figure 1. CTs phenotypic analysis. CTs express the progenitor marker ckit and the mesenchymal markers CD44 and Vimentin. Figure 2. mir-210 upregulates mitophagy. To quantify mitophagy, mouse embryonic fibroblasts (MEFs) were kindly provided by Jo Poulton s group (Oxford) who developed a transgenic mouse model expressing a red fluorescent protein (RFP) tag on the mitochondrial gene cytochrome c oxidase (COX) VIII and a green fluorescent protein (GFP) tag on the autophagosome marker LC3 (part of the autophagy machinery). MEFs were transfected with mir-210 or a control mirna then switched to culture under hypoxia (2% O2) and serum starved for 3 days to stress the cells. Mitophagy was assessed by quantifying the fraction of mitochondria co-localising with autophagosomes using the JACoP Fiji plugin. Arrows highlight co-localisation. Verifying these results on CTs isolated from this mouse model is in progress. 7

8 Figure 3. Hypothesised model. Transplantation of mir-210-transfectd CTs into the infarcted myocardium may enhance CT retention and angiogenic potential, thereby promoting further improvement in cardiac function. 8

9 References 1. Benjamin, E. J. et al. Heart Disease and Stroke Statistics 2017 Update: A Report From the American Heart Association. Circulation (2017). 2. Behfar, A., Crespo-Diaz, R., Terzic, A. & Gersh, B. J. Cell therapy for cardiac repair lessons from clinical trials. Nat. Rev. Cardiol. 11, 232 (2014). 3. Madonna, R. et al. Position Paper of the European Society of Cardiology Working Group Cellular Biology of the Heart: cell-based therapies for myocardial repair and regeneration in ischemic heart disease and heart failure. Eur. Heart J. 37, (2016). 4. Chimenti, I. et al. Relative Roles of Direct Regeneration Versus Paracrine Effects of Human Cardiosphere-Derived Cells Transplanted Into Infarcted Mice. Circ. Res. 106, 971 LP-980 (2010). 5. Carr, C. A. et al. Cardiosphere-derived cells improve function in the infarcted rat heart for at least 16 weeks - an mri study. PLoS One 6, (2011). 6. Chan, H. H. L. et al. Human Cardiosphere-Derived Cells from Patients with Chronic Ischaemic Heart Disease Can Be Routinely Expanded from Atrial but Not Epicardial Ventricular Biopsies. J. Cardiovasc. Transl. Res. 5, (2012). 7. Gharaibeh, B. et al. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat. Protoc. 3, (2008). 8. Okada, M. et al. Human Skeletal Muscle Cells With a Slow Adhesion Rate After Isolation and an Enhanced Stress Resistance Improve Function of Ischemic Hearts. Mol. Ther. 20, (2012). 9. Boštjančič, E., Zidar, N. & Glavač, D. MicroRNA Microarray Expression Profiling in Human Myocardial Infarction. Dis. Markers 27, (2009). 10. Won Kim, H., Haider, H. K., Jiang, S. & Ashraf, M. Ischemic Preconditioning Augments Survival of Stem Cells via mir-210 Expression by Targeting Caspase-8- associated Protein 2. J. Biol. Chem. 284, (2009). 11. Lou, Y.-L. et al. mir-210 activates notch signaling pathway in angiogenesis induced by cerebral ischemia. Mol. Cell. Biochem. 370, (2012). 12. Wang, N. et al. Mesenchymal stem cells-derived extracellular vesicles, via mir-210, improve infarcted cardiac function by promotion of angiogenesis. Biochim. Biophys. Acta - Mol. Basis Dis. 1863, (2017). 13. Kurihara, Y. et al. Mitophagy Plays an Essential Role in Reducing Mitochondrial Production of Reactive Oxygen Species and Mutation of Mitochondrial DNA by Maintaining Mitochondrial Quantity and Quality in Yeast. J. Biol. Chem. 287, (2012). 14. Ashrafi, G. & Schwarz, T. L. The pathways of mitophagy for quality control and clearance of mitochondria. Cell Death Differ. 20, (2012). 9