This research is funded by the North Shore Heart Research Foundation

Study 1

Title: Efficacy of genetically engineered mesenchymal stem cells (MSC) for regeneration of the post-infarct heart - assessed by cardiac magnetic resonance imaging (MRI)

Investigators: Stephen Hunyor, Mark Sussman (State University of San Diego, USA)

Funded since: 2009

Related publications: Weir C, Morel-Kopp M-C, Gill A, Tinworth K, Ladd L, Hunyor SN, Ward C. Mesenchymal stem cells: isolation, characterisation and in vivo fluorescent dye tracking. Heart, Lung and Circulation 17 (2008), pp. 395-403

Overview

Stem cell therapy and the related field of regenerative medicine have progressed at a dizzying pace since 2008. However, its safe and effective clinical application to the heart still requires meticulous research, because of the complexities of mechanical and electrical integration of newly delivered stem cells to form new heart muscle, blood vessels and conduction tissue.

Report

Despite the recent virtual revolution in ability to produce ‘embryo-like' pluripotent stem cells (iPSCs) from adult skin and fat cells, the MSCs we are using in our experiments are a good alternative for the heart. They can be obtained from the bone marrow of the eventual recipient and do not cause immune incompatibilities.

Our MSCs have been genetically engineered using the Pim-1 gene for superior survival when delivered to the ‘hostile environment' of a damaged heart. This unique collaboration with Professor Sussman's group in San Diego saw the cells travelling safely to the USA and back - itself quite an achievement. We have done the preliminary experiments on heart attack and intracoronary delivery of the transduced MSCs to areas of heart damage. We have specific equipment to allow use of the magnetic resonance imaging facility, which will be brought on stream early in 2010.

We were invited to join the new Sydney Centre for Developmental and Regenerative Medicine, presented our work at Sydney University's 2nd Sino-Australian Symposium: Active Compounds, Molecular Imaging and Clinical Trials and its 1st Regenerative Medicine Symposium, and at the Victor Chang Cardiac Research Institute's International Symposium Cardiology at the Frontier: Development, Stem cells and Heart Failure.

'Human stem cell research leads to two important areas of application:

• the development of cell-based regenerative medicine
• the development of models of disease and assays for the discovery and development of small molecule drugs, biologics, and medical devices that can be combined with drugs, biologics and cells.'

- Professor Shinya Yamanaka, the original developer of induced pluripotent stem cells (iPSCs) and recipient of the 2009 Lasker Prize, USA (quoted in World Stem Cell Summit Report - 2009).

Study 2

Title: Repair of the damaged heart and the role of Wnt growth factors

Scholarship recipient: Jason Seow

Funded since: 2007

Overview

Cardiovascular diseases remain one of Australia's major challenges, despite advances in cardiovascular medicine. Research and early clinical trials have shown that stem cells (SCs) have a pivotal role both in maintaining healthy heart muscle and repairing it after damage. A recent finding is that resident cardiac stem cells (CSCs) in the heart have an important role in damage repair. This opens up a new window of opportunity for heart regeneration. However, the mechanisms underlying this exciting development remain to be unravelled.

This project investigates the role of a specific group of growth factors (Wnts) shown to participate in normal cardiac development and tissue remodelling of the damaged heart. Wnt signalling pathways play key roles in numerous cellular activities, and are important in homeostasis (maintenance of ‘status quo') and cardiogenesis (generation of new heart cells).

Interestingly, Wnts have effects that can be agonistic or antagonistic in generating new heart muscle cells and blood vessels. Understanding Wnts will give valuable insights into differentiation of heart cells and cardiac remodelling. While previous studies have investigated the effects of Wnts in mesenchymal (MSC) and embryonic stem cells (ESC), their role in transformating resident CSCs has yet to be clarified.

Our goal is to provide an effective treatment where heart muscle is literally ‘dead' or where arteries are permanently clogged.

Report

We have worked on:

• Wnt ligands and cardiomyogenic differentiation of sheep MSCs (sMSC) and mouse CSCs (mCSCs)
• elucidating the Wnt signaling pathway in inducing cardiogenesis
• determining functional roles of the Wnt family member - Wnt16 - in stem cell cardiogenesis.

Our laboratory has shown that sMSCs can be readily trans-differentiated into cardiomyocyte lineages with 5-Azacytidine (5-Aza) - an agent with diverse anti-metabolic activities - and has been used for cardiomyogenic differentiation in stem cells. However, to bypass potential toxic effects we have sought a 5-Aza replacement. We studied wingless (Wnt) ligands acting on the Wnt receptor and its signalling pathways, and demonstrated that sMSCs display cardiomyocyte phenotype on introduction of Wnt3 and Wnt5 ligands. Using Western blotting analysis, expression of cardiac transcription markers (Troponin I, Nkx 2-5 and GATA4) indicate cardiomyogenic differentiation of MSCs. Thus Wnt ligands may serve to replace 5-Aza.

Specific resident mCSCs have also been isolated and cultured from mouse hearts. Co-culture study showed that Wnt is involved in the differentiation of CSCs into cardiomyocytes. When Wnt ligands are introduced to these CSCs, cardiogenesis was readily enhanced with upregulation of cardiomyocyte markers (MHC, ANP and α-sarcomeric actin).

Furthermore, Wnt16 exists in two different variants, and we have found its ubiquitous expression in heart. In our myocardial-infarct model, Wnt16 was upregulated in border zone, indicative of its early involvement in heart damage/repair. We also elucidated that Wnt16 plays a pivotal role in stem cell cardiogenic differentiation because of its regulation in both MSC and CSC studies.

To date, we have shown Wnt's involvement during heart damage and repair by inducing cardiogenesis in adult stem cells. We are working on creating an infarct and ischemia-reperfusion heart model in order to further understand the role of Wnt.

We have started studies to determine the molecular mechanism of action of Wnt ligands, particularly Wnt16, and its signalling pathway in stem cell cardiac regeneration.

Benefits

Understanding the effect of Wnt ligands on stem cells and cardiogenesis should enhance the use of stem cells in cellular or gene therapy to combat heart damage after a heart attack. Our goal is to provide an effective treatment where heart muscle is literally 'dead', or where arteries are permanently clogged.